NZ619023B2

NZ619023B2 - Serpin fusion polypeptides and methods of use thereof

Info

Publication number: NZ619023B2
Application number: NZ619023A
Authority: NZ
Inventors: Quinn Deveraux; Brendan P Eckelman; Grant B Guenther; Peter L Nguy; John C Timmer
Original assignee: Inhibrx Inc
Priority date: 2011-06-28
Filing date: 2012-06-28
Publication date: 2015-11-03

Abstract

Disclosed is an isolated fusion protein comprising at least one human serpin polypeptide comprising a human alpha-1 antitrypsin (AAT) polypeptide comprising the amino acid sequence of SEQ ID NO: 1 operably linked to an immunoglobulin Fc polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 6, wherein the sequences are as defined in the complete specification. Also disclosed is an isolated fusion protein comprising at least one human alpha-1 antitrypsin (AAT) polypeptide comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33 operably linked to an immunoglobulin Fc polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 6, wherein the sequences are as defined in the complete specification. Further disclosed is the use of said fusion proteins in the manufacture of a medicament for treating or alleviating inflammation or a symptom of an inflammatory disease or disorder while reducing the risk of infection, in a subject in need thereof. 98% identical to the amino acid sequence of SEQ ID NO: 6, wherein the sequences are as defined in the complete specification. Also disclosed is an isolated fusion protein comprising at least one human alpha-1 antitrypsin (AAT) polypeptide comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33 operably linked to an immunoglobulin Fc polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 6, wherein the sequences are as defined in the complete specification. Further disclosed is the use of said fusion proteins in the manufacture of a medicament for treating or alleviating inflammation or a symptom of an inflammatory disease or disorder while reducing the risk of infection, in a subject in need thereof.

Description

SERPIN FUSION POLYPEPTIDES AND METHODS OF USE THEREOF Related Applications This application claims the benefit of U.S. Provisional Application No. 61/502055, filed June 28, 201 1; U.S. Provisional Application No. 61/570394, filed December 14, 201 1; and U.S. Provisional Application No. 61/577204, filed December 19, 201 1; and U.S. Provisional Application No. 61/638168, filed April 25, 2012. The contents of each of these applications are hereby incorporated by nce in their entirety.

Field of the Invention This invention relates to molecules, particularly polypeptides, more particularly fusion proteins that e a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptides and a second ptide. onally, the invention relates to fusion proteins that include a serpin ptide or an amino acid sequence that is derived from serpin polypeptides, a second polypeptide, and a third polypeptide.

Specifically, this invention relates to fusion proteins that include at least one serpin polypeptide and a second polypeptide or fusion ns that include at least one serpin ptide, a second polypeptide, and a third polypeptide, where the second and third polypeptides of the fusion proteins of the invention can be at least one the following: an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide; a cytokine targeting polypeptide or a sequence d from a cytokine targeting polypeptide; a WAP domain containing polypeptide or a sequence derived from a WAP containing polypeptide; or an albumin polypeptide or an amino acid sequence that is derived from a serum albumin polypeptide. This invention also relates to s of using such molecules in a variety of therapeutic and diagnostic indications, as well as methods of producing such molecules.

Background of the Invention Aberrant serine protease activity or an imbalance of protease-to-protease inhibitor can lead to protease-mediated tissue destruction and inflammatory responses.

Accordingly, there exists a need for therapeutics and therapies that target aberrant serine se activity and/or imbalance of protease-to-protease inhibitor. Furthermore, enhanced therapeutic effects may be gained through the ation of aberrant cytokine signaling and serine protease activity. In addition, serpin proteins have trated anti-infective activities while targeting inflammatory cytokines has been shown to increase the risk of infection. The fusion proteins of this invention have the potential to dampen inflammatory cytokine activity and limit the risk of infection.

Summary of the Invention The fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide (Polypeptide 1) and second polypeptide (Polypeptide 2). Additionally, the fusion proteins described herein include a serpin ptide or an amino acid sequence that is derived from a serpin ptide (Polypeptide 1), a second ptide (Polypeptide 2), and a third polypeptide (Polypeptide 3). As used interchangeably herein, the terms "fusion protein" and "fusion polypeptide" refer to a serpin ptide 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 ptide. The individualized elements of the fusion protein can be linked in any of a variety of ways, including for example, direct attachment, the use of an intermediate or a spacer peptide, the use of a linker region, the use of a hinge region or the use of both a linker and a hinge . In some embodiments, the linker region may fall within the sequence of the hinge region, or alternatively, the hinge region may fall within the sequence of the linker region. Preferably, the linker region is a peptide ce. For example, the linker peptide includes anywhere from zero to 40 amino acids, e.g., from zero to 35 amino acids, from zero to 30 amino acids, from zero to 25 amino acids, or from zero to 20 amino acids. ably, the hinge region is a peptide sequence. For example, the hinge peptide includes anywhere from zero to 75 amino acids, e.g. , from zero to 70 amino acids, from zero to 65 amino acids or from zero to 62 amino acids. In embodiments where the fusion protein includes both a linker region and 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 include anywhere from zero to 90 amino acids, e.g., from zero to 85 amino acids or from zero to 82 amino acids.

In some embodiments, the serpin polypeptide and the second polypeptide can be linked through an intermediate g protein. In some embodiments, the serpin-based portion and second polypeptide portion of the fusion protein may be non-covalently linked.

In some embodiments, fusion ns according to the invention can have one of the following formulae, in an N-terminus to C-terminus direction or in a C-terminus to N-terminus direction: Polypeptide l - hingem - Polypeptide (a) 2<¾) Polypeptide l - „ - Polypeptide (a) < Polypeptide l - „ - hingem - Polypeptide 2 (a) (b) Polypeptide l - hingem - „ - Polypeptide 2(b) Polypeptide l - Polypeptide 2(b)- Polypeptide 3 (a) (C) Polypeptide l - hingem - Polypeptide 2(b)- hingem - Polypeptide 3 (a) (C) Polypeptide l - linker„ - Polypeptide 2(b)- linker„ - Polypeptide 3 (a) (C) Polypeptide l - hingem - linker„ - Polypeptide 2(b)-hinge m - linker„ - Polypeptide 3(c) Polypeptide l - linker„ - hingem - Polypeptide 2(b)- linker„ - hingem- Polypeptide 3 where n is an integer from zero to 20, where m is an integer from 1 to 62 and where a, b, and c integers of at least 1. These embodiments include the above formulations and any variation or combination thereof. For example, the order of ptides in the ae also includes Polypeptide 3 - Polypeptide l - Polypeptide 2(b), (C) (a) Polypeptide 2 - Polypeptide 3 - Polypeptide l or any variation or ation (b) (C) (a), thereof.

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 set of proteins able to inhibit proteases. Serpin proteins le for use in the fusion proteins provided herein include, by way of non-limiting example, alpha-1 antitrypsin (AAT), antitrypsin-related n NA2), alpha 1-antichymotrypsin (SERPINA3), kallistatin (SERPINA4), monocyte neutrophil elastase inhibitor (SERPINB1), PI-6 (SERPINB6), antithrombin (SERPINC1), plasminogen activator tor 1 (SERPINE1), alpha 2-antiplasmin (SERPINF2), complement 1-inhibitor (SERPING1), and erpin (SERPINI1).

In some embodiments, the Polypeptide 1 ce includes an alpha- 1 antitrypsin (AAT) polypeptide sequence or an amino acid sequence that is derived from AAT. In some embodiments, the Polypeptide 1 sequence includes a portion of the AAT protein. In some embodiments, the Polypeptide 1 sequence includes at least the reactive site loop portion of the AAT protein. In some embodiments, the reactive site loop portion of the AAT protein includes at least the amino acid sequence: GTEAAGAMFLEAI PMS IPPEVKFNK SEQ ID NO :1).

In a preferred embodiment, the AAT polypeptide sequence or an amino acid sequence that is derived from AAT is or is derived from a human AAT polypeptide In some embodiments, the fusion protein includes a full-length human AAT polypeptide sequence having the following amino acid sequence: 1 EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI FFSPVSIATA 61 FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL RTLNQPDSQL QLTTGNGLFL 121 VDKF LEDVKKLYHS FGDT EEAKKQINDY VEKGTQGKIV DLVKELDRDT 181 VFALVNYIFF KGKWERPFEV KDTEEEDFHV DQVTTVKVPM KRLG FNIQ HCKKLSSWVL 241 LMKYLGNATA IFFLPDEGKL QHLENELTHD IITKFLENED RRSASLHLPK LSITGTYDLK 301 SVLGQLGITK VFSNGADLSG LKLS KAVHKAVLTI DEKGTEAAGA MFLEAI PMS I 361 PPEVKFNKPF VFLMIEQNTK SPLFMGKVVN PTQK (SEQ ID NO: 2 ) In some embodiments, the fusion protein includes a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% cal to the amino acid sequence of SEQ ID NO: 2 .

In some embodiments, the AAT polypeptide sequence is, or the amino acid sequence derived from an AAT polypeptide is derived from, one or more of the human AAT ptide ces shown in GenBank Accession Nos. AAB59495.1, CAJ15161.1, .3, AAB59375.1, AAA5 1546.1, CAA25838.1, NP 235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001 121 179.1, NP_001 121 178.1, NP_001 121 177.1, NP_001 121 176.16, NP_001 121 175.1, NP_001 121 174.1, NP_001 121 172.1, and/or AAA51547.1.

In some embodiments, the fusion proteins contain one or more mutations.

For example, the fusion protein ns at least one mutation at a methionine (Met) residue in the serpin portion of the fusion protein. In these Met mutations, the Met e can be substituted with any amino acid. For example, the Met residue can be tuted with an amino acid with a hydrophobic side chain, such as, for example, leucine (Leu, L). t wishing to be bound by theory, the Met mutation(s) prevent oxidation and subsequent inactivation of the inhibitory activity of the fusion proteins of the invention. In some embodiments, the Met residue can be substituted with a d residue, such as, for example, glutamate (Glu, E). In some embodiments, the Met mutation is at position 358 of an AAT polypeptide. For example, the Met mutation is Met358Leu (M358L). In some embodiments, the Met mutation is at position 35 1 of an AAT polypeptide. For example, the Met mutation is Met35 lGlu (M35 IE). In some embodiments, the Met mutation is at position 351 and at position 358 of an AAT polypeptide, for example, the Met mutation is Met351Glu ) and Met358Leu (M358L). For example, the reactive site loop of this variant of the fusion AAT polypeptide has the following sequence: GTEAAGAEFLEAI PLS IPPEVKFNK (SEQ ID NO: 32) . In some embodiments, the Met mutation is at position 351 and at position 358 of an AAT polypeptide, for example, the Met mutation is Met351Leu ) and Met358Leu (M358L). For example, the reactive site loop of this variant of the fusion AAT polypeptide has the following sequence: GTEAAGALFLEAI PLS FNK (SEQ ID NO: 33).

In some embodiments, the second polypetide (Polypeptide 2) of the serpin fusion protein is an Fc polypeptide or derived from an Fc polypeptide. These embodiments are referred to tively herein as "serpin-Fc fusion proteins." The serpin-Fc fusion proteins described herein e at least a serpin polypeptide or an amino acid sequence that is derived from a serpin and an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide. In some ments, the serpin-Fc fusion protein includes a single serpin polypeptide. In other embodiments, the serpin-Fc fusion proteins includes more than one serpin polypeptide, and these ments are collectively referred to herein as "serpin(a')-Fc fusion protein," wherein (a') is an integer of at least 2 . In some embodiments, each serpin polypeptides in a serpin(a')-Fc fusion protein includes the same amino acid sequence. In other embodiments, each serpin polypeptide in a serpin ')-Fc fusion protein includes serpin polypeptides with distinct amino acid sequences. The serpin polypeptides of serpin ')-Fc fusion proteins can be d at any position within the fusion protein.

] In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein includes at least the amino acid sequence of the reactive site loop portion of the AAT protein. In some embodiments, the reactive site loop portion of the AAT protein includes at least the amino acid ce of SEQ ID NO: 1. In some embodiments, the serpin ptide of the serpin-Fc fusion n includes at least the amino acid sequence of a variant of the reactive site loop portion of the AAT protein. In some embodiments, the variant of the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In some embodiments, the serpin ptide of the -Fc fusion protein includes at least the full-length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 2 . In some embodiments the serpin polypeptide of the serpin-Fc fusion protein includes human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33.

In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein includes the AAT polypeptide sequence is or the amino acid sequence derived from an AAT ptide is derived from one or more of the human AAT polypeptide sequences shown in GenBank Accession Nos. AAB59495 .1, CAJ 15161.1, P01009.3 , AAB59375 .1, AAA5 1546.1, CAA25838.1, NP 001002235.1, CAA34982.1, NP 001002236.1, NP_000286.3, NP_001 121 179.1, NP_001 121 178. 1, NP_001 121 177.1, NP_001 121 , NP_001 121 175. 1, NP_001 121 174.1, NP_001 121 172.1, and/or AAA51547.1.

In some embodiments, the Fc polypeptide of the fusion protein is a human Fc polypeptide, for e, a human IgG Fc polypeptide sequence or an amino acid ce that is derived from a human IgG Fc ptide sequence. For example, in some embodiments, the Fc polypeptide is a human IgGl Fc polypeptide or an amino acid sequence that is derived from a human IgGl Fc polypeptide sequence. In some embodiments, the Fc polypeptide is a human IgG2 Fc polypeptide or an amino acid sequence that is 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 that is 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 that is derived from a human IgG4 Fc polypeptide sequence. In some embodiments, the Fc ptide is a human IgM Fc polypeptide or an amino acid sequence that is derived from a human IgM Fc polypeptide sequence.

In some embodiments where the fusion protein of the invention es an Fc polypeptide, the Fc polypeptide of the fusion n includes a human IgGl Fc polypeptide sequence having the following amino acid sequence: 1 APELLGGPSV FLFPPKPKDT PEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK 61 PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 121 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL 181 TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 3 ) In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgGl Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3 .

In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide is mutated or modified to enhance FcRn binding. In these embodiments the mutated or modified Fc polypeptide es the ing mutations: Met252Tyr, Ser254Thr, Glu (M252Y, S256T, T256E) or Met428Leu and Asn434Ser , N434S) using the Kabat numbering system. In some embodiments the Fc ptide portion is mutated or otherwise modified so as to disrupt Fc-mediated dimerization. In these ments, the fusion protein is monomeric in nature.

] In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG2 Fc polypeptide sequence having the following amino acid sequence: 1 APPVAGPSVF LFPPKPKDTL MISRTPEVTC HEDP EVQFNWYVDG VEVHNAKTKP 61 REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL 121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT 181 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 4 ) In some embodiments where the fusion protein of the invention includes an Fc ptide, the Fc polypeptide of the fusion protein includes a human IgG2 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4 .

In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion n includes a human IgG3 Fc polypeptide sequence having the following amino acid sequence: 1 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD GVEVHNAKTK 61 PREEQYNSTF TVLH QDWLNGKEYK CKVSNKALPA PIEKTISKTK GQPREPQVYT 121 EMTK CLVK GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL 181 TVDKSRWQQG NIFSCSVMHE ALHNRFTQKS LSLSPGK (SEQ ID NO: 5 ) In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion n includes a human IgG3 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 5 .

In some embodiments where the fusion n of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG4 Fc polypeptide sequence having the following amino acid sequence: 1 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED WYVD GVEVHNAKTK 61 PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK QVYT 121 LPPSQEEMTK NQVSLTCLVK GFYPDIAVEW ENNY KTTPPVLDSD GSFFLYSRLT 181 VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK (SEQ ID NO: 6 ) In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG4 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6 .

In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgM Fc polypeptide sequence having the following amino acid sequence: 1 KVSV FVPPRDGFFG NPRKSKLICQ ATGFSPRQIQ VSWLREGKQV GSGVTTDQVQ 61 AEAKESGPTT YKVTSTLTIK ESD LGQS F TCRVDHRGLT FQQNASSMCV IRVF 121 ASIF LTKSTKLTCL VTDLTTYDSV TISWTRQNGE AVKTHTNISE SHPNATFSAV 181 GEASICEDDW NSGERFTCTV THTDLPSPLK QTISRPKG (SEQ ID NO: 7 ) In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgM Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7 .

In some embodiments of the fusion proteins provided herein, the second polypeptide (Polypeptide 2) of the serpin fusion protein is a cytokine ing polypeptide or derived from a cytokine targeting polypeptide. These ments are referred to collectively herein as n-cytokine targeting polypeptide fusion proteins." The serpin- cytokine targeting polypeptide fusion proteins described herein include at least a serpin ptide or an amino acid sequence that is derived from a serpin polypeptide and a cytokine targeting polypeptide, or derivation thereof. In some embodiments, the serpin- cytokine targeting polypeptide fusion protein includes a single serpin polypeptide. In other ments, the serpin-cytokine targeting ptide fusion protein includes more than one serpin polypeptide, and these embodiments are collectively referred to herein as "serpin(a')-cytokine targeting polypeptide fusion proteins," wherein (a') is an integer of at least 2 . In some embodiments, each serpin polypeptide in a serpin ')-cytokine targeting polypeptide fusion protein includes the same amino acid sequence. In other ments, each serpin polypeptide of a serpin -cytokine targeting polypeptide fusion n es serpin polypeptides with distinct amino acid sequences.

In some embodiments, the cytokine ing polypeptide of the serpin- ne targeting polypeptide fusion protein is a cytokine receptor or d from a cytokine receptor. In a preferred embodiment, the cytokine targeting polypeptide or an amino acid ce that is d from the cytokine receptor is or is derived from a human ne receptor sequence. In other embodiments, the cytokine targeting polypeptide is an antibody or an antibody fragment, for example an anti-cytokine antibody or anti-cytokine antibody fragment. In a preferred embodiment, the cytokine targeting polypeptide or an amino acid sequence that is derived from the dy or antibody fragment is derived from a chimeric, humanized, or fully human antibody sequence. The term antibody fragment includes single chain, Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.

In other ments, the cytokine targeting polypeptide binds a cytokine receptor and prevents binding of a cytokine to the or. In other ments, the cytokine targeting polypeptide is an antibody or an dy fragment, for example an anti- cytokine or antibody or anti-cytokine receptor antibody fragment.

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

In some embodiments, the serpin ptide of the serpin-cytokine targeting fusion n includes an AAT polypeptide sequence or an amino acid sequence d from an AAT polypeptide that is or is derived from one or more of the human AAT polypeptide sequences shown in GenBank Accession Nos. AAB59495.1, CAJ15 161.1, P01009.3, AAB59375.1, AAA5 1546.1, CAA25838.1, NP 001002235.1, CAA34982.1, NP_001002236.1, 286.3, NP_001 121 179.1, NP 001 121 178. 1, NR OOI 121 177.1, NP_001 121 176.16, NP_001 121 175. 1, NP_001 121 174.1, NP_001 121 172.1, and/or AAA51547.1.

The serpin-cytokine ing polypeptide fusion protein can incorporate a portion of the serpin-Fc fusion protein. For example, an dy contains an Fc polypeptide. Therefore, in some embodiments where the cytokine targeting ptide is a ne -targeting antibody, the serpin-cytokine targeting polypeptide fusion protein will incorporate a n of the serpin-Fc fusion protein. Furthermore, most receptor fusion proteins that are of eutic utility are Fc fusion proteins. Thus, in some embodiments, wherein the serpin-cytokine targeting polypeptide fusion protein is a serpin-cytokine receptor fusion protein, the serpin-cytokine targeting polypeptide fusion protein may incorporate an Fc polypeptide in addition to the serpin portion and the cytokine receptor portion.

In some embodiments, where the serpin-cytokine targeting polypeptide fusion protein includes an Fc polypeptide sequence, the Fc polypeptide sequence includes or is derived from the amino acid sequence of any one of SEQ ID NO: 3, 4, 5, 6, or 7 . In some ments where the serpin-cytokine targeting fusion protein includes an Fc polypeptide sequence, the Fc polypeptide ce has at least 50%>, 60%>, 65%>, 70%>, 75%>, 80%>, 85%>, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, or 7 . In some embodiments, the serpin polypeptide and the cytokine targeting polypeptide are operably linked via a linker region, for example, a glycine-serine linker or glycine-serine based linker. In some embodiments, the serpin polypeptide and the cytokine targeting polypeptide are operably linked via a hinge region.

In some embodiments, the serpin polypeptide and the cytokine targeting polypeptide are operably linked via a linker region and a hinge region. In other ments, the serpin polypeptide and the cytokine targeting polypeptide are directly attached.

In some embodiments of the fusion proteins provided herein, the second polypeptide eptide 2) of the serpin fusion protein is a whey acidic protein (WAP) domain containing polypeptide, or an amino acid sequence that is derived from a WAP domain containing polypeptide. These embodiments are referred to collectively herein as "serpin-WAP domain fusion ns." The serpin-WAP domain fusion proteins include at least a serpin polypeptide or at least an amino acid sequence that is derived from a serpin, a WAP domain-containing ptide or an amino acid sequence that is derived from a WAP domain-containing polypeptide. In some embodiments, the -WAP domain fusion protein includes a single serpin polypeptide. In other embodiments, the serpin-WAP targeting polypeptide fusion protein includes more than one serpin polypeptide. These embodiments are collectively referred to herein as "serpin(a')-WAP domain fusion proteins," wherein (a') is an integer of at least 2 . In some embodiments, serpin ptides of the serpin(a')-WAP domain fusion protein includes the same amino acid sequence. In other embodiments, the serpin polypeptides of the serpin ')-cytokine targeting polypeptide fusion n, includes serpin ptides with distinct amino acid sequences.

] These serpin-WAP domain fusion proteins include a WAP domain containing polypeptide or polypeptide sequence that is or is derived from a WAP domain containing polypeptide. The WAP domain is an evolutionarily conserved sequence motif of 50 amino acids containing eight cysteines found in a characteristic lfide core arrangement (also called a four-disulfide core motif). The WAP domain ce motif is a functional motif characterized by serine protease inhibition activity in a number of proteins.

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

In some embodiments, the WAP domain-containing polypeptide sequence of the fusion protein includes a secretory yte protease inhibitor (SLPI) polypeptide sequence or an amino acid sequence that is derived from SLPI. These embodiments are referred to herein as "serpin-SLPI-derived fusion proteins." In some embodiments, the SLPI ptide sequence comprises a portion of the SLPI n, such as for example, the WAP2 domain or a sub-portion thereof. In a preferred embodiment, the SLPI polypeptide sequence or an amino acid sequence that is derived from SLPI is or is derived from a human SLPI polypeptide sequence.

In some embodiments of the serpin-SLPI fusion proteins of the invention, the SLPI sequence or a SLPI-derived sequence of the fusion protein es a full-length human SLPI polypeptide ce having the ing amino acid sequence: 1 MKSSGLFPFL VLLALGTLAP GKSF KAGVCPPKKS AQCLRYKKPE CQSDWQCPGK 61 KRCCPDTCGI DTPN PTRRKPGKCP VTYGQCLMLN PPNFCEMDGQ CKRDLKCCMG 121 MCGKSCVSPV KA (SEQ ID NO :8) In some embodiments of the serpin-SLPI fusion protein of the invention, the SLPI sequence or a SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide ce 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.

In some embodiments of the serpin-SLPI fusion protein of the invention, the SLPI sequence or a SLPI-derived sequence of the fusion n includes a portion of the full-length human SLPI polypeptide sequence, where the n has the following amino acid sequence: 1 SGKSFKAGVC PPKKSAQCLR YKKPECQSDW QCPGKKRCCP DTCGIKCLDP VDTPNPTRRK 61 PGKCPVTYGQ CLMLNPPNFC KRDL CGKS CVSPVKA (SEQ ID NO: 9 ) In some embodiments of the serpin-SLPI fusion protein of the invention, the SLPI sequence or a SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid ce of SEQ ID NO: 9 .

In some embodiments of the serpin-SLPI fusion protein of the invention, the SLPI sequence or a SLPI-derived sequence of the fusion protein includes the WAP2 domain of the full-length human SLPI polypeptide sequence, where the WAP2 domain has the following amino acid sequence: 1 TRRKPGKCPV TYGQCLMLNP PNFCEMDGQC KRDLKCCMGM CGKSCVSPVK A (SEQ ID NO: 10) In some embodiments of the -SLPI fusion protein of the invention, the SLPI sequence or a SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide ce 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.

In some embodiments of the -SLPI fusion proteins of the invention, the SLPI polypeptide sequence or the amino acid sequence derived from an SLPI polypeptide is or is derived from, one or more of the human SLPI polypeptide sequences shown in GenBank Accession Nos. CAA28187.1, NP_003055.1, EAW75869.1, P03973.2, AAH20708.1, CAB64235.1, CAA28188.1, AAD19661.1, and/or BAG35125.1.

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

In other embodiments, the WAP -containing polypeptide sequence of the fusion protein includes an elafin polypeptide sequence or an amino acid sequence that is derived from elafin. These embodiments are referred to herein as "serpin-elafm fusion proteins. In some embodiments, the elafin polypeptide sequence includes a portion of the elafin n, such as for example, the WAP domain or a sub-portion thereof. In a preferred embodiment, the elafin polypeptide ce or an amino acid sequence that is derived from elafin is or is derived from a human elafin polypeptide sequence.

In some embodiments of the serpin-elafm fusion proteins, the fusion protein includes a full-length human elafin polypeptide sequence having the ing amino acid 1 LIVV VFLIAGTLVL EAAVTGVPVK GQDTVKGRVP FNGQDPVKGQ VSVKGQDKVK 61 AQEPVKGPVS TKPGSCPIIL IRCAMLNPPN RCLKDTDCPG IKKCCEGSCG MACFVPQ (SEQ ID NO: 11) In some embodiments of the serpin-elafm fusion proteins, the fusion protein includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 11.

In some embodiments of the serpin-elafm fusion proteins, the fusion protein includes a portion of the full-length human elafin polypeptide sequence, where the portion has the following amino acid sequence: 1 VKGQ DTVKGRVPFN GQDPVKGQVS VKGQDKVKAQ EPVKGPVSTK ILIR 61 CAMLNPPNRC LKDTDCPGIK KCCEGSCGMA CFVPQ (SEQ ID NO: 12) In some embodiments of the serpin-elafm fusion proteins, the fusion protein includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12.

In some embodiments of the serpin-elafm fusion proteins, the fusion protein es the WAP domain of the full-length human elafin polypeptide sequence, where the WAP domain has the following amino acid sequence: 1 VSTKPGSCPI ILIRCAMLNP PNRCLKDTDC PGIKKCCEGS CGMACFVPQ (SEQ ID NO: 13) In some embodiments of the serpin-elafm fusion proteins, the fusion protein includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13.

In some embodiments of the serpin-elafm fusion ns, the elafin ptide sequence or the amino acid sequence derived from an elafin polypeptide is derived from one or more of the human elafin polypeptide sequences shown in GenBank Accession Nos. P19957.3, NP 002629.1, BAA02441.1, 14.1, EAW75813.1, . 1, and/or NP_542 181.1.

In other embodiments, the WAP -containing polypeptide sequence of the fusion protein es an Eppin polypeptide sequence or an amino acid sequence that is d from Eppin. These embodiments are referred to herein as "serpin (a')-Eppin fusion proteins. In some embodiments, the Eppin polypeptide sequence of the serpin-Eppin fusion protein includes a portion of the Eppin n, such as for e, the WAP domain or a sub-portion thereof. In a preferred embodiment, the Eppin polypeptide sequence or an amino acid sequence that is derived from Eppin is or is d from a human Eppin polypeptide sequence.

In some embodiments of the -Eppin fusion proteins, the Eppin polypeptide sequence or amino acid sequence derived from an Eppin ptide is or is derived from one or more of the human Eppin polypeptide sequences shown in GenBank Accession Nos. 095925.1, NP_065131.1, AAH44829.2, AAH53369.1, AAG00548.1, AAG00547.1, and/or AAG00546.1.

In some embodiments, the serpin polypeptide of the serpin-WAP domain fusion protein includes at least the amino acid sequence of the reactive site loop portion of the AAT protein. In some embodiments, the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 1. In some embodiments, the serpin polypeptide of the serpin-WAP fusion protein includes at least the amino acid sequence of a variant of the reactive site loop n of the AAT protein. In some embodiments, the variant of the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In some embodiments, the serpin polypeptide of the serpin-WAP domain fusion protein es at least the fulllength human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 2 .

In some embodiments the serpin polypeptide of the serpin-WAP domain fusion protein includes human AAT polypeptide ce 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.

In some embodiments, the serpin polypeptide of the -WAP domain fusion protein includes the AAT polypeptide sequence is, or the amino acid sequence derived from an AAT polypeptide is derived from, one or more of the human AAT polypeptide sequences shown in GenBank Accession Nos. AAB59495.1, CAJ15 161.1, P01009.3, 75.1, AAA5 1546.1, CAA25838.1, NP 001002235.1, CAA34982.1, 002236.1, NP_000286.3, NP_001 121 179.1, NP 001 121 178. 1, NR OOI 121 177.1, NP_001 121 176.16, NP_001 121 175. 1, NP_001 121 174.1, NP_001 121 172.1, and/or AAA5 1547.1.

In some embodiments, the serpin-WAP domain fusion protein can also include an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide.

These embodiments are referred to collectively herein as "serpin-Fc-WAP domain fusion proteins." In these embodiments, no particular order is to be construed by this terminology.

For example, the order of the fusion protein can be serpin-Fc-WAP domain, serpin-WAP domain-Fc, or any variation combination thereof. The serpin-Fc-WAP domain fusion proteins described herein include at least a serpin polypeptide or an amino acid ce that is derived from a serpin, WAP domain-containing polypeptide or an amino acid sequence that is derived from a WAP domain-containing polypeptide, and an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide.

In some ments, where the serpin-WAP domain fusion protein includes an Fc polypeptide sequence, the Fc polypeptide sequence can have the amino acid sequence of SEQ ID NO: 3-7. In other embodiments, where the serpin-WAP domain fusion protein includes an Fc polypeptide ce, the Fc polypeptide sequence can have at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% cal to the amino acid sequence of SEQ ID NOs. 3-7. In some embodiments, the serpin-WAP domain fusion protein can also include an albumin polypeptide, or an amino acid sequence that is d from an albumin polypeptide. These embodiments are ed to collectively herein as "serpin-albumin-WAP domain fusion proteins." In these embodiments, no particular order is to be construed by this terminology. For example, the order of the fusion protein can be serpin-albumin-WAP domain, serpin-WAP albumin , or any variation combination thereof. The serpin-albumin-WAP domain fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin, WAP -containing polypeptide, or an amino acid sequence that is derived from a WAP domain-containing polypeptide, and an albumin polypeptide, or an amino acid sequence that is derived from an albumin polypeptide.

] In some embodiments where the -WAP domain fusion n includes an n polypeptide sequence, the albumin polypeptide sequence includes the amino acid sequence of SEQ ID NO: 14-15, described herein. In other embodiments, where the serpin-WAP domain fusion protein includes an albumin polypeptide sequence, the n ptide sequence has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the any one of the amino acid sequences having SEQ ID NO: 14 or 15.

In some embodiments, the second polypeptide (Polypeptide 2) of the serpin fusion protein is an albumin polypeptide or is derived from an albumin polypeptide. These embodiments are referred to collectively herein as n(a')-albumin fusion proteins." The serpin-albumin fusion proteins bed herein e at least a serpin polypeptide or an amino acid sequence that is derived from a serpin and an albumin polypeptide or an amino acid ce that is derived from an n polypeptide. In addition this invention relates to serpin albumin binding polypeptide fusion proteins, wherein the albumin is operably linked to the serpin via an intermediate binding molecule. Herein, the serpin is non- covalently or covalently bound to human serum albumin.

] In ments where the fusion protein of the invention includes an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein is a human serum albumin (HSA) ptide or an amino acid sequence derived from HSA. In some embodiments, the fusion protein includes a HSA polypeptide sequence having the following amino acid sequence: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAEN CDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV MCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLD ELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTE CCHGDLLECADDRADLAKYICENQDS ISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSL AADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVS RNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCF SALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMD DFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 14) In embodiments where the fusion protein of the invention includes an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein includes a human serum albumin polypeptide sequence that is at least 50%, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14.

In embodiments where the fusion n of the invention includes an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein fusion n includes a domain 3 of human serum albumin polypeptide sequence having the following amino acid sequence: EEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCK HPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVP KEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCC KADDKETCFAEEGKKLVA (SEQ ID NO: 15) In embodiments where the fusion protein of the invention includes an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein includes a human serum albumin polypeptide sequence that is at least 50%, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 15.

In some embodiments where the fusion protein of the ion es an albumin polypeptide sequence, the fusion protein is linked to the human serum albumin via an intermediate albumin binding polypeptide. The albumin binding polypeptide can be an antibody or an antibody fragment or derived from an antibody or antibody nt. In a red ment, the albumin binding polypeptide or an amino acid ce that is derived from the antibody or antibody fragment is derived from a chimeric, humanized, or fully human antibody sequence. The term antibody nt includes single chain, Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In addition, the albumin binding polypeptide can be an albumin binding e. Another embodiment of the invention is a serpin albumin binding polypeptide fusion, wherein the albumin g polypeptide is domain 3 of Streptococcal protein G or a sequence derived from domain 3 of Streptococcal protein G.

In some embodiments, the serpin polypeptide of the (a')-albumin fusion proteins includes at least the amino acid sequence of the reactive site loop portion of the AAT protein. In some embodiments, the ve site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 1. In some embodiments, the serpin polypeptide of the serpin-albumin fusion protein includes at least the amino acid sequence of a t of the reactive site loop portion of the AAT protein. In some embodiments, the variant of the reactive site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In some embodiments, the serpin polypeptide of the serpin-albumin fusion proteins includes at least the full-length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 2 . In some ments the serpin polypeptide of the serpin-albumin fusion proteins includes human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% cal to the amino acid sequence ofSEQ ID NO: 2 or 32 or 33.

In some embodiments, the serpin polypeptide of the -albumin fusion proteins includes the AAT polypeptide sequence or the amino acid sequence derived from an AAT polypeptide is or is derived from one or more of the human AAT polypeptide sequences shown in GenBank ion Nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA5 1546.1, CAA25838.1, NP 001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001 121 179.1, NP 001 121 178. 1, NR OOI 121 177.1, NP_001 121 176.16, NP_001 121 175. 1, NP_001 121 174.1, NP_001 121 172.1, and/or AAA51547.1.

In some embodiments, the fusion proteins are modified to increase or otherwise inhibit proteolytic cleavage, for example, by mutating one or more proteolytic ge sites. In some embodiments, the fusion proteins are modified to alter or otherwise modulate an Fc effector function of the fusion protein, while simultaneously retaining binding and inhibitory function as compared to an unaltered fusion protein. Fc effector functions include, by way of non-limiting examples, Fc receptor binding, prevention of proinflammatory mediator e upon binding to the Fc receptor, phagocytosis, modified antibody-dependent cell-mediated cytotoxicity (ADCC), ed complement-dependent cytotoxicity (CDC), modified glycosylation at Asn297 residue (EU index of Kabat numbering, Kabat et al 1991 Sequences of Proteins of Immunological Interest) of the Fc ptide. In some embodiments, the fusion proteins are mutated or otherwise modified to influence Fc or binding. In some embodiments, the Fc polypeptide is modified to enhance FcRn binding. Examples of Fc polypeptide mutations that e binding to FcRn are Met252Tyr, Ser254Thr, Glu , S256T, T256E) (Kabat ing, Dall'Acqua et al 2006, J . Biol Chem Vol 281(33) 23514-23524), or Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159).

(EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest). In some embodiments the Fc polypeptide portion is mutated or otherwise modified so as to disrupt Fc-mediated dimerization (Ying et al 2012 J . Biol Chem 287(23): 19399-19408). In these ments, the fusion protein is monomeric in nature.

] The fusion proteins and variants thereof provided herein exhibit inhibitory ty, for example by inhibiting a serine protease such as human neutrophil elastase (NE), a rypsin-fold serine protease that is secreted by neutrophils during an inflammatory response. The fusion proteins provided herein completely or partially reduce or otherwise modulate serine protease expression or activity upon binding to, or otherwise cting with, a serine protease, e.g., a human serine protease. The reduction or modulation of a biological function of a serine protease is complete or partial upon interaction between the fusion proteins and the human serine protease protein, polypeptide and/or e. The fusion proteins are considered to completely inhibit serine se expression or activity when the level of serine protease expression or activity in the presence of the fusion protein is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of serine protease expression or activity in the absence of ction, e.g., binding, with a fusion protein described herein. The fusion proteins are considered to partially inhibit serine se expression or activity when the level of serine protease expression or activity in the presence of the fusion protein is decreased by less than 95%, e.g., 10%, 20%, %, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of serine protease expression or activity in the absence of interaction, e.g., binding, with a fusion protein described herein.

The fusion proteins described herein are useful in a variety of therapeutic, diagnostic and prophylactic tions. For example, the fusion proteins are useful in treating a variety of diseases and disorders in a subject. In some embodiments, the serpin fusion proteins, including, fusion proteins described herein, are useful in ng, alleviating a symptom of, ameliorating and/or delaying the progression of a disease or disorder in a subject suffering from or identified as being at risk for a disease or disorder selected from alphaantitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary e (COPD), acute respiratory distress sydrome (ARDS), allergic asthma, cystic fibrosis, cancers of the lung, ischemia-reperfusion injury, including, e.g. , ischemia/reperfusion injury following cardiac transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic tis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and/or type II diabetes, bacterial ions, fungal infections, viral infections, pneumonia, sepsis, graft versus host disease (GVHD), wound healing, Systemic lupus erythematosis, and le sclerosis. 1001118691 Pharmaceutical compositions according to the invention include a fusion protein of the invention, ing modiﬁed fusion proteins and other variants, along with a suitable carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits.

As used herein, the term "comprise" and variations of the term, such as "comprising" "comprises" and ”comprised", are not intended to exclude other additives, 3 components, integers or steps. [00074b] Reference to any prior art in the speciﬁcation is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in New d or any other jurisdiction.

Brief Description of the Drawings Figure 1A is a schematic representation of some embodiments of serpin—Fc fusion proteins according to the ion. The serpin can be located at any position within the fusion protein. Serpin—Fc fusion protein incorporating more than one scrpin polypeptide are also represented. Figure 1B is a photograph ofa SDS—PAGE gel showing serum derived AAT (lane 1), AAT—Fc 1 (lane 2, human lgGl Fe), and AA'llEL—Fcl (lane 3, Met3SlGlu, Met358Leu mutations within AAT, human lgGl Fe). Figure 1C is a graph showing the inhibition of phil se activity by AAT—Fe fusion proteins. Figure 1D is a raph of a SDS~FAGE gel showing tetravalent AAT—Fc—AAT, having two AAT polypeptides per Fe polypeptide. Figure IE is a graph showing the inhibition of neutrophil elastase activity by a alent AAT—Fc—AAT fusion protein. Figure 1F is a graphing demonstrating the effect of low pH elution from protein A resin, wherein the NE inhibiting capacity of the AAT-Fc fusion n eluted at low pH is drastically reduced. Figure 1G is a graph showing that the double mutant, AAT-EL—Fc (Met351Glu, Met358Leu mutations) is resistant to H202 inactivation (cone), compared to wild type AAT and the single mutant —Fc (Met351Glu). Figure lH is a graph depicting the serum clearance rates of serum derived AAT (sdAAT) compared to AAT-Fc in rats dosed with lOmg/kg protein (3 1001118691 rats/test protein). The half life of AAT-Fc is substantially longer than that of sdAAT.

] Figure 2A is a schematic representation of some embodiments of the serpin- ne targeting fusion ns of the invention. The serpin can be fused to either the heavy chain, the light chain, or both of an antibody. Serpin-cytokine receptor fusion proteins are also depicted. Figure 2B is a photograph of a SDS-PAGE gel showing the D2E7 antibody (lane 1), and the D2E7 antibody with—AAT fused to heavy chain (lane 2).

Figure 2C is a graph showing the inhibition of neutrophil elastase activity by a D2E7 antibody fused to AAT. Serum derived AAT is shown as a positive control, whereas the D2E7 antibody alone is shown as a negative control for NE inhibition.

Figure 3A is a schematic representation of some embodiments of the serpin- Fc-WAP fusion proteins. Figure 3B is a photograph of a SDS-PAGE gel showing AAT-Fc- ELAFIN (lane 1) and AAT-Fc-SLPI (lane 2). Figure 3C is a graph showing the inhibition of neutrophil elastase activity by an AAT-Fc-ELAFIN fusion protein and an AAT-Fc-SLPI fusion protein. An AAT-Fc fusion protein and serum derived AAT are included for comparison.

Figure 4A is a schematic entation of some embodiments of the AATHSA fusion proteins. Figure 4B is a photograph of a SDS-PAGE gel showing an AAT- HSA fusion. Figure 4C is a graph showing the inhibition of neutrophil elastase activity by an AAT-HSA compared to serum derived AAT.

Detailed Description of the Invention Human neutrophil elastase (NE) is a chymotrypsin-fold serine protease, secreted by neutrophils during mation. Aberrant activity of NE results in a ssive degradation of elastin s and the slow ction of the alveolar structures of the lungs leading to emphysema and lung fibrosis (Lungarella et al 2008 Int. J . Biochem Cell Biol 40: 1287). Often, misguided NE ty is due to an imbalance of the protease with its natural inhibitor, alpha 1-antitrypsin (AAT). This imbalance can result from ed neutrophil infiltration into the lungs, as observed in the lungs of smokers and patients with Cystic Fibrosis (CF), or Acute Respiratory Distress Syndrome (ARDS).

Conversely, a deficiency of AAT, usually as a result of a point mutation that causes ATT to aggregate and late in the liver, leaves the lungs exposed to ked NE activity.

Individuals with AAT deficiencies are at sed the risk of emphysema, COPD, liver disease, and us other conditions.

AAT deficiency affects approximately 100,000 Americans (according to estimates from the Alpha-1 Foundation), and many of the afflicted people die in their 30's and 40's. There are currently only a few FDA-approved drugs for treatment of ATT deficiency (Prolastin®, Aralast™, Zemaira®, Glassia™). Each drug is the natural AAT derived from pooled human , which appears to be insufficient to meet the anticipated clinical demand. Furthermore, these products have short serum half lives (T 2 of approximately 5 days) and require high dose (60 mg/kg body weight) weekly ons. The current market for these drugs is estimated at approximately $400 million. The market for AAT-like drugs is likely substantially larger, based on the estimation that as many as 95% of individuals with AAT-deficiencies go undiagnosed, and the fact that these drugs have the potential to be effective therapies for pathologies characterized by enhanced NE activity in duals that are not AAT-deficient (e.g., cystic fibrosis (CF), acute respiratory distress syndrome (ARDS), g-induced emphysema and/or COPD).

AAT has been suggested to have broad spectrum anti-inflammatory activity (Tilg et al 1993 J Exp Med 178:1629 -1636, Libert et al 1996 Immunol 157:5126 -5129, Pott et al, Journal of Leukocyte Biology 85 2009, Janciauskiene et al 2007 J . Biol Chem 282(12): 8573-8582, Nita et al 2007 Int J Biochem Cell Biol 39:1 165 -1 176). Recently, evidence has mounted that AAT may be useful in treating numerous human pathologies, outside of the ly ted inflammatory pulmonary ions. Human AAT has shown to protect mice from clinical and histopathological signs of experimental autoimmune encephalomyelitis (EAE), suggesting it could be a potential treatment of autoimmune diseases, such as multiple sclerosis or ic lupus erythematosus (SLE) (Subramanian et al 20 11Metab Brain Dis 26: 107-1 13). Serum AAT has shown ty in rodent models of Graft Versus Host Disease (GVHD) (Tawara et al 201 1 Proc. Natl. Acad.

Sci. USA 109: 564-569, Marcondes t 201 1 ov 3;118(1 8):503 1-9), which has lead to a human clinical trial using AAT to treat individuals with Steroid Non-responsive Acute GVHD (NCT01523821). Additionally, AAT has been effective in animal models of type I and type II diabetes, dampening inflammation, protecting islet cells from sis and enabling durable islet cell allograft (Zhang et al 2007 Diabetes 6-1323, Lewis et al 2005 Proc Natl Acad Sci USA 102: 12153-12158, Lewis et al 2008 Proc Natl Acad Sci USA 105:16236 , Kalis et al 2010 Isl ts 2:185 - 1 89). Currently, there are numerous early human clinical trials of type I diabetes using serum derived AAT products (NCT01 183468, NCT01319331, NCT01304537).

The current serum-derived AAT products undergo extensive cation 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 tion capacity of serum derived AAT. Attempts to address the concerns of serum derived ts and production issues have been aimed at the expression of recombinant AAT. However, after 20 years of work, the generation of a therapeutically viable recombinant AAT has yet to reach the market (Kamaukhova et al 2006 Amino Acids : 317). Like the plasma-derived products, recombinant versions of AAT suffer from short serum half-lives, low tion yields, and poor lung distribution.

] The fusion proteins of the present invention have enhanced functionalities compared to the unmodified AAT molecule. The fusion of an AAT polypeptide with a second polypeptide that interacts with the neonatal Fc receptor (FcRn), serves to increase the serum half life, providing a much needed dosing benefit for patients. These FcRn interacting polypeptides of the fusion protein include immunoglobulin (Ig) Fc polypeptides derived from human IgGl, IgG2, IgG3, IgG4, or IgM, and derivatives of human albumin.

In some embodiments, the fusion protein incorporates mutations with the AAT portion that render the molecule more resistant to inactivation by oxidation. For example Met35 lGlu, Met358Leu (AAT-EL-Fc), demonstrates resistance vation by H20 2 ion (Figure 1G). While AAT is a l anti-inflammatory protein, some embodiments of the invention provide enhanced inflammation dampening capacity through the fusion of an AAT polypeptide and a cytokine ing polypeptide. The coupling of dual anti inflammatory functionalities from AAT and a second polypeptide, will provide more a potent therapeutic protein than either polypeptide on their own. Additionally, the coupling the nfective ty of AAT will mitigate the infection risk of most cytokine targeting biologies. Some ments e for more potent anti-inflammatory and anti-infective proteins through the fusion an lypeptide and WAP domain contain polypeptide.

The fusion proteins of the present invention are ed to be a great therapeutic utility and be superior to the current serum derived AAT products.

] To extend the half life of recombinant AAT, recombinant DNA technology was used to create a AAT gene fusion with the Fc domain of human IgGl , IgG2, IgG3, IgG4, IgM, or HSA, such that the expected protein product would be AAT followed by an Fc domain ((AAT-Fc (IgGl), AAT-Fc (IgG2), AAT-Fc (IgG3), AAT-Fc (IgG4), AAT-Fc (IgM)) or AAT followed by HSA. While it was known that fusion of Fc domains of HSA to some proteins, protein domains or peptides could extend their half-lives (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 unknown if an Fc domain or HSA fused to AAT would allow for proper folding and maintenance of NE inhibitory activity, or could extend the half-life of recombinant AAT. The fusion proteins of the present invention are shown to be potent inhibitors of NE, have extended serum half lives, and in some embodiments resistant to oxidation. In other embodiments, the fusion proteins described herein have distinct properties by the incorporation of other functional polypeptides, including cytokine targeting polypeptides, and WAP domain ning polypeptides.

The fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin and a second ptide. In some ments, for example, the invention provides a serpin polypeptide fused to human IgGl-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or HSA derivatives. The serpin-fusion described herein are expected to be useful in treating a y of indications, including, by way of non-limiting example, alphaantitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress sydrome (ARDS), allergic asthma, cystic fibrosis, cancers of the lung, ischemia-reperfusion injury, including, e.g., ischemia/reperfusion injury following cardiac transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and/or type II diabetes, bacterial infections, fungal infections, viral infections, pneumonia, sepsis, graft versus host disease (GVHD), wound healing, ic lupus erythematosis, and Multiple sclerosis.

In some embodiments, the fusion proteins bed herein include at least an alpha- 1-antitrypsin (AAT) ptide or an amino acid sequence that is d from AAT and second polypeptide. For example, the invention provides alpha- 1-antitrypsin (AAT) fused to human IgGl-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or HSA tives.

In some embodiments, the fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide and a cytokine targeting polypeptide or an amino acid sequence that is d from a cytokine targeting polypeptide. For example, the invention provides serpin polypeptide or a ce d from a serpin polypeptide fused to a human cytokine or or derivative thereof. Another embodiment of the invention es serpin polypeptide or a sequence derived from a serpin polypeptide fused to a cytokine targeting antibody, e.g., an anti-cytokine antibody, or a sequence d from of a cytokine targeting antibody, e.g. , an anti-cytokine antibody, or sequence derived from a fragment of ne targeting antibody, e.g., a fragment of an anti-cytokine antibody. For example, the invention es a serpin polypeptide or a sequence derived from a serpin ptide fused to a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds any of the following human cytokines: TNFa, IgE, IL-12, IL-23, IL-6, IL-la, IL- I b, IL-17, IL-13, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

For example, in some embodiments, the cytokine targeting polypeptide targets TNFa and includes any of the ing TNFa-targeting polypeptide or sequences derived from the following TNFa-targeting polypeptides: Remicade®, Humira®, Simponi®, Cimiza®, Enbrel® or 3 and ATN-192.

For example, in some embodiments, the cytokine targeting polypeptide s IgE and includes any of the following IgE-targeting polypeptide or sequences derived from the following IgE-targeting polypeptides: Xolair or FcsRI.

For example, in some embodiments, the cytokine targeting polypeptide targets the shared p40 t of IL-12 and IL-23 and includes the Stelara® polypeptide or sequences d from the a® ptide.

For example Stelara® the cytokine targeting polypeptide targets IL-13 and includes the 6 polypeptide or sequences derived from the CDP7766 polypeptide.

In some embodiments, the fusion proteins described herein include at least a alpha- 1-antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a cytokine targeting polypeptide or an amino acid ce that is derived from a cytokine targeting polypeptide. For example, the invention provides alpha- 1-antitrypsin inhibitor (AAT) fused a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds any of the following human nes: TNFa, IgE, IL-6, IL-la, IL- I b, IL-12, IL-17, IL-13, IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

In some embodiments the cytokine targeting polypeptide binds a cytokine receptor and prevents binding of the cytokine. For example, the present invention includes a serpin fused to a cytokine receptor targeting antibody. For example, the ion provides alpha- 1-antitrypsin inhibitor (AAT) fused a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds the or of any of the ing human cytokines: TNFa, IgE, IL-6, IL-la, IL- I b, 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.

For example, in some embodiments, the cytokine targeting polypeptide targets the IL-6 receptor and includes the Actemra® polypeptide (as described in patent publication 639), or the ALX-0061 polypeptide (as described in WO20 10/1 15998), or sequences derived from the Actemra® polypeptide, or ALX-0061 polypeptide.

For example, Actemra® the cytokine targeting polypeptide targets the IL-6 receptor and includes the zumab polypeptide or sequences derived from the tocilizumab polypeptide.

The ing of inflammatory cytokines and immune-stimulating agents by protein therapeutics has demonstrated clinical success in us inflammatory conditions. The most common proteins used as cytokine targeting agents are the soluble cytokine ors and monoclonal antibodies and fragments f. A significant drawback with targeting cytokines is the increased risk of infection in these patients, as evidenced by the TNFa targeting biologies, Remicade®, Humira®, Simponi®, Cimiza®, and Enbrel®, and the IL- 12/23 p40 ing antibody, Stelara®. This is likely to be a common problem of targeting inflammatory cytokines leading to immune suppression in patients. AAT and other serpin proteins are interesting in that they demonstrate both anti- infective and anti-inflammatory activities. Thus, the -cytokine targeting polypeptide fusion proteins of this ion can dampen aberrant cytokine activities while alleviating the risk of infections.

In some embodiments, the fusion ns described herein include a serpin polypeptide or an amino acid sequence that is derived from a serpin, a WAP domaincontaining polypeptide or an amino acid sequence that is derived from a WAP domaincontaining polypeptide, and an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide. For example, the ion provides a serpin polypeptide, a WAP domain-containing polypeptide and human IgGl-Fc, IgG2-Fc, c, c or IgM-Fc derivatives operably linked together in any functional combination. In some embodiments, the WAP domain containing protein is human SLPI or derived from human SLPI. In other embodiments, the WAP domain containing protein is human ELAFIN or derived from human ELAFIN. In some embodiments, the fusion proteins bed herein include at least an alpha- 1-antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a SLPI polypeptide or an amino acid ce that is derived from SLPI. In some embodiments, the fusion proteins described herein include at least an AAT polypeptide or an amino acid sequence that is derived from AAT and an ELAFIN polypeptide or an amino acid sequence that is derived from Elafin.

SPLI and Elafin are WAP domain containing proteins that display serine protease inhibitory activity. Both of these ns are anti-inflammatory in function. In addition these proteins possess broad anti-infective capacities toward numerous strains of bacteria, viruses, and fungi.

In some embodiments, the fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin and a human serum albumin (HSA) polypeptide or an amino acid sequence that is derived from a HSA polypeptide. Further embodiments of invention include -albumin binding polypeptide fusion proteins, wherein the albumin binding polypeptide is responsible for the association of the serpin and HSA. Thereby the invention includes both covalent and non-covalent linkages of the serpin polypeptide and the HSA polypeptide or sequences derived from the serpin polypeptide or a HSA polypeptide. For example, the invention provides a serpin ptide fused to human HSA, or HSA derivatives, or HSA binding e or ptides.

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

In some embodiments, the fusion proteins bed herein include a serpin polypeptide or an amino acid sequence that is derived from a serpin, a HSA polypeptide or or an amino acid sequence that is derived from a HSA polypeptide, and a WAP domaincontaining ptide or an amino acid ce that is derived from a WAP domaincontaining polypeptide. In some embodiments, the fusion proteins described herein include at least an alpha- 1-antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a HSA ptide or an amino acid sequence that is derived from a HSA polypeptide, and a SLPI polypeptide or amino acid sequence derived from SLPI. In other embodiments, the fusion proteins described herein include at least an alpha- trypsin (AAT) polypeptide or an amino acid sequence that is d from AAT and a HSA polypeptide or an amino acid ce that is derived from a HSA polypeptide, and an Elafin polypeptide or amino acid sequence derived from Elafin.

The fusion proteins of the present invention can be readily produced in mammalian cell expression systems. For example Chinese Hamster Ovary (CHO) cells, Human nic Kidney (HEK) 293 cells, COS cells, PER.C6®, NS0 cells, SP2/0, YB2/0 can readily be used for the sion of the serpin fusion proteins described herein.

Importantly, mammalian cell expression s produce proteins that are generally more optimal for therapeutic use. In contrast to bacterial, insect, or yeast-based expression systems, mammalian cell expression systems yield proteins with glycosylation patterns that are r or the same as those found in natural human ns. Proper gylcosylation of a protein can greatly influence serum stability, pharmacokinetics, biodistribution, protein folding, and functionality. Therefore, the ability to produce therapeutic proteins in ian expression systems has distinct advantages over other systems. rmore, most of the mammalian cell expression systems (e.g., CHO, NSO, PER.C6® cells) can be readily scaled in commercial manufacturing facilities to produce therapeutic proteins to meet clinical demands. The fusion ns described herein have enhanced functionalities over the l form of AAT and can be produced in mammalian expression systems for clinical and commercial supply. Some embodiments of the invention include a purification system that enables the isolation of serpin fusion proteins that retain their ability to inhibit NE. Importantly, the purification process of the present invention can be readily incorporated into today's cial mammalian cell-based manufacturing processes.

Unless ise defined, scientific and cal terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the ar. Generally, nomenclatures utilized in tion with, and ques of, cell and tissue culture, molecular biology, and protein and oligo- or cleotide chemistry and hybridization described herein are those 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, ction). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed ing to conventional methods well known in the art and as described in various l and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. rd techniques are used for chemical syntheses, chemical analyses, ceutical preparation, formulation, and delivery, and treatment of patients. The term patient includes human and nary subjects.

It will be appreciated that administration of therapeutic entities in accordance with the invention will be administered with suitable carriers, buffers, excipients, and other agents that are orated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, ous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax thylene glycols of various molecular weights), sem i solid gels, and semi-solid es ning carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W.

"Lyophilization and pment 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 eral ations" PDA J Pharm Sci Technol. 52:238-31 1 (1998) and the citations n for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

] Therapeutic formulations of the invention, which e a fusion protein of the invention, are used to treat or alleviate a m associated with a disease or disorder associated with aberrant serine protease activity in a subject. The present invention also provides methods of treating or alleviating a symptom associated with a disease or er associated with aberrant serine se activity in a subject. A therapeutic regimen is carried out by identifying a t, e.g., a human patient suffering from (or at risk of developing) a disease or disorder associated with aberrant serine protease activity, using standard methods, including any of a variety of clinical and/or laboratory procedures. The term patient includes human and nary subjects. The term subject includes humans and other mammals.

Efficaciousness of treatment is determined in association with any known method for diagnosing or ng the particular disease or disorder associated with nt serine protease activity. Alleviation of one or more ms of the disease or disorder ated with aberrant serine se activity indicates that the fusion protein confers a clinical t.

Methods for the screening of fusion proteins that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA), enzymatic assays, flow cytometry, and other immunologically mediated techniques known within the art. 8] The fusion proteins described herein may be used in methods known within the art relating to the zation and/or quantitation of a target such as a serine protease, e.g., for use in ing levels of these targets within appropriate logical s, for use in diagnostic s, for use in imaging the protein, and the like). The terms "physiological sample" and "biological sample," used interchangeably, herein are intended to include tissues, cells and biological fluids isolated from a subject, as well as s, cells and fluids present within a subject. Included within the usage of the terms "physiological sample" and "biological sample", therefore, is blood and a on or component of blood including blood serum, blood plasma, or lymph.

In a given embodiment, fusion proteins specific for a given target, or derivative, fragment, analog or homolog thereof, that contain the target-binding domain, are utilized as pharmacologically active compounds (referred to hereinafter as "Therapeutics").

A fusion protein of the invention can be used to isolate a particular target using standard techniques, such as immunoaffinity, chromatography or 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, b-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of le fluorescent materials include umbelliferone, fluorescein, scein isothiocyanate, rhodamine, rotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material es luminol; es of bioluminescent materials e luciferase, luciferin, and aequorin, and es of suitable radioactive material include 125I, 131I, 35S or 3H.

A therapeutically effective amount of a fusion protein of the invention relates generally to the amount needed to achieve a therapeutic ive. As noted above, this may be a binding interaction between the fusion protein and its target that, in certain cases, interferes with the functioning of the target. The amount required to be administered will furthermore depend on the binding ty of the fusion protein for its specific target, and will also depend on the rate at which an administered fusion protein is depleted from the free volume other subject to which it is administered. Common ranges for eutically effective dosing of an fusion protein or fragment thereof invention may be, by way of nonlimiting example, from about 0 .1 mg/kg body weight to about 250 mg/kg body weight.

Common dosing frequencies may range, for example, from twice daily to once a month.

Where fusion protein fragments are used, the smallest inhibitory fragment that ically binds to the target is preferred. For example, peptide molecules can be designed that retain the ability to bind the . Such peptides can be synthesized chemically and/or ed by recombinant DNA technology. (See, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. atively, or in addition, the composition can se an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, growth-inhibitory agent, an anti-inflammatory agent or anti-infective agent. Such molecules are suitably present in combination in s that are effective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile. This is readily lished by filtration through sterile filtration membranes.

Sustained-release preparations can be prepared. Suitable es of ned-release preparations include semipermeable matrices of solid hydrophobic rs ning the fusion protein, which matrices are in the form of shaped articles, e.g., films, or microcapsules. es of sustained-release matrices include polyesters, hydrogels (for example, -hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 919), copolymers of L-glutamic acid and g ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)hydroxybutyric acid.

While polymers such as ethylene-vinyl e and lactic acid-glycolic acid enable e of les for over 100 days, certain hydrogels release proteins for shorter time periods.

Pharmaceutical compositions The fusion proteins of the invention (also referred to herein as "active compounds"), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the fusion rotein and a pharmaceutically acceptable carrier.

As used herein, the term aceutically acceptable r" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying , and the like, compatible with pharmaceutical stration.

Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.

Preferred examples of such carriers or ts include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., tion), ermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; idants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediammetetraacetic acid (EDTA); buffers such as acetates, es or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

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 dispersion. For enous administration, suitable carriers include physiological , bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate ed saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the inating action of microorganisms such as ia and fungi.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for e, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of tants. tion of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, cohols such as manitol, ol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be t about by including in the composition an agent which delays absorption, for example, aluminum monostearate and n. e injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterilefiltered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier.

They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid r for use as a mouthwash, wherein the nd in the fluid r is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the ition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum anth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as dal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as mint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from red container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. 2] Systemic administration can also be by transmucosal or transdermal means.

For transmucosal or transdermal administration, ants appropriate to the r to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for e, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are ated into nts, salves, gels, or creams as generally known in the art.

The nds can also be prepared in the form of itories {e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery s.

Biodegradable, biocompatible rs can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. mal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,81 1.

] It is especially ageous to formulate oral or parenteral compositions in dosage unit form for ease of stration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the d eutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique teristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

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

The invention will be further described in the following es, which do not limit the scope of the invention described in the claims.

EXAMPLES Example 1: AAT-Fc Fusion Proteins and Variants Exemplary, but non-limiting examples of AAT-Fc fusion proteins according to the ion include the following sequences. While these examples include a hinge sequence and/or a linker sequence, fusion proteins of the ion can be made using any hinge sequence and/or a linker sequence suitable in length and/or flexibility. Alternatively fusion proteins can be made without using a hinge and/or a linker sequence. For e, the ptide components can be directly attached. 1001118691 An exemplary AAT-Fc fusion protein is the AAT—hFcl (human IgG1 Fc) described herein. As shown below, AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), the hinge region is shown in normal text (SEQ ID NO: 43), and the lgG-Fe polypeptide n of the fusion protein is italicized (SEQ ID NO: 3).

AAT—hFcl (human IgG1 Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO:16) An ary AAT~FC fusion protein is the AAT—hFcZ (human IgG2 Fc), described herein. As shown below, AAT polypeptide portion of the fusion n is underlined (SEQ ID NO: 2), the hinge region is shown in normal text (SEQ ID NO: 44), and the lgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 4).

AAT—hFcZ (human IgG2 Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP 1001118691 WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 17) An ary AAT—Fc fusion protein is the AAT—MM—EL-hFcl (human IgG1 Fc, Met351Glu/Met358Leu), described herein. As shown below, AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 34), the hinge region is shown in normal text (SEQ ID NO: 43), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 3), and the Met351Glu mutation is boxed, and the Met358Leu on is shaded in grey.

AAT—MM—EL—hFcl(human IgGl Fc, Met351Glu/Met358Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE KFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGNEFLEAIPLSIPPEVK FNKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 18) An exemplary AAT-Fc fusion protein is the AAT-MM—EL—hFcZ (human IgGZ Fc, Met35 l Glu/Met358Leu), described herein. As shown below, AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 34), the hinge region is shown in normal text (SEQ ID NO: 44), the IgG—Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 4), the Met3SIGlu mutation is boxed, and the Leu mutation is shaded in grey.

AAT—MM—EL—hFcZ (human IgG2 Fc, Glu/Met358Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY 1001118691 LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAEFLEAIPLSIPPEVK FNKPFVFLMIEQNTKSPLFMGKVVNPTQKERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD WLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN LSLSPGK (SEQ ID NO: 19) An exemplary AAT—Fc fusion n is the AAT—MM-LL—hFcl(human IgGl Fc, Met351Leu/Met358Leu), described herein. As shown below, AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 35), the hinge region is shown in normal text (SEQ ID NO: 43), the lgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 3), the Met351Leu mutation is shaded in black, and the Met358Leu mutation is shaded in grey.

AAT—m/I—LL-hFcl(human IgGl Fc, Leu/Met358Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAEFLEAIPLSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFTEYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK(SEQ ID NO:36) An exemplary AAT-Fc fusion protein is the AAT-MM:LL—hFcZ(human IgGZ Fc, Met351Leu/Met358Leu), described herein. As shown below, AAT polypeptide portion of the fusion protein is ined (SEQ ID NO: 35), the hinge region is shown in normal text (SEQ ID NO: 44), the IgG—Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 4), the Met351Leu on is shaded in black, and the Met358Leu mutation is shaded in grey. 1001118691 AAT—MM:LL—hFc2(human IgGZ Fc, Met351Leu/Met358Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY IFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAEFLEAIPLSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ :D NO:20) An exemplary AAT-Fe fusion protein is the AAT—hFel —AAT (human IgGl), described herein. As shown below, AAT polypeptide n of the fusion protein is underlined (SEQ ID NO: 2), the hinge region is shown in normal text (SEQ ID NO: 43), the ASTGS linker is shown in normal text (SEQ ID NO: 45), and the IgG—Fe ptide portion of the fusion protein is italicized (SEQ ID NO: 3).

AAT—hFcl—AAT (human IgG1) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQ LAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLR TLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVE KGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKR 1001118691 LGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSA SLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGT EAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK ( SEQ ID NO : 2 1) These exemplary AAT—Fc fusion proteins were made using the following techniques.

The gene encoding human AAT was PCR ampliﬁed from human liver cDNA (Zyagen). Speciﬁc point mutations within the gene encoding AAT or the Fc region were generated by overlapping PCR. The AAT ng gene was cloned in frame with a gene encoding the hinge region, followed by a CH2 domain, and a CH3 domain ofhuman lgGl , IgGZ, IgG3, IgG4, or IgM into a mammalian expression vector, containing a ian secretion signal sequence up stream of the AAT gene insertion site. These expression s were ected into mammalian cells (speciﬁcally HEK293 or CHO cells) and grown for l days in 8% C02 at 37° C. The recombinant AAT~FC fusion proteins were puriﬁed from the expression cell supernatant by protein A chromatography. hnportantly, a near neutral pH buffer was used (Gentle Ag/Ab Elution Buffer, Thermo Scientiﬁc) to elute the AAT~FC fusion protein from the protein A resin. The AAT-Fc fusion protein can not be eluted from protein A resin using a standard low pH elution, as the ability ofAAT to inhibit NE is compromised following low pH treatment, likely due to a low pH mediated oligomerization ofAAT. Figure 1F shows the s of low pH elution on the y of AAT to inhibit neutrophil elastase. AAT—Fc fusion protein can be puriﬁed either by protein A and a near neutral pH elution buffer, by CaptureSelect® Alpha—l Antitrypsin afﬁnity matrix (BAC BV). 8] The puriﬁed AAT—Fc fusion ns were tested for activity by determining their ability to inhibit neutrophil elastase (NE). Figure 1B and 1D show a reducing SDS- PAGE gel ofpuriﬁed serum derived AAT (sdAAT) and AAT—Fc ﬁision proteins (Fig 1B— lane 1: sdAAT, lane 2: AAT—Fc (SEQ ID NO: 16), lane 31AAT-EL—Fc (SEQ ID NO: 18), Fig 1D AAT—Fc-AAT (SEQ ID NO: 20). The proteins were visualized by staining with coomassie blue.

To monitor human Neutrophil Elastase (NE) activity a fluorescent microplate assay was used. Inhibitory activity was measured by a itant decrease in the residual NE activity using the following assay. This assay buffer is composed of 100 mM Tris pH 7.4, 500 mM NaCl, and % Triton X-100. Human NE is used at a final tration of 5 nM (but can also be used from 1-20 nM). The fluorescent peptide substrate AAVPAMC is used at a final concentration of 100 mM in the assay. The Gemini EM plate reader from Molecular Devices is used to read the assay kinetics using excitation and emission wavelengths of 370 nm and 440 nm respectively, and a cutoff of 420 nm. The assay is read for 10 min at room ature scanning every 5 to 10 seconds. The Vmax per second corresponds to the residual NE activity, which is plotted for each concentration of inhibitor.

The intercept with the x-axis tes the concentration of inhibitor needed to fully inactivate the starting concentration of NE in the assay. Human serum derived AAT (sdAAT) was used as a positive control in these assays. The AAT-Fc fusion proteins display potent NE inhibitory activity as shown in Figure 1C. The fusion wherein there are two AAT polypeptides fused to single Fc polypetide (AAT-Fc-AAT) displays enhanced potency over both sdAAT and the AAT-Fc fusion protein comprising a single AAT polypeptide (Figure IE). These findings ted here demonstrate for the first time the AAT can be fused to an Fc region and maintain its ability to inhibit NE. Of particular interest, the AAT-Fc-AAT fusion protein was found to be a more potent NE inhibitor.

Figure IF demonstrates the resistance of the AAT-EL-Fc (M35 IE, M358L) fusion protein to inactivation by ion. AAT fusion proteins, AAT-Fc (wt), AAT-ELFc , M358L), and AAT-EM-Fc (M351E), were treated with 33mM H20 2 and compared to untreated fusion proteins in the NE inhibition assays. The inhibition of NE by AAT-EL-Fc was not comprised by oxidation, converse to the other proteins tested.

Furthermore, AAT-Fc fusion protein displayed a longer serum half life in rats compared to serum derived AAT (Figure 1H). In these s 3 rats per each test protein were injected I.V. with lOmg/kg of sdAAT or AAT-Fc. Serum sample were taken at various time points over a 48 period. The serum ATT concentration was using an ELISA. These findings demonstrate that the fusion proteins of the invention have improved pharmacokinetic properties and are a superior eutic format over serum d AAT, for treating numerous human inflammatory ions and ious diseases. 1001118691 Example 2: AAT-TNFQ. Targeting Molecule Fusion ns The s presented herein describe several, non-limiting examples of recombinant AAT derivatives comprising human AAT fused to an anti-TNFOL antibody or a tive of a TNFoc receptor. These examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not and are not intended to limit the claimed invention.

The ﬁision proteins below include ne targeting polypeptide sequences that are from or are derived from (i) the anti—TNch antibody D2137 (also known as Adalimumab or Humira®), or (ii) the extracellular domain ofType 2 TNFOL Receptor (TNFR2-ECD). The AAT ptide portion ofthe fusion protein is underlined, the antibody constant regions (CH1-hinge—CH2-CH3, or CL) are italicized, and D2E7—VH, D2E7-VK, and TNFR2~ECD are denoted in bold text. While these examples include a hinge sequence and/or a linker sequence, ﬁision proteins of the invention can be made using any hinge sequence and/or a linker sequence suitable in length and/or ﬂexibility. Alternatively fusion proteins can be made t using a hinge and/or a linker sequence.

An exemplary AAT—TNFOt fusion protein is D2E7—Light Chain—AAT (G3 S)2 Linker, described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7—VK is denoted in bold text (SEQ ID NO: 37), the (G3S)2 linker is shown in normal text (SEQ ID NO: 46), and the antibody constant regions are ized (SEQ ID NO: 38) D2E7—Light Chain~AAT (G3S)2 Linker DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSR FSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSD TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVKACEVTHQGLSSPVTKSFNRGECGGGSGGGSEDPQGDAAQKTDTSHHDQDHPT FNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFN LTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFT VNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEE QVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENE LTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPL 1001118691 KLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKV VNPTQK (SEQ ID NO:22) An exemplary AAT—TNFOL fusion protein is D2E7-Light Chain-AAT ASTGS Linker, described herein. As shown below, the AAT polypeptide portion of the fusion n is underlined (SEQ ID NO: 2), D2E7-VK is denoted in bold text (SEQ ID NO: 37), the ASTGS linker is shown in normal text (SEQ ID NO: 45), and the antibody constant regions is italicized (SEQ ID NO: 38) D2E7—Light Chain—AAT ASTGS Linker DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSR GTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECASTGSEDPQGDAAQKTDTSHHDQDHPTFNK ITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTE IPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNF GDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDF HVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTH DIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLS VLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNP TQK (SEQ ID NO:23) An ary AAT~TNFOt fusion protein is D2E7—Heavy Chain—AAT (G3S)2 Linker, described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7—VH is denoted in bold text (SEQ ID NO: 39), the (G3S)2 linker is shown in normal text (SEQ ID NO: 46), and the antibody constant s is ized (SEQ ID NO: 40) D2E7—Heavy Chain—AAT (G3S)2 Linker EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYA DSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ 1001118691 VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSEDPQGDAAQKTDTSHHDQDHPTFNKITPN LAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEA QELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTE EAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIIT KFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVH KAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:24) An ary AAT-TNFOL fusion protein is D2E7—Heavy Chain—AAT ASTGS Linker, described herein. As shown below, the AAT polypeptide portion of the ingonpnndnisumkﬂnmd(SEQlDlﬂDﬂD,D2E74HlBdemﬁuﬁnbothﬂ(SEQlD NO: 39), the ASTGS linker is shown in normal text (SEQ ID NO: 45), and the antibody constant s is italicized (SEQ ID NO: 40) D2E7—Heavy Chain—AAT ASTGS Linker EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYA DSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAE FAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIH EGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAK KQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTT VKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFL ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAV LTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NOz25) 1001118691 An exemplary AAT—TNFOL fusion protein is TNFR2-ECD—Fcl- AAT(G38)2 Linker, described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), TNFR2-ECD is denoted in bold text (SEQ ID NO: 41), the hinge region is shown in normal text (SEQ ID NO: 43), the (G38); linker is shown in normal text (SEQ ID NO: 46), and the antibody constant regions is italicized (SEQ ID NO: 42) TNFRZ—ECD—Fcl—AAT(G38)2 Linker LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTY TQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCR PGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSP TRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVH NAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSEDPQGDAAQKTDT SHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHD NFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVK KLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWER PFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDE GKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADL SGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNT KSPLFMGKVVNPTQK (SEQ ID NOz26) An exemplary AAT—TNFOL fusion protein is TNFRZ—ECD—FCl—AAT ASTGS Linker, described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), TNFR2—ECD is denoted in bold text (SEQ ID NO: 41), the hinge region is shown in normal text (SEQ ID NO: 43), the ASTGS linker is shown in normal text (SEQ ID NO: 45), and the antibody nt s is italicized (SEQ ID NO: 42) TNFRZ—ECD—Fcl—AAT ASTGS Linker LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTY TQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCR 1001118691 PGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSP TRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKENWYVDGVQVH NAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHH DQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEIL EGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLY HSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFE VKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKL QHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGV TEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSP LFMGKVVNPTQK (SEQ ID NO:27) These exemplary AAT—TNFOt targeting le fusion proteins were made using the following ques.

The genes encoding the variable heavy (VH) and variable kappa (VK) regions of the NFOL antibody, D2137, 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 a CH] domain, a hinge , a CH2 domain, and a CH3 domain, into a mammalian expression vector, ning a mammalian secretion signal sequence up stream of the VH domain insertion site (D2E7—HC). The D2137~VK gene was cloned in frame with a human antibody kappa light chain constant (CL) domain, into a ian expression , containing a mammalian secretion signal sequence up stream of the VK domain insertion site (D2E7~LC). The AAT encoding gene and the adjacent 5’ linker sequence were cloned in frame into the 3 ’ end of either, the CH3 domain of the D2E7 heavy chain gene (D2E7—HC—AAT), or the CL domain ofthe D2137 light chain gene (D2E7~ LC-AAT) coding sequences in the above described mammalian expression vectors. The extracellular domain of the TNFoc Receptor 2 (TNFRZ—ECD) was generated by gene synthesis and cloned in frame with a gene encoding the hinge region, followed by a CH2 domain and a CH3 domain ofhuman IgG1 (hFcl) into a mammalian expression, containing a mammalian secretion signal sequence up stream of the TNFR2-ECD insertion site. The AAT encoding gene and the adjacent 5 ’ linker sequence were cloned in frame into the 3 ’ end ofthe gene encoding TNFRZ-ECD—hFcl into a mammalian expression vector (TNFR2— ECD—hFcl-AAT). 0] The D2E7-HC-AAT expression vector was co-transfected with either the D2E7-LC or the D2E7-LC-AAT expression vector into mammalian cells (specifically HEK293 or CHO cells) to generate the D2E7 antibody with AAT fused to the C-terminus of the heavy chain or to the C-terminus of both the heavy chain and light chain, respectively.

The D2E7-LC-AAT was co-transfected with the D2E7-HC expression vector into mammalian cells to generate the D2E7 antibody with AAT fused to the C-terminus of the light chain. The TNFR2-hFcl-AAT expression vector was transfected into mammalian cells. Transfected cells were grown for several days in 8% C0 2 at 37° C.

The recombinant AAT-TNFa ing fusion proteins were purified from the expression cell supernatant by protein A chromatography. A near neutral pH buffer was used (Gentle Ag/Ab Elution Buffer, Thermo ific) to elute the AAT-TNFa targeting fusion ns from the protein A resin.

Figure 2B shows an SDS-PAGE gel of the D2E7 antibody alone (lane 1) and variant wherein AAT is fused to the heavy chain of D2E7 (lane 2). The proteins were visualized by ng with coomassie blue.

The purified AAT-TNFa targeting molecule fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase. Human serum derived AAT ) was used as a positive control in these assays. NE inhibitory assay were conducted as described above. Figure 2C demonstrates relative to sdAAT, the AAT-TNFa targeting le fusion protein shows similar inhibition of neutrophil elastase, indicating that the inhibitory ty of AAT has not been compromised by its fusion to an antibody.

Example 3 AAT-Fc-SLPI and AAT-Fc-Elafin The studies presented herein describe l, non-limiting examples of recombinant AAT tives comprising human AAT fused a WAP domain containing n. These examples are provided below to further illustrate different es of the present invention. The examples also illustrate useful methodology for practicing the invention. The AAT polypeptide portion of the fusion protein is underlined, the Fc portion is italicized, and the WAP domain containing polypeptide is in bold font. While these examples include a hinge sequence and/or a linker sequence, fusion proteins of the invention can be made using any hinge ce and/or a linker sequence suitable in length 1001118691 and/or ﬂexibility. Alternatively fusion ns can be made without using a hinge and/or a linker sequence. For example, the polypeptide components can be directly attached.

An exemplary AAT-Fc—SLPI fusion protein is AAT-hFcl ~SLPI (human IgGl F0), described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), the hinge region is shown in normal text (SEQ ID NO: 43), the ASTGS linker is shown in normal text (SEQ ID NO: 45), the Fc portion is italicized (SEQ ID NO: 3), and the WAP domain containing ptide is in bold font (SEQ ID NO: 9) AAT—hFcl—SLPI (human IgG1 Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE KFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY :FFLPDEGKLQHLENELTHDI:TKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLM:EQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKASTGSSGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCP DTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGMCGKSC VSPVKA (SEQ ID NO:28) An exemplary AAT~Fc—Elaﬁn fusion n is AAT—hFcl—Elaﬁn (human IgG1 F0), described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), the hinge region is shown in normal text (SEQ ID NO: 43), the ASTGS linker is shown in normal text (SEQ ID NO: 45), the Fc portion is italicized (SEQ ID NO: 3), and the WAP domain containing polypeptide is in bold font (SEQ ID NO: 12) AAT—hFcl—Elafin (human IgG1 Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE 1001118691 GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA FKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTIMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALRAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKASTGSAVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQ EPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ (SEQ ID NO:29) The genes encoding the SLPT and Elaﬁn were PCR ed from human spleen cDNA (Zyagen). These genes were cloned into the mammalian expression vectors of example 1, n the SLPl or Elaﬁn gene was inserted in frame with the AAT—Fc gene.

These sion vectors were transfected into mammalian cells (speciﬁcally HEK293 or CHO cells) and grown for several days in 8% C02 at 37° C. The recombinant AAT-Fc— WAP domain fusion proteins were puriﬁed from the expression cell supernatant by protein A chromatography. A near neutral pH buffer was used (Gentle Ag/Ab Elution Buffer, Thermo Scientiﬁc) to elute the AAT—Fc—WAP domain fusion protein from the protein A resin.

Figure 3B shows an GE gel of the AAT—FC~WAP fusion proteins (lane 1 AAT-Fc—Elaﬁn, lane 2 AAT~FC~SLPI). The proteins were Visualized by staining with coomassie blue. The puriﬁed AAT—Fc-WAP domain fusion proteins were tested for activity by ining their ability to inhibit neutrophil elastase. NE inhibitory assays were conducted as described above. Human serum derived AAT ) and the AAT—Fc fusion protein were used as a positive control in these assays. Relative to sdAAT, the AAT- Fc—WAP targeting molecule fusion proteins display enhanced potency ofNE inhibition of neutrophil elastase (Figure 3C). e 4 AAT—Albumin The studies presented herein describe several, non—limiting examples of recombinant AAT derivatives comprising human AAT fused an albumin ptide. These examples are provided below to further illustrate different features of the t invention.

The examples also illustrate useful methodology for practicing the invention. These examples do not and are not intended to limit the claimed invention. The AAT portion is 1001118691 underlined and the albumin portion is italicized. For example, the polypeptide components can be directly attached.

An exemplary AAT—Albumin fusion protein is AAT—HSA, described herein.

As shown below, the AAT polypeptide portion of the fusion protein is ined (SEQ ID NO: 2), the ASTGS linker is shown in normal text (SEQ ID NO: 45), and the albumin polypeptide is italicized (SEQ ID NO: 14) AAT—HSA EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNY:FFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTH?"ITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKASTGSDAHKSEVAHRFKDLGEENFKALVLIAFA QYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMAD CCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAF KAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSIS SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYE DYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCEL FEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTL SEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA GL (SEQ ID NO:30) An exemplary AAT—Albumin fusion protein is A Domain 3, described herein. As shown below, the AAT polypeptide n of the fusion protein is underlined (SEQ ID NO: 2), the ASTGS linker is shown in normal text (SEQ ID NO: 45), and the albumin polypeptide is italicized (SEQ ID NO: 15) AAT—HSA Domain 3 EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE 1001118691 GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKASTGSEFPQNLIKQNCELFEQLGEYKFQNALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVS CTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVE LVKHKPKATKEQLKAVMDDEAAFVEKCCKADDKETCEAEEGKKLVA (SEQ ID NO:3l) The gene encoding human serum albumin (HSA) was PCR ampliﬁed from human liver cDNA (Zyagen). A mammalian expression vector was generated, wherein gene encoding HSA or the domain 3 ofHSA, was cloned in frame to the 3’ end of the AAT encoding gene, containing a mammalian secretion signal sequence up stream of AAT.

These expression vectors were transfected into ian cells (speciﬁcally HEK293 or CHO cells) and grown for several days in 8% C02 at 37° C. The recombinant AAT—HSA fusion proteins were d from the expression cell atant using the CaptureSelect® Alpha—l Antitrypsin afﬁnity matrix (BAC BV), wherein the binding buffer ted of 20mM Tris, 150mM NaCl, pH 7.4 and the elution buffer consisted of 20mM Tris, 2M MgClg pH 7.4.

Figure 4B shows an SDS—PAGE gel of the AAT—HSA fusion protein The proteins were visualized by staining with coomassie blue. The puriﬁed A fusion proteins were tested for activity by ining their ability to inhibit neutrophil elastase.

NE inhibitory assays were conducted as described above. Human serum derived AAT ) was used as a positive control in these assays. Relative to sdAAT, the AAT-HS fusion protein displays similar potency ofNE inhibition, demonstrating that the fusion to n does not dampen the capacity ofAAT to inhibit NE (Figure 4C.) Other Embodiments While the invention has been described in conjunction with the ed description thereof, the foregoing description is intended to illustrate and not limit the scope ofthe invention, which is deﬁned by the scope ofthe appended claims. Other aspects, advantages, and modiﬁcations are Within the scope ofthe following claims. 1001118691

Claims

The claims deﬁning the invention are as follows:

1. An isolated fusion protein sing at least one human serpin polypeptide comprising a human I antitrypsin (AAT) polypeptide comprising the amino acid sequence of SEQ ID NO:1 operably linked to an immunoglobulin Fe polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid ce of SEQ ID NO: 6.

2. The isolated fusion protein of claim 1, wherein AAT polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

3. The isolated fusion n ofclaim l or 2, wherein the serpin polypeptide and the immunoglobulin Fc polypeptide are operably linked Via a hinge region, a linker region, or both a hinge region and linker region.

4. The isolated fusion protein of claim 3, wherein the hinge region, the linker region or both the hinge region and the linker region comprise a peptide sequence.

5. The isolated fusion protein of claim 4, wherein the peptide sequence ses the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, or SEQ ID NO: 46.

6. The isolated fusion n of any one of claims 1—5, wherein the fusion protein further ses a WAP domain—containing polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 9-13.

7. The isolated fusion protein of any one of claims 1—6, wherein the immunoglobulin Fc polypeptide is modified to enhance FeRn binding.

8. The isolated fusion protein of any one of claims 1—7, wherein the immunoglobulin Fe polypeptide comprising at least one of the following mutations: Tyr, Ser254Thr, Thr256Glu, Met428Leu or Asn434Ser. 1001118691

9. The isolated fusion protein of any one of claims 1-7, wherein the immunoglobulin Fc ptide comprises at least one on at a position selected from the group consisting of: Met252, Ser254, Thr256, Met428, and Asn434.

10. An isolated fusion protein comprising at least one human 1 antitrypsin (AAT) polypeptide sing the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33 operably linked to an immunoglobulin Fc polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 6.

l l. The isolated fusion protein of claim 10, n the AAT polypeptide and the immunoglobulin Fe polypeptide are operably linked Via a hinge region, a linker region, or both a hinge region and linker region.

12. The isolated fusion protein of claim 10 or I 1, wherein the fusion protein further comprises a SLPI polypeptide comprising the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

13. The isolated fusion n of claim 1 1, wherein the hinge region, the linker region or both the hinge region and the linker region se a peptide sequence.

14. The isolated fusion protein of any one of claims 10—13, wherein the immunoglobulin Fe polypeptide is modiﬁed to enhance FcRn binding.

15. The isolated fusion protein of any one of claims 10—14, wherein the globulin Fc polypeptide comprises at least one mutation at a position selected from the group consisting of: Met252, Ser254, Thr256, Met428, and Asn434.

16. The isolated fusion protein of any one of claims 10-15, wherein the immunoglobulin Fc polypeptide comprises at least one of the following mutations: Met252Tyr, Thr, Thr256Glu, Met428Leu or Asn434Ser. 1001118691

17. Use of the fusion protein of any one of claims 1—16, in the manufacture of a medicament for treating or alleviating a symptom of a disease or disorder associated with aberrant serine se expression or activity in a subject in need thereof.

18. Use of the fusion protein of any one of claims 1—16, or claim 10 in the manufacture of a ment for treating or alleviating ation or a symptom of an inﬂammatory disease or disorder while reducing the risk of infection, in a subject in need thereof.

19. The use of the fusion protein of claim 18, wherein the atory disease or er is selected from the following: emphysema, chronic obstructive pulmonary disease , acute respiratory distress syndrome (ARDS), allergic asthma, cystic ﬁbrosis, cancers of the lung, ischemia—reperfusion injury, ischemia/reperfusion injury ing cardiac transplantation, myocardial tion, rheumatoid arthritis, septic arthritis, psoriatic arthritis, sing spondylitis, Crohn's disease, psoriasis, type 1 and/or type II diabetes, pneumonia, sepsis, graft versus host disease , wound healing, Systemic lupus erythematosus, and Multiple sclerosis.

20. Use of the fusion protein of any one of claims l—16, in the manufacture of a medicament for reducing the risk of infection in a subject in need thereof.

21. The use of the fusion protein of claim 20, wherein the infection is selected from bacterial infections, fungal infections, or viral infections.

22. The use of the fusion protein of any one of claims l7—21, wherein the subject is a human.

23. An isolated fusion protein according to claim 1, ntially as hereinbefore described.

24. An isolated fusion protein according to claim 10, substantially as hereinbefore described.

25. Use according to any one of claims 17 to 21, substantially as hereinbefore described.