NZ727846B2 - Serpin fusion polypeptides and methods of use thereof - Google Patents

Abstract

Disclosed is an isolated fusion protein comprising at least one human serpin polypeptide operably linked to a second polypeptide wherein the second polypeptide comprises an immunoglobulin Fc polypeptide, a cytokine targeting polypeptide, a WAP domain containing polypeptide and/or an albumin polypeptide. The human serpin polypeptide is preferably alpha-1 antitrypsin (AAT). Also disclosed herein is a method of purifying a fusion protein comprising AAT operably linked to a human immunoglobulin Fc peptide by eluting it from protein A with a near-neutral pH elution buffer to prevent AAT oligomerisation. ide. The human serpin polypeptide is preferably alpha-1 antitrypsin (AAT). Also disclosed herein is a method of purifying a fusion protein comprising AAT operably linked to a human immunoglobulin Fc peptide by eluting it from protein A with a near-neutral pH elution buffer to prevent AAT oligomerisation.

Description

SERPIN FUSION POLYPEPTIDES AND METHODS OF USE THEREOF Related Applications This application is a divisional of New Zealand patent application no. 708965, which is a divisional of New Zealand patent application no. 619023, the entire disclosures of which are incorporated herein by reference. [0001A] This application claims the benefit of U.S. ional Application No. 61/502055, filed June 28, 2011; U.S. Provisional Application No. 394, filed December 14, 2011; and U.S. Provisional Application No. 61/577204, filed December 19, 2011; and U.S. Provisional Application No. 61/638168, filed April25, 2012. The contents of each of these applications are hereby incorporated by reference 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 polypeptide. Additionally, the ion relates to fusion ns that include a serpin polypeptide or an amino acid sequence that is derived from serpin polypeptides, a second polypeptide, and a third polypeptide. Specifically, this invention s to fusion proteins that include at least one serpin polypeptide and a second polypeptide or fusion ns that include at least one serpin polypeptide, a second polypeptide, and a third polypeptide, where the second and third polypeptides of the fusion proteins of the invention can be at least one the following: an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide; a cytokine targeting polypeptide or a sequence derived from a cytokine targeting polypeptide; a WAP domain containing polypeptide or a sequence derived from a WAP ning ptide; or an albumin polypeptide or an amino acid sequence that is derived from a serum albumin polypeptide. This invention also relates to methods of using such molecules in a variety of therapeutic and diagnostic indications, as well as methods of producing such molecules.

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 protease activity and/or imbalance of protease-to-protease tor. rmore, enhanced therapeutic effects may be gained through the attenuation of aberrant cytokine signaling and serine protease activity. In addition, serpin proteins have demonstrated 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 ty and limit the risk of infection.

Summary of the ion 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). onally, the fusion proteins described herein include a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide eptide 1), a second ptide (Polypeptide 2), and athird polypeptide (Polypeptide 3). As used interchangeably herein, the terms "fusion protein" and "fusion polypeptide" refer to a serpin polypeptide or an amino acid ce derived from a serpin polypeptide operably linked to at least a second polypeptide or an amino acid sequence derived from at least a second polypeptide. The individualized elements ofthe fusion protein can be linked in any of a variety of ways, including for e, direct attachment, the use of an intermediate or a spacer peptide, the use of a linker region, the use of a hinge region or the use of both a linker and a hinge region. In some embodiments, the linker region may fall within the sequence of the hinge region, or alternatively, the hinge region the linker region. may fall within the sequence of Preferably, the linker region is apeptide ce. For example, the linker peptide includes anywhere from zero to 40 amino acids, from zero to 30 amino acids, from zero to 25 amino acids, e.g., from zero to 35 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 62 amino acids. In to 70 amino acids, from zero to 65 amino acids or from zero 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 re from zero to 90 amino acids, acids or from zero to 82 amino acids. e.g., from zero to 85 amino In some embodiments, the serpin polypeptide and the second polypeptide can be linked through an intermediate binding protein. In some embodiments, the serpin-based linked. portion and second polypeptide portion ofthe fusion n may be non—covalently In some embodiments, fusion proteins according to the invention can have one of the following formulae, in an N—terrninus to C—terminus direction or in a C—terminus to N-terminus ion: ptide 1(a) — hingem — Polypeptide 20)) Polypeptide 1(a) — linker" — Polypeptide 2(1)) Polypeptide 1(a) — " — hingem — Polypeptide 20,) Polypeptide 1(a) — hingem — linker" —— Polypeptide 2(1)) Polypeptide ha) — Polypeptide 2(b)— Polypeptide 3(a) Polypeptide 1(a) — hingem — Polypeptide 203— hingem — Polypeptide 3(6) Polypeptide 1(a) — " — Polypeptide 2(b)— linkern — Polypeptide 3(a) Polypeptide 1(a) — hingem — linker" ~ Polypeptide 2(b)—~hingem ~ n — Polypeptide 3(0) Polypeptide 1(a) — linker" — hingem — Polypeptide 209— linkern — hinge,"— Polypeptide 3(6) where n is an integer from zero to 20, Where m is an r 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 formulae also es Polypeptide 3(a) — Polypeptide 1(a)—- Polypeptide 20,), Polypeptide 20,) — Polypeptide 3(c)~ Polypeptide 1(a), or any variation or combination thereof.

In some embodiments, the Polypeptide 1 sequence includes a serpin polypeptide. Serpins are a group ofproteins with similar structures that were first identified as a set ofproteins able to inhibit proteases. Serpin proteins suitable for use in the fusion proteins provided herein include, by way ofnon-limiting example, alpha—l antitrypsin (AAT), ypsin-related protein (SERPINAZ), alpha l—antichymotrypsin (SERPINA3), kallistatin (SERPINA4), monocyte neutrophil elastase tor (SERPINBI), (SERPINB6), antithrombin (SERPINCI), plasminogen activator inhibitor 1 (SERPINEI), alpha 2—antiplasmin (SERPINF2), complement l-inhibitor (SERPING l), and neuroserpin (SERPINII).

In some embodiments, the Polypeptide 1 sequence includes an alpha-1 antitrypsin (AAT) polypeptide sequence or an amino acid sequence that is d 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 n of the AAT protein. In some ments, the reactive site loop portion of the AAT protein includes at least the amino acid sequence: GTEAAGAMFLEAI PMS I PPEVKFNK SEQ ID NO: 1) .

In a red ment, the AAT polypeptide sequence or an amino is or is derived from a human AAT polypeptide ce that is derived from AAT sequence.

In some embodiments, the fusion protein includes a full~length human polypeptide sequence having the ing amino acid sequence: 1 EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ STNI FFSPVSIATA 61 FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL RTLNQPDSQL GLFL 121 SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT EEAKKQINDY VEKGTQGKIV DLVKELDRDT MKRLGMFNIQ HCKKLSSWVL 181 VFALVNYIFF KGKWERPFEV KDTEEEDFHV DQVTTVKVPM 241 LMKYLGNATA IFFLPDEGKL QHLENELTHD IITKFLENED RRSASLHLPK LSITGTYDLK 301 SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA MFLEAIPMSI PPEVKFNKPF VFLMIEQNTK SPLFMGKVVN PTQK (SEQ ID NO: 2) In some embodiments, the fusion protein includes a human AAT polypeptide 85%, 90%, 91%, 92%, 93%, sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, of SEQ ID NO: 2. 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence amino acid In some embodiments, the AAT polypeptide sequence is, or the is derived from, one or more of the human sequence derived from an AAT polypeptide AAT polypeptide sequences shown in GenBank Accession Nos. AABS9495.1, CA115161.1,P01009.3,AAB59375.1,AAA51546.1, CAA25838.1,NP_001002235.1, 82.1,NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1,NP_001121176.16,NP_001121175.1,NP_001121174.1, NP_001121172.1, and/or AAA51547.1. mutations.

In some embodiments, the fusion proteins contain one or more methionine (Met) residue For e, the fusion protein contains at least one mutation at a in the serpin portion of the fusion protein. In these Met mutations, the Met residue can be substituted with an substituted with any amino acid. For example, the Met residue can be leucine (Leu, L). Without amino acid with a hydrophobic side chain, such as, for example, and subsequent wishing to be bound by theory, the Met mutation(s) prevent oxidation invention. In some vation of the tory activity of the fusion proteins of the such as, for embodiments, the Met residue can be tuted with a charged e, is at position 358 of example, glutamate (Glu, E). In some embodiments, the Met mutation Met mutation is Leu ). In some an AAT polypeptide. For example, the embodiments, the Met mutation is at position 351 of an AAT polypeptide. For example, the Met mutation is Met351Glu (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 (M351E) and Met358Leu (M358L). For example, the reactive site loop of this t of the fusion AAT polypeptide has the following sequence: GTEAAGAEFLEAI PLS I PPEVKFNK (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 (M351L) and Met358Leu (M3 58L). For e, the reactive site loop of this t of the fusion AAT polypeptide has the following sequence: GTEAAGALFLEAI PLS I PPEVKFNK (SEQ ID NO: 33).

] In some embodiments, the second polypetide (Polypeptide 2) of the serpin fusion protein is an F0 ptide or derived from an F0 polypeptide. These embodiments are ed to collectively herein as "serpin-Po fusion proteins." The serpin-Fc filSIOIl proteins described herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin and an F0 polypeptide or an amino acid sequence that is derived from an Fc polypeptide. In some embodiments, the serpin-Fe fusion n includes a single serpin polypeptide. In other embodiments, the serpin-Fe fusion proteins es more than one serpin ptide, and these embodiments are collectively referred to herein as "serpin(a,)—Fc fusion protein," wherein (a’) is an r of at least 2. In some embodiments, each serpin polypeptides in a serpin(av)-Fc fusion protein includes the same amino acid sequence. In other embodiments, each serpin polypeptide in a (a’)-Fc fusion protein includes serpin polypeptides with distinct amino acid sequences. The serpin polypeptides of serpirnavac fusion proteins can be located at any position within the fusion protein.

In some ments, the serpin polypeptide of the serpin—F0 fiision 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—Fe 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 n 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 serpin-Fc fusion protein includes at least the full—length human AAT polypeptide sequence having amino acid sequence of SEQ ID NO: 2. In some embodiments the serpin polypeptide of the serpin—Fc fusion protein es 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 ments, the serpin polypeptide ofthe serpin-Fc fiision n includes the AAT polypeptide sequence is or the amino acid ce derived from an AAT polypeptide is derived from one or more of the human AAT polypeptide sequences shown in GenBank Accession Nos. AAB59495.1, CAJ15161.1, P010093, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA349821, NP_001002236. 1, NP__000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, 121 176.16, NP_001121175.1,NP_001121174.1, NP_001121172.1, and/or AAA51547.1.

In some embodiments, the Fc polypeptide of the fusion protein is a human Fc polypeptide, for example, a human IgG Fc polypeptide sequence or an amino acid sequence that is derived from a human IgG Fc polypeptide sequence. For example, in some embodiments, the Fc polypeptide is a human IgG1 Fc polypeptide or an amino acid In some sequence that is derived from a human IgG1 Fc polypeptide sequence. ments, the Fc ptide is a human IgG2 Fc polypeptide or an amino acid In some sequence that is derived from a human IgG2 Fc polypeptide sequence. embodiments, the FC polypeptide is a human IgG3 Fc polypeptide or an amino acid In some sequence that is derived from a human IgG3 Fc polypeptide sequence. ments, the Fc polypeptide is a human IgG4 Fc polypeptide or an amino acid Fc ptide sequence. In some sequence that is d from a human IgG4 embodiments, the Fc polypeptide 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 includes an F0 polypeptide, the Fc polypeptide of the fusion protein includes a human IgG1 Fc polypeptide sequence having the following amino acid sequence: 1 APELLGGPSV FLFPPKPKDT LMISRTPEVI‘ CVVVDVSHED PEVKFNWYVD GVEVHNAKTK 61 PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 121 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL 181 WQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 3) In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc ptide of the fusion protein includes a human IgG1 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3.

In some embodiments where the quIOIl protein of the invention includes an F0 polypeptide, the Fc polypeptide is mutated or modified to enhance FcRn binding. In these embodiments the mutated or modified Fc polypeptide includes the following mutations: Met252Tyr, Ser254Thr, Thr256Glu (M252Y,.S256T, T256E) or Met428Leu and Ser (M428L, N434S) using the Kabat numbering system. In some embodiments the Fc polypeptide portion is mutated or otherwise modified so as to disrupt iated dimerization. In these ments, the fusion protein is monomeric in nature.

In some embodiments where the fusion n of the ion includes an F0 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 F0 ptide, the Fc ptide 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 protein includes a human IgG3 Fc polypeptide sequence having the following amino acid sequence: 1 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD GVEVHNAKTK 61 PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKTK QVYT 121 LPPSREEMTK NQVSLTCLVK IAVE PENN YNTTPPMLDS DGSFFLYSKL 181 WQQG NIFSCSVMHE ALHNRFTQKS LSLSPGK (SEQ ID NO: 5) In some embodiments where the fusion n of the invention includes an F0 polypeptide, the Fc polypeptide of the fusion protein includes a human IgG3 Fc polypeptide sequence that is at least 50%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 91%, acid sequence of SEQ 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino ID NO: 5. includes an In some embodiments where the fusion protein of the invention human IgG4 Fc F0 polypeptide, the Fc polypeptide ofthe fusion protein includes a polypeptide sequence having the following amino acid sequence: PEVQFNWYVD GVEVHNAKTK 1 APEFLGGPSV FLFPPKPKDT PEVT CVVVDVSQED SIEKTISKAK GQPREPQVYT 61 PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS KTTPPVLDSD GSFFLYSRLT 121 EMTK NQVSLTCLVK GFYPDIAVEW ESNGQPENNY 181 VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK (SEQ ID NO: 6) includes an In some embodiments where the fusion protein of the invention human IgG4 Fc F0 polypeptide, the Fc polypeptide of the fusion protein includes a polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, amino acid sequence of SEQ 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the ID NO: 6. invention includes an ] In some embodiments where the fusion protein of the includes a human IgM Fc EC polypeptide, the Fc polypeptide of the fusion protein polypeptide sequence having the following amino acid sequence: VSWLREGKQV GSGVTTDQVQ 1 IAELPPKVSV FVPPRDGFFG NPRKSKLICQ ATGFSPRQIQ TCRVDHRGLT FQQNASSMCV IRVF 61 AEAKESGPTT YKVTSTLTIK ESDWLGQSMF AVKTHTNISE FSAV 121 AIPPSFASIF LTKSTKLTCL VTDLTTYDSV TISWTRQNGE (SEQ ID NO: 7) 181 GEASICEDDW NSGERFTCTV THTDLPSPLK QTISRPKG the invention includes an In some embodiments where the fusion n of includes a human IgM Fc F0 polypeptide, the Fe polypeptide of the fusion protein 90%, 91%, ptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, the amino acid ce of SEQ 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to ID NO: 7. the second In some embodiments of the fusion proteins provided herein, polypeptide polypeptide (Polypeptide 2) of the serpin fusion protein is a cytokine targeting These embodiments are referred to or derived from a cytokine targeting polypeptide. fusion proteins." The serpin- collectively herein as n-cytokine targeting ptide include at least a serpin cytokine ing polypeptide fusion proteins described herein and a polypeptide or an amino acid sequence that is d from a serpin polypeptide embodiments, the serpin- cytokine targeting polypeptide, or derivation thereof. In some In other cytokine targeting polypeptide fusion protein includes a single serpin ptide. includes more than embodiments, the serpin—cytokine targeting polypeptide fusion protein one serpin polypeptide, and these embodiments are collectively referred to herein as "serpin(av)—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 ptide fusion protein includes the same amino acid sequence. In other embodiments, each serpin polypeptide of a serpin(a)~cytokine targeting polypeptide fusion protein es serpin polypeptides with distinct amino acid ces.

In some embodiments, the cytokine targeting polypeptide of the serpin— cytokine ing polypeptide fusion protein is a cytokine receptor or derived from a cytokine receptor. In a preferred embodiment, the cytokine targeting polypeptide or an amino acid sequence that is derived from the cytokine receptor is or is derived from a human ne receptor sequence. In other ments, the cytokine targeting ptide is an antibody or an antibody fragment, for example an anti-cytokine antibody or anti-cytokine antibody nt. In a preferred embodiment, the cytokine targeting polypeptide or an amino acid sequence that is derived from the antibody or antibody nt is derived from a chimeric, zed, 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 embodiments, the cytokine targeting polypeptide binds a cytokine receptor and prevents binding of a cytokine to the receptor. In other embodiments, the cytokine targeting polypeptide is an antibody or an antibody fragment, for example an anti- cytokine receptor 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 ve site loop portion of the AAT protein. In some ments, 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 qulOI’l ns 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 ve site loop portion of the AAT protein includes at least the amino acid sequence of SEQ ID N032 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 ptide sequence having amino acid sequence of SEQ ID NO: 2. In some embodiments the serpin polypeptide of the serpin-cytokine ing fusion protein es 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 N022 or 32 or 33.

In some embodiments, the serpin polypeptide of the serpin-cytokine targeting fusion protein includes an AAT polypeptide sequence or an amino acid sequence derived from an AAT polypeptide that is or is d from one or more of the human AAT polypeptide sequences shown in GenBank Accession Nos. AAB59495.1, CA115161.1, 3, AAB59375.1, 46.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1,NP_001121178.1, NP_001121177.1, NP_001121176.16,NP_001121175.1, NP_001121174.1, NP_001121172.1, and/or AAAS 1547.1.

The serpin-cytokine targeting polypeptide fusion protein can incorporate a portion of the -Fe fusion protein. For example, an antibody contains an EC polypeptide. Therefore, in some embodiments where the cytokine ing polypeptide is a cytokine-targeting antibody, the -cytokine targeting polypeptide fusion protein will incorporate a portion of the serpin-Fe fusion protein. Furthermore, most receptor fusion proteins that are of therapeutic utility are Fc fusion ns. Thus, in some embodiments, wherein the serpin—cytokine ing polypeptide fusion protein is a serpin~cytokine receptor fusion protein, the serpin—cytokine targeting polypeptide fusion protein may incorporate an EC 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 F0 ptide 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 embodiments where the serpin—cytokine targeting fusion protein es an F0 polypeptide sequence, the Fc polypeptide sequence has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequence of SEQ ID NO: 3, 4, 5, 6, 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 embodiments, the serpin polypeptide and the cytokine targeting polypeptide are directly attached.

In some ments of the fusion proteins provided herein, the second polypeptide (Polypeptide 2) of the serpin fusion protein is a whey acidic protein (WAP) domain containing polypeptide, or an amino acid sequence that is derived from a WAP domain containing polypeptide. These embodiments are referred to tively herein as "serpimWAP domain fiision 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 —containing polypeptide or an amino acid sequence that is derived from a WAP domain—containing polypeptide. In some ments, the serpin—WAP domain fusion protein includes a single serpin polypeptide. In other embodiments, the serpin-WAP targeting polypeptide fusion protein includes more than one serpin polypeptide. These embodiments are collectively referred to herein as n(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 ce, In other embodiments, the serpin polypeptides of the serpinpvycytokine targeting polypeptide fusion protein, includes serpin polypeptides with distinct amino acid sequences.

These serpin—WAP domain fusion proteins include a WAP domain containing polypeptide or polypeptide sequence that is or is derived from a WAP domain containing polypeptide. The WAP domain is an evolutionarily conserved sequence motif of 50 amino acids containing eight cysteines found in a characteristic 4—disulfide core arrangement (also called a four-disulfide core motif). The WAP domain sequence motif is a functional motif characterized by serine se inhibition ty 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 (S LPI) 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 polypeptide sequence comprises a portion of the SLPI n, such as for example, the WAPZ 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 ments ofthe serpin—SLPI fusion proteins of the invention, the SLPI sequence or a SLPI—derived sequence of the fusion n includes a full—length human SLPI polypeptide sequence having the following amino acid ce: 1 MKSSGLFPFL VLLALGTLAP WAVEGSGKSF KAGVCPPKKS AQCLRYKKPE CQSDWQCPGK 61 KRCCPDTCGI KCLDPVDTPN 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 ce of the fusion protein includes a human SLPI polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8.

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

In some embodiments of the —SLPI fusion protein of the ion, the SLPI sequence or a SLPI—derived sequence of the fusion protein includes the WAPZ domain of the full—length human SLPI polypeptide sequence, where the WAP2 domain has the following amino acid sequence: 1 TRRKPGKCPV MLNP PNFCEMDGQC KRDLKCCMGM CGKSCVSPVK A (SEQ ID NO: 10) In some embodiments ofthe serpin—SLPI fusion n of the invention, the SLPI ce or a SLPI—derived sequence of the fusion protein includes a human SLPI polypepfidesequencethatisatleast5096,6096,6596,7096,7596,8096,8596,9096,9l9@ 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 serpin——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, P039732, AAH20708.1, CA864235.1, CAA28]88.], 61.1, and/or BAG35125.1.

In some embodiments of the —SLPI fusion proteins of the invention, the SLPI polypeptide sequence or a SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide sequence that is modified at a Methoine (Met) residue. In these Met mutations, 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 Wa1, V). Without wishing to be bound by theory, the Met mutation(s) t oxidation and subsequent inactivation of the inhibitory activity of the fusion ns of the ion. In some embodiments, the Met mutation is at on 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 domain—containing polypeptide ce of the fusion protein includes an elafin ptide sequence or an amino acid sequence that is derived from elafin. These embodiments are referred to herein as "serpin—elafin fusion proteins. In some embodiments, the elafin polypeptide sequence includes a portion of the elafin protein, such as for example, the WAP domain or a sub-portion thereof. In a preferred embodiment, the elafin ptide sequence or an amino acid ce that is derived from elafin is or is derived from a human elafin polypeptide sequence.

In some embodiments of the serpin—elafin fusion proteins, the fusion protein includes a full—length human elafin polypeptide sequence having the following amino acid sequence: 1 MRASSFLIVV TLVL EAAVTGVPVK GQDTVKGRVP FNGQDPVKGQ DKVK 61 AQEPVKGPVS TKPGSCPIIL NPPN RCLKDTDCPG IKKCCEGSCG MACFVPQ (SEQ ID NO: ll) In some ments of the serpinflelafin fusion proteins, the fusion n 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—elafin fusion proteins, the fusion protein includes a n of the full-length human elafin polypeptide sequence, where the n has the following amino acid sequence: 1 AVTGVPVKGQ DTVKGRVPFN GQDPVKGQVS VKGQDKVKAQ EPVKGPVSTK PGSCPIILIR 61 CAMLNPPNRC LKDTDCPGIK KCCEGSCGMA CFVPQ (SEQ ID NO: 12) In some embodiments of the serpin—elafin 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: l2.

In some embodiments of the serpin—elafin fusion proteins, the fusion protein includes the WAP domain of the full—length human elafin polypeptide sequence, where the WAP domain has the following amino acid sequence: 1 VSTKPGSCPI ILIRCAMLNP PNRCLKDTDC PGIKKCCEGS CGMACFVPQ (SEQ ID NO: 13) In some ments of the serpin—elafln 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—elafin fusion proteins, the elafin polypeptide sequence or the amino acid sequence derived from an elafin ptide is derived from one or more of the human elafin polypeptide sequences shown in GenBank Accession Nos. P199573, NP_002629.1, BAA02441.1, EAW75814.1, EAW75813.1, Q8IUBZ.1, and/or NP_542181.1.

In other embodiments, the WAP —containing polypeptide sequence of the fiision protein includes an Eppin polypeptide sequence or an amino acid sequence that is derived from Eppin. These embodiments are referred to herein as "serpin(,’)—Eppin fusion proteins. In some embodiments, the Eppin polypeptide sequence of the serpin—Eppin fusion n includes a portion of the Eppin protein, such as for example, 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 derived from a human Eppin polypeptide sequence.

In some embodiments of the serpin—Eppin fusion proteins, the Eppin polypeptide sequence or amino acid sequence derived from an Eppin polypeptide is or is derived from one or more of the human Eppin polypeptide sequences shown in GenBank Accession Nos. 0959251, NP_065131.1, AAH44829.2, AAH53369. 1, AAG00548. 1, AAG00547. 1, and/or AAG00546.1.

In some embodiments, the serpin ptide of the serpin-WAP domain fusion n 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 es at least the amino acid sequence of SEQ ID NO:1. In some ments, the serpin polypeptide of the serpin—WAP fusion protein includes at least the amino acid sequence of a variant of the reactive site loop portion of the AAT protein. 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 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-WAP domain fusion protein includes human AAT ptide 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-WAP domain fusion n 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 ion Nos. AAB59495.1, CA]15161 . 1, P010093, 75.1, AAA51546.1, 38.1, NP_001002235.1, CAA34982.1, NP_001002236.1,NP_000286.3, NP~001121179.1, NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, and/or AAAS 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 EC polypeptide.

These ments 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 —Fc—WAP , serpin—WAP —Fe, or any variation combination thereof. The serpin—Fc-WAP domain fusion proteins described herein e 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 EC polypeptide or an amino acid sequence that is derived from an EC polypeptide.

In some embodiments, where the serpin—WAP domain fusion protein includes an F0 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 EC polypeptide sequence, the Fc polypeptide sequence can have at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NOS. 3—7. In some embodiments, the serpin—WAP domain fusion protein can also include an albumin polypeptide, or an amino acid sequence that is derived from an albumin polypeptide. These embodiments are referred to tively herein as "serpin-albumin—WAP domain fusion proteins." In these embodiments, no particular order is to be construed by this terminology. For example, the order of the fusion protein can be serpin—albumin—WAP domain, serpin-WAP domain— n, or any variation combination f. The serpin-albumin—WAP domain fusion proteins bed herein include at least a serpin ptide or an amino acid sequence that is derived from a serpin, WAP domain~containing polypeptide, or an amino acid and an albumin sequence that is derived from a WAP domain-containing polypeptide, polypeptide, or an amino acid sequence that is d from an albumin polypeptide.

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

] In some embodiments, the second polypeptide (Polypeptide 2) of the serpin fusion protein is an n polypeptide or is derived from an albumin polypeptide. These embodiments are referred to collectively herein as "serpin(as)-albumin fusion proteins." The serpin-albumin fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence that is d from a serpin and an albumin polypeptide or an amino acid sequence that is derived from an albumin 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 le. , the serpin is non- covalently or covalently bound to human serum albumin.

In embodiments where the fusion protein of the invention includes an albumin polypeptide sequence, the albumin ptide sequence of the fusion n is a human serum albumin (HSA) polypeptide or an amino acid sequence derived from HSA. In some embodiments, the fusion n includes a HSA polypeptide sequence having the following amino acid sequence: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAEN CDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV MCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLD ELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTE CCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSL AADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADP HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVS GSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCF 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 fiision 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 protein of the invention includes an albumin polypeptide ce, the albumin polypeptide sequence of the fusion protein fusion protein includes a domain 3 of human serum albumin polypeptide sequence having the following amino acid sequence: 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 ce 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: 15.

In some embodiments where the fusion protein of the invention includes an albumin polypeptide sequence, the fusion protein is linked to the human serum albumin via an intermediate albumin binding polypeptide. The albumin binding polypeptide can be an antibody or an antibody fragment or derived from an antibody or antibody fragment. In a preferred embodiment, the albumin binding polypeptide or an amino acid sequence that is derived from the antibody or antibody fragment is d 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 addition, the albumin binding polypeptide can be an albumin binding e. Another embodiment of the ion is a serpin albumin g polypeptide fusion, wherein the albumin binding polypeptide is domain 3 of Streptococcal protein G or a sequence derived from domain 3 of ococcal protein G.

In some embodiments, the serpin polypeptide of the (ay)—albumin fusion proteins es 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 polypeptide of the serpin-albumin fusion protein includes at least the amino acid ofthe AAT protein. In some sequence of a t of the reactive site loop portion embodiments, the variant of the reactive site loop portion of the AAT protein includes at least the amino acid ce of SEQ ID NO:32 or SEQ ID N0133. 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 embodiments the serpin polypeptide of the serpin-albumin fusion proteins es 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 ofSEQ ID NO: 2 or 32 or 33.

In some embodiments, the serpin polypeptide of the -albumin fusion proteins includes the AAT ptide sequence or the amino acid sequence derived from an AAT polypeptide is or is derived from one or more of the human AAT polypeptide sequences shown in GenBank Accession Nos. AAB59495. 1, CA115161 .1, P010093, AAB59375.1, AAA51546.1, CAA25838.1,NP~001002235.1,CAA34982.1, NP_001002236.1, NP_000286.3, NPa001121179.1, NP~001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, 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 cleavage sites. In some embodiments, the fusion proteins are modified to alter or otherwise modulate an F0 effector function of the fusion protein, while simultaneously retaining binding and inhibitory function as compared to an unaltered fusion protein. F0 effector functions include, by way of non-limiting examples, Fc receptor binding, prevention of lammatory mediator e upon binding to the Fc receptor, phagocytosis, modified dy—dependent cell—mediated cytotoxicity , modified complement-dependent cytotoxicity (CDC), modified glycosylation at Asn297 residue (EU index of Kabat numbering, Kabat er a1 1991 Sequences ofProteins ofImmunological Interest) of the Fc polypeptide. In some embodiments, the fusion proteins are mutated or otherwise modified to influence Fc receptor binding. In some embodiments, the Fc polypeptide is modified to enhance FcRn binding. Examples of Fe polypeptide mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Glu (M252Y, S256T, T256E) (Kabat numbering, Dall’Acqua et a1 2006, J. Biol Chem V01281(33) 23514—23524), or Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157—159).

(EU index of Kabat et a1 1991 Sequences ofProteins ofImmunological Interest). In some embodiments the Fc polypeptide n is mutated or otherwise modified so as to disrupt iated zation (Ying et al 2012 J. Biol Chem 287(23): 19399—19408). In these embodiments, the fusion protein is monomeric in nature.

] The fusion proteins and variants thereof provided herein exhibit inhibitory activity, for example by inhibiting a serine protease such as human phil elastase (NE), a chemotrypsin-fold serine protease that is secreted by neutrophils during an inflammatory otherwise response. The fusion proteins provided herein completely or partially reduce or modulate serine protease expression or activity upon binding to, or otherwise interacting with, a serine protease, e.g., a human serine protease. The reduction or modulation of a biological fimction of a serine protease is complete or partial upon interaction between the fusion proteins and the human serine protease n, polypeptide and/or peptide. The fusion proteins are considered to completely inhibit serine protease sion or activity when the level of serine se expression or activity in the presence of the fusion protein is decreased by at least 95%, e.g., by 96%, 97%, 98%, 99% or 100% as compared to the level of serine protease expression or activity in the absence of interaction, e. g., binding, with a firsion protein described herein. The fusion proteins are considered to partially inhibit serine protease sion or activity when the level of serine protease expression or ty 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 ed 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 described herein are useful in a variety of therapeutic, diagnostic and prophylactic indications. For e, the fusion proteins are useful in treating a variety of diseases and ers in a subject. In some embodiments, the serpin fusion proteins, including, fiision proteins bed herein, are useful in treating, alleviating a symptom of, ameliorating and/or delaying the progression of a disease or disorder in subject ing from or fied as being at risk for a disease or disorder selected from alpha—1-antitrypsin(AAT) deficiency, emphysema, chronic obstructive pulmonary disease , acute respiratory distress sydrome (ARDS), allergic asthma, cystic fibrosis, cancers of the lung, ischemia—reperfusion injury, including, e.g., ischemia/reperfiision 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, fiingal infections, viral infections, pneumonia, , graft versus host disease (GVHD), wound healing, ic lupus erythematosis, and Multiple sclerosis. 1001118691 Pharmaceutical compositions according to the invention include a fusion protein of the invention, including modified fusion ns 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" 3 "comprises" and "comprised", are not intended to exclude other additives, 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 Zealand or any other jurisdiction.

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

Figure 2A is a schematic representation of some embodiments of the serpin— cytokine targeting fusion proteins of the invention. The serpin can be fused to either the heavy chain, the light chain, or both of an antibody. -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 phil 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—Fo- ELAFIN (lane 1) and —SLPI (lane 2). Figure 3C is a graph showing the tion of neutrophil elastase activity by an AAT-Fc-ELAFIN fusion protein and an AAT—Fc~SLPl fusion protein. An AAT—Fe fusion n and serum derived AAT are included for comparison.

Figure 4A is a schematic representation of some embodiments of the AAT- HSA fusion proteins. Figure 4B is a raph 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 ption of the Invention Human neutrophil elastase (NE) is a chymotrypsin-fold serine se, secreted by neutrophils during inflammation. Aberrant activity ofNE results in a progressive degradation of elastin tissues and the slow destruction of the alveolar structures of the lungs leading to emphysema and lung fibrosis (Lungarella er al 2008 Int. J. Biochem Cell Biol 40: 1287). Often, misguided NE activity is due to an imbalance of the protease with its natural inhibitor, alphal -antitrypsin (AAT). This imbalance can result from enhanced neutrophil ation into the lungs, as observed in the lungs of smokers and patients with Cystic is (CF), or Acute Respiratory Distress Syndrome (ARDS).

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

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

AAT deficiency affects approximately 0 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 tly only a few FDA—approved drugs for treatment of ATT deficiency (Prolastin®, AralastTM, Zemaira®, GlassiaTM). Each drug is the natural AAT derived from pooled human plasma, which appears to be insufficient to meet the anticipated clinical demand. Furthermore, these products have short serum half lives (T [/2 of approximately 5 days) and require high dose (60 mg/kg body weight) weekly infusions. The current market for these drugs is estimated at approximately $400 million. The market AAT-like drugs is likely ntially larger, based on the estimation that as many as 95% of individuals with AAT-deficiencies go undiagnosed, and the fact that these drugs have the potential to be effective therapies for pathologies characterized by enhanced NE activity in individuals that are not AAT-deficient (e.g., cystic s (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-~-l636, Libert er al 1996 Immunol lS7:5126---5129, Pott et al, l of Leukocyte Biology 85 2009, Janciauskiene et al 2007 J. Biol Chem 282(12): 8573—8582, Nita er al 2007 Int J Bloc/28m Cell Biol 39:11.65-~1176). Recently, evidence has mounted that AAT may be useful in treating numerous human pathologies, e of the commonly suggested inflammatory pulmonary conditions. Human AAT has shown to protect mice from clinical and histopathological signs of experimental autoimmune encephalomyelitis (EAR), suggesting it could be a potential treatment of autoimmune diseases, such as multiple sclerosis or systemic lupus erythematosus (SLE) (Subramanian er al 2011 Metab Brain Dis 26: 107—1 13). Serum AAT has shown activity in rodent models of Graft Versus Host Disease (GVHD) (Tawara et al 2011 Proc. Natl. Acad.

Sci. USA 109: 564—569, Marcondes et al 2011 Blood Nov 18):5031-9), which has lead to a human clinical trial using AAT to treat individuals with d Non-responsive Acute GVHD (NCT01523 821). Additionally, AAT has been effective in animal models of type I and type II diabetes, dampening inflammation, protecting islet cells from apoptosis and enabling e islet cell allograft (Zhang et al 2007 Diabetes 56:1316—1323, Lewis et al 2005 Proc Natl Acad Sci USA 1.02:12153—12158, Lewis et' al 2008 Proc Natl Acad Sci USA 236-~16241, Kalis et a1 2010 Islets 2:185~—189). Currently, there are numerous early human clinical trials of type I diabetes using serum derived AAT products (NCT01183468, NCT01319331, NCT01304537).

The current serum—derived AAT products undergo extensive purification and testing to ensure the removal of pathogenic viruses, however, the risk of transmission of infectious agents cannot be completely eliminated. Moreover, serum is d, which limits the production capacity of serum d AAT. Attempts to address the ns of serum derived products and production issues have been aimed at the expression of recombinant AAT. r, after 20 years ofwork, the generation of a therapeutically viable recombinant AAT has yet to reach the market (Kamaukhova et a12006 Amino Acids : 317). Like the plasma-derived products, recombinant ns of AAT suffer from short serum ives, low production 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 ptide with a second polypeptide that interacts with the al 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 (lg) Fc polypeptides derived from human lgGl or lgM, and derivatives of human albumin.

, IgGZ, IgG3, IgG4, In some embodiments, the fusion protein incorporates mutations with the AAT portion that render the molecule more resistant to inactivation by oxidation. For example Met351Glu, Met358Leu (AAT—EL-Fc), demonstrates resistance inactivation by H202 oxidation (Figure 1G). While AAT is a natural anti-inflammatory protein, some embodiments of the invention provide enhanced ation dampening capacity through the fusion of an AAT polypeptide and a cytokine targeting 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 anti-infective activity of AAT will mitigate the infection risk of most cytokine ing biologics. Some embodiments provide for more potent anti-inflammatory and anti-infective proteins through the fusion an AAT—polypeptide and WAP domain contain polypeptide.

The filSlOIl proteins of the present ion are expected 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 IgG1, lgGZ, IgG3, IgG4, IgM, or HSA, such that the expected protein product would be AAT ed by an F0 domain ((AAT—Fc (lgGl), AAT-Fe (lgGZ), AAT—Fe (IgG3), AAT-Fe , AAT-Fe (lgM)) or AAT followed by HSA. While it was known that fusion of Fe domains of HSA to some proteins, protein domains or peptides could extend their half-lives (see e.g., Jazayeri et a1. BioDrugs 22, 11-26, Huang et al. (2009) Curr Opin hnol 20, 692-699, mann et a1. (2009) BioDrugs 23, 93—109, Schmidt et a1. (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 ofNE inhibitory activity, or could extend the half—life of recombinant AAT. The fusion proteins of the present invention are shown to be potent inhibitors ofNE, have extended serum half lives, and in some embodiments resistant to oxidation. In other embodiments, the fusion proteins described herein have ct properties by the incorporation of other functional polypeptides, including cytokine targeting polypeptides, and WAP domain containing 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 polypeptide. In some embodiments, for example, the invention provides a serpin polypeptide fused to human IgGl-Fc, IgGZ—Fc, c, IgG4-Fc, IgM-Fc, or HSA derivatives. The serpin-fusion described herein are expected to be useful in treating a variety of tions, including, by way limiting example, I—antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress sydrome (ARDS), allergic asthma, cystic fibrosis, cancers of the lung, ischemia~reperfusion injury, ing, e.g., ischemia/reperfusion injury following c 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, nia, , graft versus host disease (GVI—ID), wound healing, Systemic lupus erythematosis, and Multiple sclerosis.

] In some embodiments, the fusion proteins described herein include at least an alpha-l—antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and second ptide. For example, the invention provides alpha-l-antitrypsin (AAT) fused to human IgGl-Fc, IgGZ-Fc, c, IgG4-Fc, IgM-Fc, or HSA derivatives.

In some ments, the fusion proteins bed herein include at least a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide and a cytokine targeting polypeptide or an amino acid sequence that is derived from a cytokine targeting polypeptide. For example, the invention provides serpin polypeptide or a sequence derived from a serpin polypeptide fiised to a human cytokine receptor or derivative thereof. Another embodiment of the invention provides serpin polypeptide or a sequence derived from a serpin polypeptide fused to a cytokine targeting antibody, e.g, an anti—cytokine antibody, or a sequence derived from of a cytokine targeting antibody, e.g., an anti—cytokine antibody, or ce derived from a fragment of cytokine targeting dy, e.g., a fragment of an anti—cytokine antibody. For example, the ion provides a serpin polypeptide or a ce derived from a serpin polypeptide fused to a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds any of the following human cytokines: TNFOL, IgE, IL-12, IL-23, IL—6, IL-1 0t, IL—1 [3, IL—17, IL—13, IL-4, IL—1 0, IL—2, IL—l8, IL—27, or IL-32.

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

For example, in some embodiments, the cytokine targeting ptide targets IgE and includes any of the following IgE—targeting polypeptide or sequences derived from the following IgE—targeting polypeptides: Xolair or chR1.

For example, in some embodiments, the ne targeting ptide targets the shared p40 subunit of IL—12 and IL-23 and includes the Stelara® polypeptide or sequences derived from the Stelara® polypeptide.

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

In some embodiments, the fusion proteins described herein include at least a alpha—l—antitrypsin (AAT) polypeptide or an amino acid sequence that is d from AAT and a cytokine targeting polypeptide or an amino acid sequence that is derived from a cytokine targeting polypeptide. For example, the invention provides alpha—l—antitrypsin inhibitor (AAT) fused a cytokine targeting polypeptide in which the cytokine ing polypeptide binds any of the following human cytokines: TNFd, IgE, IL—6, IL—l 0L, IL—lB, IL-lZ, IL—l7, IL-l3, IL—23, IL—4, IL-10, IL—2, IL—18,IL-27, or IL—32.

In some embodiments the cytokine targeting polypeptide binds a ne receptor and ts binding of the cytokine. For example, the present invention includes a serpin fused to a cytokine receptor targeting dy. For example, the invention provides alpha-l-antitrypsin inhibitor (AAT) fiised a ne targeting polypeptide in which the cytokine targeting polypeptide binds the receptor of any of the following human cytokines: TNFoc, IgE, IL-6, IL—la, IL—1 [3, IL-12, IL—17, IL-13, IL—23, the p40 t of IL—12 and IL—23, IL—4, IL-10, IL-2, IL-l8, IL-27, or IL—32.

For example, in some embodiments, the cytokine targeting polypeptide targets the lL-6 receptor and includes the Actemra® polypeptide (as described in patent publication EP0628639), or the ALX-0061 polypeptide (as described in W02010/115998), or sequences d from the Actemra® polypeptide, or 6l polypeptide.

For e, Actemra® the cytokine targeting ptide targets the IL—6 receptor and es the tocilizumab polypeptide or sequences derived from the tocilizumab polypeptide.

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

] In some embodiments, the fiision proteins described herein include a serpin polypeptide or an amino acid sequence that is derived from a serpin, a WAP domain- containing polypeptide or an amino acid sequence that is derived from a WAP domain— containing polypeptide, and an Fe polypeptide or an amino acid ce that is derived from an F0 polypeptide. For e, the invention provides a serpin polypeptide, a WAP domain~containing polypeptide and human IgGl-Fc, IgGZ—Fc, IgG3—Fc, IgG4—Fc 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 described herein include at least an alpha-l—antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a SLPI polypeptide or an amino acid sequence that is derived from SLPI. In some embodiments, the fusion proteins described herein e 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 proteins 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 serpin-albumin binding polypeptide fusion proteins, wherein the n 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 ptide or sequences derived from the serpin polypeptide or a HSA polypeptide. For example, the invention es a serpin polypeptide fitsed to human HSA, or HSA derivatives, or HSA binding peptide or polypeptides.

In some embodiments, the fusion proteins described herein include at least an 1-antitrypsin(AAT) polypeptide or an amino acid ce 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-l-antitrypsin (AAT) fused to HSA or a fragment derived from HSA, or an albumin binding polypeptide.

In some embodiments, the fusion proteins described herein include a serpin polypeptide or an amino acid ce 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 - containing polypeptide or an amino acid sequence that is derived from a WAP domain— containing polypeptide. In some embodiments, the fusion proteins described herein include at least an alpha—l-antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a HSA polypeptide or an amino acid sequence that is derived from a HSA polypeptide, and a SLPI polypeptide or amino acid sequence derived from SLPI. In other ments, the fusion proteins described herein include at least an alpha—l-antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a HSA polypeptide or an amino acid sequence that is derived from a HSA polypeptide, and an Elafin polypeptide or amino acid sequence d from Elafin.

The fusion proteins of the present invention can be readily ed in mammalian cell expression s. For example Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) 293 cells, COS cells, PER.C6®, NSO cells, SP2/O, YBZ/O can readily be used for the expression of the serpin fusion proteins described herein. lmportantly, mammalian cell expression systems produce proteins that are lly more optimal for therapeutic use. In contrast to bacterial, insect, or yeast-based expression s, mammalian cell expression systems yield proteins with glycosylation patterns that are similar or the same as those found in natural human proteins. Proper gylcosylation of a protein can greatly ce serum stability, pharmacokinetics, biodistribution, protein folding, and functionality. Therefore, the y to produce therapeutic proteins in mammalian expression systems has distinct advantages over other systems. Furthermore, most of the mammalian cell expression systems (e.g., CHO, NSO, PER.C6® cells) can be readily sealed in commercial manufacturing facilities to produce eutic proteins to meet clinical demands. The fusion proteins described herein have enhanced onalities over the natural form ofAAT and can be produced in mammalian expression systems for clinical and commercial supply. Some ments of the invention include a purification system that enables the isolation of serpin fusion proteins that retain their ability to inhibit NE. Importantly, the purification process of the present invention can be readily incorporated into today’s commercial mammalian cell-based manufacturing processes.

Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ry skill in the art. Further, unless otherwise required by t, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques 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. rd techniques are used for inant DNA, oligonucleotide sis, and tissue culture and transformation (e.g., oporation, lipofection). Enzymatic reactions and purification techniques are performed ing to manufacturer's specifications or as commonly lished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in s general 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, NY. (1989)). The latures 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. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. The term patient includes human and veterinary ts.

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 incorporated into formulations to provide improved transfer, ry, 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, , PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, nts, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LipofectinTM), DNA conjugates, anhydrous absorption pastes, oil—in—water and water—in—oil emulsions, emulsions carbowax thylene glycols of various molecular weights), semi— solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing es 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 pment: the need for preclinical guidance." Regul. Toxicol Pharmacol. 32(2):210—8 (2000), Wang W.

"Lyophilization and development of solid protein pharmaceuticals." Int. J. Pharm. 203(1— 2):1-60 , Charman WN "Lipids, lipophilic drugs, and oral drug delivery-some emerging ts." J Pharm Sci. 89(8):967~78 (2000), Powell et a1. "Compendium of ents for parenteral formulations" PDA J Pharm Sci Technol. 52:23 8-3 11 (1998) and the citations n for additional information related to formulations, ents and carriers well known to pharmaceutical chemists.

Therapeutic formulations of the invention, which include a fusion n 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 es methods of treating or alleviating a symptom associated with a disease or disorder ated with aberrant serine protease activity in a subject. A therapeutic n is d out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a disease or disorder associated with aberrant serine protease ty, using standard methods, including any of a variety of clinical and/or laboratory procedures. The term patient includes human and veterinary subjects. The term subject es humans and other mammals.

Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular disease or disorder associated with aberrant serine protease activity. ation of one or more symptoms of the disease or disorder associated with aberrant serine protease activity indicates that the fusion protein confers a clinical benefit.

Methods for the screening of fusion proteins that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA), enzymatic , flow try, and other immunologically mediated techniques known within the art.

The fusion proteins described herein may be used in methods known within the art relating to the localization and/or quantitation of a target such as a serine protease, e.g., for use in measuring levels of these targets within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). The terms "physiological sample" and "biological sample," used interchangeably, herein are ed to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. ed within the usage of the terms "physiological sample" and "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood , or lymph.

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

A fusion protein of the invention can be used to e a particular target using standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. Detection can be facilitated by coupling (216., physically g) the fusion protein to a detectable substance. es of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive als. 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 suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine cein, dansyl chloride or rythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and es of suitable radioactive material include 125 35S I, 131I, or 3H. [00011 1] A therapeutically effective amount of a fusion protein of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding ction between the fusion protein and its target that, in certain cases, interferes with the functioning of the target. The amount required to be administered will furthermore depend on the binding affinity of the fusion protein for its specific target, and will also depend on the rate at which an administered fusion protein is depleted from the free volume other subject to which it is stered. Common ranges for therapeutically effective dosing of an fusion protein or fragment thereof invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 250 mg/kg body. weight.

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

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

The active ingredients can also be entrapped in microcapsules ed, for example, by coacervation ques 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 viva administration must be sterile. This is readily accomplished by filtration h sterile ion membranes. ned—release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the fusion protein, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl~methacrylate), or poly(vinylalcohol)), polylactides (US. Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L- ate, non—degradable ethylene-vinyl e, able lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM (injectable microspheres composed of lactic acid—glycolic acid copolymer and leuprolide acetate), and ~(—)—3—hydroxybutyric acid.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for r 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 able carrier.

As used herein, the term "pharmaceutically able carrier" is intended to include and all solvents, dispersion media, coatings, cterial and antifungal , isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

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

Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and non- aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is plated. Supplementary active compounds can also be orated 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., inhalation), transdermal (i.e., topical), transmucosal, and rectal stration. Solutions or suspensions used for parenteral, intradermal, or aneous 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 tic ts; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium ide. The parenteral preparation can be enclosed in ampoules, able syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for inj ectable use e sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile able solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, hor ELTM (BASF, Parsippany, NJ.) or phosphate buffered 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 contaminating action of microorganisms such as bacteria and fungi.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polycl (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a g such as lecithin, by the nance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to e isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for e, aluminum monostearate and gelatin.

Sterile injectable solutions can be ed by incorporating the active nd in the required amount in an appropriate t 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 e 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 onal desired ingredient from a previously sterile- filtered solution thereof.

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

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

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

For transmucosal or transderrnal administration, penetrants appropriate to the barrier 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. ucosal stration can be accomplished through the use of nasal sprays or suppositories. For transdermal stration, the active compounds are formulated into nts, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of itories (e.g, with tional suppository bases such as cocoa butter and other ides) 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 ncapsulated delivery systems.

Biodegradable, biocornpatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be ed commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared ing to methods known to those skilled in the art, for example, as described in US. Patent No. 4,522,811.

It is ally advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration 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 desired therapeutic effect in association with the required pharmaceutical r. The specification for the dosage unit forms of the invention are dictated by and ly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

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

The invention will be further described in the following examples, 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 Fc fusion proteins according to the invention e the following sequences. While these examples include a hinge sequence and/or a linker sequence, fusion proteins of the invention can be made using any hinge sequence and/or a linker sequence suitable in length and/or flexibility. Alternatively fusion proteins can be made t using a hinge and/or a linker sequence. For example, the polypeptide components can be directly attached. 1001118691 An exemplary AAT—Fc fusion protein is the AAT-hFcl (human IgG1 Fc) bed 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 IgG—Fc ptide portion of the fusion protein is italicized (SEQ ID NO: 3).

AAT—hFcl (human IgG1 Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO:16) An exemplary AAT-Fc fusion protein is the AAT-hFcZ (human IgG2 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: 44), and the IgG-Fc polypeptide n of the fusion protein is italicized (SEQ ID NO: 4).

AAT-hFcz (human IgG2 Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE KFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM VTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDW LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP 1001118691 SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 17) An exemplary AAT-Fe fusion protein is the AAT-MM-EL—hFcl (human IgG1 Fe, Met351Glu/Met358Leu), described herein. As shown below, AAT polypeptide portion of the fusion n 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 mutation is shaded in grey.

AAT-MM—EL—hFcl(human IgGl Fc, Met351Glu/Met358Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGéEFLEAIPLSIPPEVK FNKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 18) An exemplary AAT-F0 fusion protein is the —EL-hFC2 (human IgG2 Fe, Met351G1u/Met358Leu), described herein. As shown below, AAT ptide n 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 Met351Glu mutation is boxed, and the Met358Leu mutation is shaded in grey.

AAT—MM—EL—hFcZ (human IgG2 Fc, Glu/Met358Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAG‘EFLEAIPLSIPPEVK FNKPFVFLMIEQNTKSPLFMGKVVNPTQKERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD WLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK (SEQ ID NO: 19) An exemplary AAT-Fc fusion protein is the AAT-MM-LL-hFc1(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 IgG—Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 3), the Met351Leu mutation is shaded in black, and the Met358Leu mutation is shaded in grey.

AAT—MM—LL-hFcl(human IgGl Fc, Met351Leu/Met358Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGQEFLEAIPLSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK AAT-hFcl —-AAT (human IgG1) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY IFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQ LAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLR SQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVE KGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKR 1001118691 LGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSA SLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGT EAAGAMFLEAIPMS FNKPFVFLMIEQNTKSPLFMGKVVNPTQK ( 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 encoding gene was cloned in frame with a gene ng the hinge region, followed by a CH2 domain, and a CH3 domain of human IgG1 , IgG2, IgG3, IgG4, or IgM into a ian expression vector, containing a ian secretion signal sequence up stream of the AAT gene insertion site. These expression 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 fusion proteins were purified from the expression cell supernatant by protein A tography. lmportantly, a near neutral pH buffer was used (Gentle Ag/Ab Elution Buffer, Thermo Scientific) to elute the AAT—Fc fusion protein item the n 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 ed. oligomerization ofAAT. Figure 1F shows the effects of low pH elution on the ability of AAT to inhibit neutrophil elastase. AAT-Fc fusion protein can be purified either by protein A and a near neutral pH elution buffer, by CaptureSelect® Alpha—l Antitrypsin y matrix (BAC BV).

The purified AAT—Fc fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase (NE). Figure 1B and 1D show a reducing SDS~ PAGE gel fied serum derived AAT (sdAAT) and AAT—Fc fusion proteins (Fig 1B— lane 1: sdAAT, lane 2: AAT-Fc (SEQ ID NO: 16), lane 3: AAT—EL—Fc (SEQ ID NO: 18), Fig 1D —AAT (SEQ ID NO: 20). The proteins were visualized by staining with coomassie blue.

To monitor human Neutrophil Elastase (NB) ty a fluorescent microplate assay was used. Inhibitory activity was measured by a concomitant 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 0.0005% Triton X-lOO. Human NE is used at a final concentration of 5 nM (but can also be used from 1—20 nM). The fluorescent peptide substrate AAVP— AMC is used at a final concentration of 100 uM in the assay. The Gemini EM plate reader from lar Devices is used to read the assay cs using excitation and emission wavelengths of 370 nm and 440 nm respectively, and a cutoff of 420 nm. The assay is read for 10 min at room temperature scanning every 5 to 10 seconds. The Vmax per second corresponds to the residual NE activity, which is plotted for each concentration of inhibitor.

The intercept with the x~axis indicates the tration of inhibitor needed to fully inactivate the starting tration ofNE 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 c-AAT) displays enhanced potency over both sdAAT and the AAT-Fc fusion protein comprising a single AAT polypeptide (Figure 1B). These findings presented 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 n was found to be a more potent NE inhibitor.

Figure 1F demonstrates the resistance of the AAT-EL—Fc (M351E, M358L) fusion n to inactivation by oxidation. AAT fusion proteins, AAT—Fe (wt), AAT—EL— Fc (M351E, M358L), and AAT-EM—Fc (M35133), were treated with 33mM H202 and compared to untreated fusion proteins in the NE inhibition assays. The tion ofNE by AAT—EL-Fc was not comprised by oxidation, converse to the other proteins tested.

Furthermore, AAT-Fe fusion protein displayed a longer serum half life in rats compared to serum derived AAT (Figure 1H). In these studies 3 rats per each test protein were injected TV. with lOmg/kg of sdAAT or . 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 therapeutic format over serum derived AAT, for treating numerous human atory conditions and infectious diseases. 1001118691 Example 2: AAT~TNFa Targeting Molecule Fusion Proteins The studies presented herein describe several, non-limiting examples of recombinant AAT tives comprising human AAT heed to an anti-TNFOL antibody or a derivative of a TNFon receptor. These es are provided below to further illustrate different features ofthe 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. 3] The fusion proteins below include cytokine targeting polypeptide sequences that are from or are derived from (i) the anti—TNFon antibody D2137 (also known as Adalimumab or Humira®), or (ii) the extracellular domain of Type 2 TNFOL Receptor (TNFRZ-ECD). The AAT polypeptide portion ofthe fusion protein is underlined, the antibody constant regions (CHl—hinge—CH2-CH3, or CL) are italicized, and D2E7—VH, VK, and TNFRZ—ECD are denoted in bold text. While these es include a hinge sequence and/or a linker sequence, fusion ns ofthe invention can be made using any hinge sequence and/or a linker ce suitable in length and/or flexibility. Alternatively fusion proteins can be made without using a hinge and/or a linker sequence.

An exemplary AAT—TNFoc fusion protein is D2E7—Light Chain-AAT (G3S)2 Linker, described herein. As shown below, the AAT polypeptide n of the fusion protein is ined (SEQ ID NO: 2), D2E7—VK is denoted in bold text (SEQ ID NO: 37), the (G38); linker is shown in normal text (SEQ ID NO: 46), and the antibody constant regions are italicized (SEQ ID NO: 38) D2E7—Light AAT (G38)2 Linker DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSR FSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVKACEVTHQGLSSPVTKSFNRGECGGGSGGGSEDPQGDAAQKTDTSHHDQDHPT FNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFN LTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFT VNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEE EDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENE LTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPL 1001118691 KLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKV VNPTQK (SEQ ID NO:22) An ary AAT-TNFOL fusion protein is D2E7—Light Chain—AAT ASTGS Linker, described herein. As shown below, the AAT ptide portion of the fusion protein 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 ized (SEQ ID NO: 38) D2E7—Light Chain—AAT ASTGS Linker DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSR GTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVKACEVTHQGLSSPVTKSFNRGECASTGSEDPQGDAAQKTDTSHHDQDHPTFNK ITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTE IPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNF GDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDF HVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTH DIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLS KAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNP TQK (SEQ ID NO:23) An exemplary AAT-TNFOL fusion n 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 (G3$)2 linker is shown in normal text (SEQ ID NO: 46), and the antibody constant regions is ized (SEQ ID NO: 40) D2E7-Heavy Chain—AAT (G3S)2 Linker EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYA DSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ 1001118691 VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSEDPQGDAAQKTDTSHHDQDHPTFNKITPN LAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEA QIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTE EAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ PMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIIT KFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVH KAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NOz24) An exemplary AAT-TNFOC fusion protein is D2E7-Heavy Chain-AAT ASTGS Linker, bed 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 ASTGS linker is shown in normal text (SEQ ID NO: 45), and the antibody constant regions is italicized (SEQ ID NO: 40) D2E7—Heavy Chain—AAT ASTGS Linker EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYA DSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAE FAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIH LRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAK KQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTT VKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFL ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAV LTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NOz25) 1001118691 ] An exemplary AAT—TNFoc fusion protein is ECD—Fcl- S)2 Linker, described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), TNFRZ-ECD is denoted in bold text (SEQ ID NO: 41), the hinge region is shown in normal text (SEQ ID NO: 43), the (G38)2 linker is shown in normal text (SEQ ID NO: 46), and the antibody constant regions is italicized (SEQ ID NO: 42) TNFRZ —ECD — Fcl ~AA'I' (G38) 2 Linker LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTY TQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCR PGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSP TRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVH NAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSEDPQGDAAQKTDT SHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHD EILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVK KLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWER PFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDE GKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADL SGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNT KSPLFMGKVVNPTQK (SEQ ID NO:26) 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), TNFRZ—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 TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVH NAKTKPREQQKNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHH DQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEIL LTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLY HSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFE VKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKL LTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGV TEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSP LFMGKVVNPTQK (SEQ ID NOz27) These exemplary AAT-TNFOL targeting molecule fusion proteins were made using the following techniques. {0001491 The genes encoding the variable heavy (VH) and variable kappa (VK) regions of the anti-TNFa antibody, D2E7, were generated by gene sis. The D2E7— VH gene was cloned in frame with a gene encoding a human IgG1 antibody heavy chain constant region, consisting of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, into a mammalian expression vector, containing a mammalian secretion signal sequence up stream of the VH domain insertion site (D2E7—HC). The D2E7-VK gene was cloned in frame with a human antibody kappa light chain constant (CL) domain, into a mammalian expression vector, containing a mammalian secretion signal sequence up stream of the VK domain insertion site (D2E7—LC). The AAT ng gene and the adjacent 5’ linker sequence were cloned in frame into the 3’ end of either, the CH3 domain of the D2E7 heavy chain gene (D2E7-HC-AAT), or the CL domain of the D2E7 light chain gene (D2E7- LC-AAT) coding sequences in the above described ian expression s. The extracellular domain of the TNFor Receptor 2 (TNFR2-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 of human IgG1 (hFcl) into a mammalian expression, containing a mammalian secretion signal sequence up stream of the TNFRZ-ECD ion 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 sion vector (TNFR2— ECD—hFcl—AAT).

The C—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 ofboth the heavy chain and light chain, respectively.

The D2E7-LC—AAT was co-transfected with the D2E7-HC sion vector into mammalian cells to generate the D2137 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% C02 at 37° C. 1] The recombinant AAT-TNFoc targeting fusion proteins were purified from the expression cell supernatant by protein A chromatography. A near neutral pH buffer was used (Gentle Ag/Ab Elution , Thermo Scientific) to elute the AAT—TNFoc targeting fusion ns from the protein A resin.

Figure 2B shows an SDS—PAGE gel of the D2137 antibody alone (lane 1) and variant wherein AAT is fused to the heavy chain of D2E7 (lane 2). The proteins were ized by staining with coomassie blue. 3] The purified AAT-TNFOL ing molecule fusion proteins were tested for ty by determining their y to inhibit neutrophil elastase. Human serum derived AAT (sdAAT) was used as a positive control in these assays. NE inhibitory assay were conducted as described above. Figure 2C demonstrates ve to sdAAT, the AAT-TNFd targeting molecule fusion protein shows similar inhibition of neutrophil elastase, indicating that the tory capacity 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 several, non-limiting examples of recombinant AAT derivatives comprising human AAT fused a WAP domain containing protein. 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 sequence and/or a linker sequence suitable in length 1001118691 and/or flexibility. Alternatively fusion proteins can be made without using a hinge and/or a linker sequence. For example, the polypeptide components can be directly ed.

An ary AAT-Fc—SLPI fusion protein is AAT-hFcl—SLPI (human IgG1 Fc), described . 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: 9) AAT—hFcl—SLPI (human IgG1 Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKASTGSSGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCP DTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGMCGKSC VSPVKA (SEQ ID NO:28) An exemplary -Elafin fusion protein is AAT—hFcl—Elafin (human IgG1 Fe), bed 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 LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKASTGSAVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQ EPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ (SEQ ID NO:29) The genes encoding the SLPI and Elafin were PCR amplified from human spleen cDNA (Zyagen). These genes were cloned into the ian expression vectors of example 1, wherein the SLPI or Elafin gene was inserted in frame with the AAT—Fe gene.

These expression vectors were transfected into mammalian cells (specifically HEK293 or CHO cells) and grown for several days in 8% C02 at 37° C. The inant AAT-Fe— 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 n A resin.

Figure 3B shows an SDS—PAGE gel of the AAT-Fc-WAP fission 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 ns were tested for activity by determining their y to inhibit neutrophil elastase. NE inhibitory assays AAT (sdAAT) and the AAT-Fe were conducted as described above. Human serum derived fusion n were used as a positive control in these assays. Relative to sdAAT, the AAT- inhibition of Fc-WAP targeting molecule fiision proteins display ed potency of NE neutrophil elastase (Figure 3C).

Example 4 AAT-Albumin The studies presented herein be several, non—limiting examples of These recombinant AAT derivatives comprising human AAT fused an albumin polypeptide. invention. examples are provided below to further rate different features of the present These The examples also illustrate useful methodology for practicing the invention. es do not and are not intended to limit the claimed invention. The AAT portion 1001118691 underlined and the albumin portion is italicized. For example, the polypeptide components can be directly attached. 0] An exemplary AAT—Albumin fusion n is AAT-HSA, bed herein.

As shown below, the AAT polypeptide portion 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: l4) AAT-HSA EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKASTGSDAHKSEVAHRFKDLGEENFKALVLIAFA QYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMAD CCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAF KAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSIS SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYE YARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCEL FEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTL SEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA ASQAALGL (SEQ ID NO:30) An exemplary AAT-Albumin fusion protein is AAT—HSA Domain 3, described herein. As shown below, the AAT ptide 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: 15) AAT~HSA Domain 3 EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE 1001118691 KFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKASTGSEEPQNLIKQNCELFEQLGEYKFQNALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVS DRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVE LVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA (SEQ ID NO:31) The gene encoding human serum albumin (HSA) was PCR ed 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 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—HSA fusion proteins were purified item the expression cell atant using the CaptureSelect® Alpha—l Antitrypsin affinity matrix (BAC BV), wherein the binding buffer consisted of20mM Tris, 15OrnM NaCl, pH 7.4 and the elution buffer consisted of ZOmM Tris, 2M MgClz 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 AAT—HSA fusion proteins were tested for ty by determining their ability to inhibit neutrophil elastase.

NE tory assays were conducted as described above. Human serum derived AAT (sdAAT) was used as a positive l in these assays. Relative to sdAAT, the AAT—HS fusion protein displays similar potency ofNE inhibition, demonstrating that the fusion to albumin does not dampen the capacity ofAAT to inhibit NE (Figure 4C.) Other Embodiments While the invention has been described in conjunction with the detailed description thereof, the foregoing description is ed to illustrate and not limit the scope of the invention, which is defined by the scope of the ed claims. Other aspects, advantages, and modifications are within the scope ofthe following claims. 1003777385

Claims (2)

What is claimed is:

1. A method of purifying a fusion protein and preventing oligomerization of AAT in the fusion protein, the method comprising the steps of: (a) culturing a mammalian cell comprising a nucleic acid uct that encodes the fusion protein under conditions that allow for the expression of the fusion protein, wherein the fusion protein comprises at least one human serpin polypeptide comprising an alpha-1 antitrypsin (AAT) ptide comprising the ve site loop of AAT comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 32 and 33 or an AAT ptide sing an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 34, and 35; wherein the at least one human serpin polypeptide is ly linked to a human immunoglobulin Fc polypeptide; (b) contacting a supernatant from the cultured cell with an affinity resin under conditions that allow for binding between the affinity resin and the fusion protein, wherein the affinity resin comprises protein A; and (c) eluting the fusion protein from the affinity resin using a buffer under conditions that allow for the detachment of the fusion protein from the affinity resin and prevent oligomerization of AAT in the fusion protein, wherein the buffer is at a near-neutral pH, wherein the purified fusion protein inhibits neutrophil elastase (NE) activity.

2. The method of claim 1, wherein the cell comprises a e Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) 293 cell, a COS cell, a .RTM. cell, a NS0 cell, a SP