NZ619035B2 - Wap domain fusion polypeptides and methods of use thereof - Google Patents

Abstract

Disclosed is an isolated fusion protein comprising at least one human whey acidic protein (WAP) domain-containing polypeptide comprising a human secretory leukocyte proteinase inhibitor (SLPI) polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a human elafin polypeptide comprising the amino acid sequence of SEQ ID NO: 6 operably linked to an immunoglobulin Fc polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 10, wherein the sequences are as listed in the complete specification. amino acid sequence of SEQ ID NO: 6 operably linked to an immunoglobulin Fc polypeptide comprising an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 10, wherein the sequences are as listed in the complete specification.

Description

WAP DOMAIN FUSION POLYPEPTIDES AND METHODS OF USE THEREOF Related Applications This application claims the benefit of US ional Application No. 61/502052, flied June 28, 2011; US Provisional Application No. 625, filed December 1, 2011; and US.

Provisional Application No. 168, filed April 25, 2012; and US. Provisional Application No. 61/638516, filed April 26, 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 include a whey acidic protein (WAP) —containing ptide or an amino acid sequence that is derived from a WAP domain-containing polypeptide, and second polypeptide. Additionally, the invention relates to fusion proteins that e a WAP domain— containing polypeptide or an amino acid sequence that is derived from a WAP domain-containing polypeptide, a second polypeptide, and a third polypeptide. Specifically, this invention relates to fusion proteins that include WAP domain—containing polypeptides and a second polypeptide or fusion proteins that include WAP domain-containing polypeptides, 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 of the following: an Fe polypeptide or an amino acid this derived from an Fc polypeptide; an albumin polypeptide, or an amino acid sequence that is derived from an albumin polypeptide; a cytokine ing polypeptides or an amino acid sequence that is derived from a cytokine targeting polypeptide; and a serpin polypeptide or an amino acid sequence that is derived from a serpin 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 ion

[0003] nt 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 inhibitor. 1001117592 ] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general dge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art. [0003b] As used herein, except where the context requires otherwise the term ‘comprise’ and variations ofthe term, such as ‘comprising’, ‘comprises’ and ‘comprised’, are not intended to exclude other additives, components, integers or steps. 1001117592 Summary of the Invention The fusion proteins described herein include at least a whey acidic protein (WAP) domain-containing polypeptide or an amino acid sequence that is derived from a WAP domain-containing polypeptide eptide 1), and second polypeptide (Polypeptide 2). Additionally the fusion proteins described herein include a whey acidic protein (WAP) domain-containing polypeptide or an amino acid sequence that is derived from a WAP domain-containing polypeptide (Polypeptide 1), a second polypeptide (Polypeptide 2), and a third polypeptide (Polypeptide 3). Specifically, this invention s to fusion ns that include a WAP domain-containing polypeptide and a second polypeptide or a WAP domain-containing polypeptide, a second polypeptide, and a third polypeptide, where the second and third polypeptides can include at least one of the following; an Fc polypeptide or an amino acid this derived from an Fc polypeptide; an albumin ptide or an amino acid sequence that is derived from an albumin ptide; a cytokine ing polypeptides or an amino acid sequence that is d from a cytokine targeting polypeptide; and a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide.

As used interchangeably herein, the terms "fusion protein" and "fusion ptide" refer to a WAP domain-containing polypeptide or an amino acid sequence derived from a WAP domain-containing polypeptide operably linked to at least a second polypeptide or an amino acid sequence derived from a second polypeptide. The dualized elements of the fusion protein can be linked in any of a variety of ways, including for example, direct attachment, the use of an intermediate or 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 may fall within the sequence of the linker region.

Preferably, the linker region is a e sequence. For example, the linker peptide includes anywhere from zero to 40 amino acids, e.g., from zero to 35 amino acids, from zero to 30 amino acids, from zero to 25 amino acids, or from zero to 20 amino acids. Preferably, the hinge region is a peptide sequence. For e, the hinge peptide includes anywhere from zero to 75 amino acids, e.g., from zero to 70 amino acids, from zero to 65 amino acids or from zero to 62 amino acids. In embodiments where the fusion protein includes both a linker region and hinge region, preferably each of the linker region and the hinge region is a peptide sequence. In these embodiments, the hinge peptide and the linker peptide together include anywhere from zero to 90 amino acids, e.g., from zero to 85 amino acids or from zero to 82 amino acids.

In some ments, the WAP domain-based portion and the second polypeptide-based portion of the fusion protein can be linked valently through an intermediate binding polypeptide. In some embodiments, the WAP domain-based portion and the second polypeptide-based portion of the fusion protein may be non-covalently linked.

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

In some embodiments, the Polypeptide 1 sequence includes a whey acidic protein (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 isulfide core motif). The WAP domain sequence motif is a functional motif characterized by serine protease inhibition ty in a number of proteins. WAP domain-containing polypeptides suitable for use in the fusion proteins provided herein include, by way of non-limiting example, secretory leukocyte protease inhibitor (SLPI), Elafin, and Eppin.

In some embodiments, the Polypeptide 1 sequence includes a secretory leukocyte protease inhibitor (SLPI) polypeptide sequence or an amino acid sequence that is derived from SLPI. In some embodiments, the Polypeptide 1 sequence es a portion of the SLPI protein, such as for example, the WAP2 domain or a sub-portion thereof. In a preferred embodiment, the SLPI polypeptide sequence or an amino acid sequence that is derived from SLPI that is derived from a human SLPI polypeptide sequence.

In some ments, the fusion protein es a full-length human SLPI polypeptide sequence having the following amino acid sequence: 1 MKSSGLFPFL VLLALGTLAP WAVEGSGKSF KAGVCPPKKS AQCLRYKKPE CQSDWQCPGK 61 KRCCPDTCGI KCLDPVDTPN PTRRKPGKCP VTYGQCLMLN PPNFCEMDGQ CKRDLKCCMG 121 MCGKSCVSPV KA (SEQ ID NO: 1 ) In some embodiments, the fusion protein includes a human SLPI polypeptide ce that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the fusion protein includes a portion of the full-length human SLPI polypeptide ce, where the portion has the following amino acid sequence: 1 SGKSFKAGVC PPKKSAQCLR YKKPECQSDW QCPGKKRCCP CLDP VDTPNPTRRK 61 PGKCPVTYGQ CLMLNPPNFC EMDGQCKRDL KCCMGMCGKS CVSPVKA (SEQ ID NO: 2 ) In some embodiments, the fusion n 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: 2 .

In some embodiments, the fusion protein includes the WAP2 domain of the full-length human SLPI polypeptide sequence, where the WAP2 domain has the following amino acid ce: 1 KCPV TYGQCLMLNP PNFCEMDGQC KRDLKCCMGM CGKSCVSPVK A (SEQ ID NO: In some embodiments, 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: 3 .

In some embodiments, the SLPI polypeptide sequence is or the amino acid sequence d from an SLPI ptide is derived from, one or more of the human SLPI polypeptide sequences shown in GenBank ion Nos. CAA28 187.1, NP_003055.1, EAW75869.1, P03973.2, AAH20708.1, CAB64235.1, CAA28188.1, AAD 19661.1, and/or BAG35 125.1.

In some ments, the fusion protein includes a full-length human elafin polypeptide sequence having the following amino acid sequence: 1 LIVV VFLIAGTLVL EAAVTGVPVK GQDTVKGRVP FNGQDPVKGQ VSVKGQDKVK 61 AQEPVKGPVS TKPGSCPIIL IRCAMLNPPN RCLKDTDCPG IKKCCEGSCG MACFVPQ (SEQ ID NO: 4 ) In some embodiments, 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: 4 .

In some embodiments, the fusion protein includes a portion of the full-length human elafin ptide sequence, where the portion has the following amino acid sequence: 1 AVTGVPVKGQ VPFN GQDPVKGQVS VKGQDKVKAQ EPVKGPVSTK PGSCPIILIR 61 CAMLNPPNRC LKDTDCPGIK KCCEGSCGMA CFVPQ (SEQ ID NO: 5 ) In some embodiments, 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: 5 .

In some embodiments, 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: 6 ) In some embodiments, the fusion protein includes a human elafin polypeptide ce that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6 .

In some embodiments, the Elafin polypeptide sequence is or the amino acid sequence derived from an Elafin polypeptide is derived from, one or more of the human elafin polypeptide sequences shown in GenBank Accession Nos. .3, NP 002629.1, BAA02441.1, EAW75814.1, EAW75813.1, .1, and/or NP_542 18 1.1.

In some embodiments, the Polypeptide 1 sequence includes an Eppin polypeptide sequence or an amino acid sequence that is derived from Eppin. In some embodiments, the Polypeptide 1 sequence 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 is or an amino acid sequence that is d from Eppin is derived from, a human Eppin polypeptide sequence.

In some embodiments, the Eppin polypeptide sequence is or the amino acid sequence derived from an Eppin polypeptide is derived from one or more of the human Eppin polypeptide sequences shown in k Accession Nos. 095925 .1, NP 065 131.1, AAH44829.2, AAH53369.1, AAG00548.1, 47.1, and/or AAG00546.1.

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

For e, the fusion protein contains at least one mutation at a nine (Met) e in the non-Fc portion of the fusion protein, for example in the SLPI portion of the fusion protein. In these Met ons, the Met residue can be substituted with any amino acid.

For example, the Met residue can be tuted with an amino acid with a hydrophobic side chain, such as, for example, leucine (Leu, L) or valine (Val, V). Without wishing to be bound by theory, the Met mutation(s) prevent oxidation and subsequent inactivation of the inhibitory activity of the fusion proteins of the invention. In some embodiments, the Met mutation is at position 98 of an SLPI polypeptide, for example, the Met mutation is Met98Leu (M98L) in SEQ ID NO: 8.

In some embodiments, the fusion proteins are modified to se or otherwise inhibit proteolytic cleavage, for example, by mutating proteolytic cleavage sites.

In some embodiments, the proteolytic cleavage site mutation occurs at a residue in the SLPI portion of the fusion protein. For example, the proteolytic cleavage site mutation occurs at a residue in the amino acid sequence of SEQ ID NO: 2 selected from Serl5, Alal6, Glul7, and combinations thereof.

In some embodiments, the second polypeptide (Polypeptide 2) of the WAP domain-containing fusion protein is an Fc polypeptide or derived from an Fc polypeptide.

These embodiments are referred to collectively herein as "WAP-Fc fusion ns." The WAP-Fc fusion proteins bed herein include at least a WAP domain-containing polypeptide or an amino acid ce that is derived from a WAP domain-containing polypeptide and an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide.

In some embodiments, the WAP-Fc fusion protein includes a single WAP domain containing polypeptide. In other embodiments, the WAP-Fc fusion ns include more than one WAP domain containing polypeptide, collectively referred to herein as "WAP -Fc fusion n," where (a') is an integer of at least 2 . In some embodiments, (a ) WAP domain containing polypeptides in a WAP -Fc fusion protein can include the same (a ) amino acid sequence. For example, the WAP -containing polypeptides of the WAP -Fc fusion protein can be derived from a SLPI or an Elafin polypeptide, but not both (a ) (e.g., Elafin-Fc-Elafin, or SLPI-Fc-SLPI). In other embodiments of WAP domain containing polypeptides in a WAP -Fc fusion protein can include distinct amino acid (a ) sequences. For example, the fusion protein orates amino acid sequences derived from both SLPI and Elafm (e.g., SLPI-Fc-Elafm, or Elafm-Fc-SLPI).

In some embodiments, the WAP domain-containing polypeptide of the WAP-Fc fusion protein is derived from any one of the amino acid sequences of SEQ ID NOs 1-6. In some embodiments, the WAP domain-containing polypeptide of the WAP-Fc fusion n has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the amino acid ces having SEQ ID NO. 1, 2, 3, 4, 5 or 6 .

In some embodiments, the WAP domain-containing polypeptide sequence of the WAP-Fc fusion protein is or is derived from sequences shown in k Accession Nos. CAA28187.1, NP_003055.1, EAW75869.1, P03973.2, AAH20708.1, CAB64235.1, CAA28188.1, AAD19661.1, BAG35125.1., P19957.3, NP_002629.1, BAA02441.1, EAW758 14. 1, EAW758 13.1, Q8IUB2. 1, and/or NR_542 181.1., 095925 .1, NR_065 131.1, AAH 44829.2, AAH53369.1, AAG00548.1, AAG00547.1, and/or AAG00546.1.

In some embodiments, the Fc polypeptide of the WAP-Fc fusion protein is a human Fc polypeptide, for example, a human IgG Fc polypeptide sequence or an amino acid sequence that is d from a human IgG Fc polypeptide ce. In some embodiments, the Fc ptide is a human IgGl Fc polypeptide or an amino acid sequence that is derived from a human IgGl Fc polypeptide ce. In some embodiments, the Fc polypeptide is a human IgG2 Fc polypeptide or an amino acid sequence that is derived from a human IgG2 Fc polypeptide sequence. In some embodiments, the Fc polypeptide is a human IgG3 Fc polypeptide or an amino acid sequence that is derived from a human IgG3 Fc polypeptide sequence. In some embodiments, the Fc polypeptide is a human IgG4 Fc polypeptide or an amino acid sequence that is derived from a human IgG4 Fc polypeptide sequence. In some embodiments, the Fc ptide is a human IgM Fc polypeptide or an amino acid sequence that is derived from a human IgM Fc polypeptide sequence.

In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgGl Fc polypeptide sequence having the following amino acid sequence: 1 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED WYVD GVEVHNAKTK 61 PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 121 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL 181 TVDKSRWQQG NVFSCSVMHE TQKS K (SEQ ID NO: 7 ) In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein es a human IgGl Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid ce of SEQ ID NO: 7 .

In some embodiments where the fusion protein of the invention includes an Fc ptide, the Fc polypeptide of the fusion protein includes a human IgG2 Fc polypeptide sequence having the following amino acid sequence: 1 APPVAGPSVF KDTL MISRTPEVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKP 61 REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL 121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT 181 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 8 ) In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG2 Fc polypeptide sequence 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 ments where the fusion n of the ion includes an Fc polypeptide, the Fc polypeptide of fusion protein includes a human IgG3 Fc polypeptide sequence having the following amino acid ce: 1 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD GVEVHNAKTK 61 PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKTK GQPREPQVYT 121 LPPSREEMTK CLVK IAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL 181 TVDKSRWQQG VMHE ALHNRFTQKS LSLSPGK (SEQ ID NO: 9 ) 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 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 where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG4 Fc polypeptide ce having the following amino acid sequence: 1 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK 61 PREEQFNSTY TVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT 121 LPPSQEEMTK NQVSLTCLVK GFYPDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSRLT 181 VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK (SEQ ID NO: 10) In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG4 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10.

In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgM Fc polypeptide sequence having the following amino acid sequence: 1 IAELPPKVSV FVPPRDGFFG LICQ ATGFS PRQIQ GKQV GSGVTTDQVQ 6 1 GPTT YKVTSTLT I K E SD LGQS F TCRVDHRGLT FQQNAS S CV PDQDTAIRVF 1 2 1 AI PPSFAS I F LTKSTKLTCL VTDLTTYDSV T I SWTRQNGE AVKTHTNI SE SHPNATFSAV 1 8 1 GEAS ICEDDW NSGERFTCTV THTDLPS PLK QT I SRPKG ( SEQ I D NO : 1 1 ) In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgM Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 11.

In some embodiments of the fusion proteins provided , the second polypeptide (Polypeptide 2) of the WAP domain containing fusion protein is a cytokine ing polypeptide, or derived from a cytokine ing polypeptide. These embodiments are referred to collectively herein as "WAP-cytokine ing polypeptide fusion proteins." The WAP-cytokine targeting polypeptide fusion proteins described herein e at least a WAP domain ning polypeptide or an amino acid sequence that is derived from a WAP domain containing polypeptide and a ne targeting polypeptide, or derivation thereof. In some embodiments, the tokine targeting polypeptide fusion protein includes a single WAP domain containing polypeptide. In other embodiments, the WAP- cytokine targeting polypeptide fusion protein includes more than one WAP domain ning polypeptide, and these embodiments are collectively referred to herein as "WAP(a-)- cytokine targeting polypeptide fusion ns," wherein (a') is an integer of at least 2 . In some embodiments, each WAP domain containing polypeptide in a WAP ne targeting polypeptide fusion protein can include the same amino acid sequence. In other embodiments, each WAP domain containing polypeptide of a WAP - cytokine (a ) targeting polypeptide fusion protein can include distinct amino acid sequences.

In some embodiments, the WAP domain-containing polypeptide of the WAP-cytokine targeting polypeptide fusion protein is derived from any one of the amino acid sequences of SEQ ID NOs 1-6. In some embodiments, the WAP domain-containing polypeptide of the WAP-cytokine targeting polypeptide fusion protein has at least 50%>, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the amino acid sequences having SEQ ID NO. 1, 2, 3, 4, 5 or 6 .

In some embodiments, the WAP domain-containing polypeptide ce of the WAP-cytokine targeting polypeptide fusion protein is or is derived from sequences shown in GenBank Accession Nos. CAA28187.1, NP_003055.1, EAW75869.1, P03973.2, AAH20708.1, CAB64235.1, CAA28188.1, AAD19661.1, BAG35125.1., P19957.3, NP_002629.1, BAA02441.1, 14.1, EAW75813.1, Q8IUB2.1, and/or NP_542181.1., 095925.1, NP_065131.1, AAH44829.2, AAH53369.1, AAG00548.1, AAG00547.1, and/or AAG00546.1.

In some embodiments, the cytokine targeting polypeptide of the WAP- cytokine targeting polypeptide fusion n is a ne receptor or derived from a ne receptor. In a preferred embodiment, the cytokine targeting polypeptide is or an amino acid sequence that is derived from the cytokine receptor is derived from a human cytokine receptor sequence. In other embodiments, the cytokine targeting polypeptide is an antibody or antibody fragment, for e an ytokine antibody or anti-cytokine antibody fragment. 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 a preferred embodiment, the cytokine targeting polypeptide is or an amino acid sequence that is d from the antibody or dy fragment is derived from a chimeric, zed, or fully human antibody sequence. In other embodiments, the cytokine targeting ptide binds a cytokine or and prevents binding of a cytokine to the receptor.

The WAP-cytokine targeting ptide fusion protein can incorporate a portion of a WAP-Fc fusion protein. For example, an antibody contains an Fc polypeptide. ore, in some embodiments, where the cytokine targeting polypeptide is a cytokine- targeting antibody, the WAP-cytokine targeting polypeptide fusion protein will incorporate a portion of the WAP-Fc fusion protein. Furthermore, most or fusion proteins that are of therapeutic utility, are Fc fusion proteins. Thus, in some embodiments, wherein the WAP-cytokine targeting polypeptide fusion protein is a WAP-cytokine receptor fusion protein, the WAP-cytokine ing polypeptide fusion protein may incorporate an Fc polypeptide in addition to the WAP domain-containing polypeptide portion and the cytokine receptor portion.

In some embodiments, where the WAP-cytokine targeting polypeptide fusion protein includes an Fc polypeptide sequence, the Fc polypeptide sequence is derived from any one of the amino acid sequences having the SEQ ID NO. 7, 8, 9, 10, or 11. In some embodiments, where the WAP-cytokine targeting fusion protein includes an Fc polypeptide ce, 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% sequence identity to the amino acid sequence of any one of the sequences having the SEQ ID NO. 7, 8, 9, 10, or 11.

In some embodiments, the WAP domain containing 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 WAP domain containing polypeptide and the cytokine targeting ptide are operably linked via a hinge region.

In some ments, the WAP domain ning 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 embodiments of the fusion proteins provided herein, the second polypeptide (Polypeptide 2) of the WAP domain containing fusion n is a serpin polypeptide, or derived from a serpin polypeptide. These embodiments are referred to collectively herein as "WAP-serpin fusion proteins." The rpin fusion proteins described herein include at least a WAP domain containing polypeptide or an amino acid ce that is derived from a WAP domain containing polypeptide, and a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide.

In some embodiments, the WAP -containing polypeptide of the WAP-serpin fusion protein is derived from any one of the amino acid sequences of SEQ ID NOs 1-6. In some ments, the WAP domain-containing polypeptide of the WAP- serpin fusion protein has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence ty to any one of the amino acid sequences having SEQ ID NO. 1, 2, 3, 4, 5 or 6 .

In some embodiments, the WAP domain-containing ptide sequence of the WAP-serpin fusion protein is or is derived from sequences shown in GenBank Accession Nos. CAA28 187.1, NP 003055 .1, EAW75869. 1, P03973 .2, AAH20708. 1, CAB64235.1, CAA28188.1, AAD19661.1, BAG35125.1., P19957.3, NP_002629.1, BAA02441.1, EAW75814.1, EAW75813.1, .1, and/or NP_542 18 1.1., 095925.1, NP 065131.1, AAH44829.2, AAH53369.1, AAG00548.1, 47.1, and/or AAG00546.1.

The rpin fusion proteins described herein include a WAP - ning polypeptide and a serpin ptide or an amino acid sequence that is derived from a serpin polypeptide. s are a group of proteins with similar structures that were first identified as a set of proteins able to inhibit proteases. Serpin proteins suitable for use in the fusion ns provided herein include, by way of non-limiting example, 1 antitrypsin (AAT), antitrypsin-related protein (SERPINA2), alpha 1-antichymotrypsin (SERPINA3), tatin (SERPINA4), monocyte neutrophil elastase inhibitor (SERPINBl), PI-6 (SERPINB6), antithrombin (SERPINC1), plasminogen activator inhibitor 1 (SERPINE1), alpha plasmin (SERPINF2), complement 1-inhibitor (SERPING1), and neuroserpin (SERPINI1).

In some embodiments, the serpin polypeptide sequence comprises an alpha- 1 antitrypsin (AAT) polypeptide ce or an amino acid sequence that is d from AAT. In some embodiments, the serpin polypeptide sequence comprises a portion of the AAT protein. In some embodiments, the serpin polypeptide sequence comprises at least the reactive site loop portion of the AAT protein. In some embodiments where the fusion protein of the invention includes a serpin polypeptide, the serpin polypeptide of the fusion protein includes the reactive site loop portion of the AAT protein or includes at least the amino acid sequence GTEAAGAMFLEAIPMSIPPEVKFNK (SEQ ID NO: 12). In a preferred embodiment, the AAT polypeptide sequence is or an amino acid sequence that is derived from AAT is derived from a human AAT polypeptide sequence. In some embodiments where the fusion protein of the invention includes a serpin polypeptide, the serpin polypeptide of the fusion n includes a modified variant of the reactive site loop portion of the AAT protein or includes at least the amino acid sequence GTEAAGAEFLEAIPLSIPPEVKFNK (SEQ ID NO: 38).

In some ments of the WAP-serpin fusion proteins, the serpin polypeptide includes a full-length human AAT polypeptide sequence having the ing amino acid sequence: 1 EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI FFSPVSIATA 61 FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL RTLNQPDSQL QLTTGNGLFL 121 SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT EEAKKQINDY VEKGTQGKIV DLVKELDRDT 181 VFALVNYIFF KGKWERPFEV KDTEEEDFHV DQVTTVKVPM KRLG FNIQ HCKKLSSWVL 241 LMKYLGNATA IFFLPDEGKL QHLENELTHD ENED RRSASLHLPK LSITGTYDLK 301 SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA MFLEAI P SI 361 PPEVKFNKPF VFLMIEQNTK KVVN PTQK (SEQ ID NO: 13) In some embodiments of the WAP-serpin fusion proteins, the serpin polypeptide includes a human AAT polypeptide sequence that is at least 50%, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13.

In some embodiments of the rpin fusion proteins, the serpin polypeptide includes the AAT polypeptide sequence or the amino acid sequence derived from an AAT polypeptide or is derived from one or more of the human AAT ptide sequences shown in GenBank Accession Nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA5 1546.1, CAA25838.1, NP 235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001 121 179.1, NP 001 121 178. 1, NR OOI 121 177.1, NP 001 121 176.16, NP_001 121 175.1, NP_001 121 174.1, NP_001 121 172.

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

These embodiments are referred to collectively herein as "WAP-Fc-serpin 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 WAP-Fc-serpin, serpin-WAP-Fc, or any variation or combination thereof. The -serpin fusion proteins described herein include at least a WAP domain-containing polypeptide or an amino acid sequence that is derived from a WAP -containing polypeptide, a serpin polypeptide or an amino acid sequence that is d from a serpin polypeptide, and an Fc polypeptide or an amino acid sequence that is d from an Fc polypeptide.

In some embodiments, where the WAP-serpin ptide fusion protein includes an Fc polypeptide sequence, the Fc polypeptide sequence is derived from any one of the amino acid sequences having the SEQ ID NO. 7, 8, 9, 10, or 11. In some embodiments, where the WAP-serpin fusion protein includes an Fc polypeptide sequence, the Fc polypeptide sequence has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of the sequences having the SEQ ID NO. 7, 8, 9, 10, or 11.

In some embodiments, the WAP-serpin domain fusion protein can also include an albumin polypeptide or an amino acid sequence that is derived from an albumin polypeptide. These embodiments are ed to collectively herein as "WAP-albumin- serpin 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 WAP-albumin-serpin, serpin-albumin-WAP, or any variation combination f. The WAP-albumin-serpin fusion proteins described herein e at least a WAP domain-containing polypeptide or an amino acid sequence that is derived from a WAP -containing polypeptide, a serpin polypeptide or an amino acid sequence that is derived from a serpin, and an albumin polypeptide or an amino acid sequence that is derived from an albumin polypeptide.

In some embodiments, where the WAP-serpin domain fusion protein includes an albumin polypeptide sequence, the albumin polypeptide sequence is derived from any one of the amino acid sequences of SEQ ID NO. 14-15, described herein. In other embodiments, where the serpin-WAP domain fusion protein includes an n ptide ce, 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% sequence identity to any one of the amino acid sequences having SEQ ID NO. 14 or 15.

In some embodiments of the fusion protein provided , the second polypeptide (Polypeptide 2) of the WAP domain containing fusion protein is an albumin polypeptide or is d from an albumin polypeptide. These ments are referred to collectively herein as "WAP-albumin fusion proteins." The WAP-albumin fusion proteins bed herein include at least a WAP domain containing polypeptide or an amino acid sequence that is d from a WAP domain ning polypeptide and an albumin polypeptide or an amino acid sequence that is derived from an albumin polypeptide. In addition this invention relates to WAP domain containing polypeptide albumin binding polypeptide fusion proteins, wherein the albumin is operably linked to the WAP domain containing polypeptide via an intermediate binding molecule. Herein, the WAP domain containing polypeptide is non-covalently or covalently bound to human serum In some embodiments, the WAP domain-containing polypeptide of the WAP-albumin fusion protein is derived from any one of the amino acid sequences of SEQ ID NOs 1-6. In some embodiments, the WAP domain-containing polypeptide of the WAP- albumin fusion protein has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of the amino acid sequences having SEQ ID NO. 1, 2, 3, 4, 5 or 6 .

In some embodiments, the WAP domain-containing polypeptide sequence of the WAP-albumin fusion protein is or is derived from sequences shown in GenBank Accession Nos. CAA28 187.1, NP 003055 .1, EAW75869. 1, P03973 .2, AAH20708. 1, CAB64235.1, CAA28188.1, AAD19661.1, BAG35125.1., P19957.3, NP_002629.1, BAA02441.1, EAW75814.1, EAW75813.1, Q8IUB2.1, and/or NP_542 18 1.1., 095925.1, NP .1, 29.2, AAH53369.1, AAG00548.1, AAG00547.1, and/or AAG00546.1.

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

In some ments of the WAP-albumin fusion proteins, the albumin polypeptide sequence 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 some embodiments of the WAP-albumin fusion proteins, the albumin polypeptide sequence includes a human serum albumin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 15.

In some embodiment the WAP domain-containing ptide 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 n binding polypeptide is or an amino acid sequence that is derived from the antibody or antibody nt is derived from a chimeric, humanized, or fully human antibody sequence. The term antibody fragment includes single chain, Fab fragment, a F(ab')2 fragment, a scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody. In addition the albumin binding polypeptide can be an albumin binding e.

Another embodiment of the this invention is a WAP domain-containing polypeptide - albumin binding polypeptide fusion, wherein the albumin g polypeptide is the domain 3 of streptococcal n G or a sequence derived from domain 3 of streptococcal protein G. In other embodiments the WAP domain-containing polypeptide and the human serum n is directly attached.

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

For e, the fusion protein contains at least one mutation at a nine (Met) residue in the non-Fc portion of the fusion protein, for example in the SLPI portion of the fusion n. In these Met mutations, the Met residue can be substituted with any amino acid.

For example, the Met residue can be tuted with an amino acid with a hydrophobic side chain, such as, for example, leucine (Leu, L) or valine (Val, V). Without wishing to be bound by theory, the Met mutation(s) prevent oxidation and subsequent inactivation of the inhibitory activity of the fusion proteins of the invention. In some embodiments, the Met mutation is at on 98 of an SLPI polypeptide. For e, a Met mutation that occurs at a residue in the amino acid sequence of SEQ ID NO: 8 is Met98Leu (M98L).

In some embodiments, the fusion proteins are modified to increase or otherwise inhibit proteolytic ge, for example, by mutating proteolytic cleavage sites.

In some embodiments, the proteolytic cleavage site mutation occurs at a residue in the SLPI portion of the fusion n. For example, the proteolytic cleavage site mutation occurs at a residue in the amino acid sequence of SEQ ID NO: 2 selected from Serl5, Alal6, Glul7, and combinations thereof.

In some embodiments, the fusion ns are modified to alter or otherwise modulate an Fc effector function of the fusion protein, while simultaneously retaining binding and tory function as compared to an unaltered fusion protein. Fc effector functions e, by way of non-limiting examples, Fc or binding, prevention of proinflammatory mediator release upon binding to the Fc receptor, phagocytosis, modified antibody-dependent cell-mediated cytotoxicity (ADCC), modified complement-dependent cytotoxicity (CDC), ed glycosylation at Asn297 residue (EU index of Kabat numbering, Kabat et al 1991 Sequences of Proteins of Immunological Interest) of the Fc polypeptide. In some ments, 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 Fc polypeptide mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu , S256T, T256E) (Kabat numbering, Dall'Acqua et al 2006, J . Biol Chem Vol 281(33) 23514-23524), or Met428Leu and Asn434Ser , N434S) (Zalevsky et al 2010 Nature Biotech, Vol 28(2) 157-159).

(EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest).

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

The fusion proteins described herein are useful in a variety of therapeutic, diagnostic and prophylactic tions. For example, the fusion proteins are useful in treating a variety of diseases and disorders in a t. In some embodiments, the fusion proteins bed herein, are useful in treating, alleviating a symptom of, rating and/or delaying the progression of a disease or disorder in a subject suffering from or identified as being at risk for a disease or disorder selected from 1-antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, cancers of the lung, ischemia- reperfusion injury, including, e.g., ischemia/reperfusion injury following cardiac transplantation, myocardial infarction, arthritis, rheumatoid arthritis, septic tis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and/or type II diabetes, bacterial infections, fungal infections, viral infections, pneumonia, sepsis, graft versus host disease , wound healing, Systemic lupus erythematosis, and Multiple sclerosis.

The fusion proteins and variants thereof provided herein exhibit inhibitory activity, for e by inhibiting a serine protease. The fusion proteins provided herein completely or partially reduce or otherwise modulate serine protease expression or activity upon binding to, or otherwise cting with, a serine protease, e.g., a human serine se. The reduction or modulation of a biological on of a serine se is complete or partial upon interaction between the fusion proteins and the human serine protease n, polypeptide and/or peptide. The fusion ns are considered to completely inhibit serine protease expression or activity when the level of serine protease expression or activity in the presence of the fusion protein is decreased by at least 95%, e.g., by 96%, 97% , 98%>, 99% or 100% as compared to the level of serine protease expression or activity in the absence of interaction, e.g., binding, with a fusion n described herein.

The fusion proteins are considered to partially inhibit serine protease expression or activity when the level of serine protease expression or activity in the presence of the fusion protein is decreased by less than 95%, e.g., 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90% as compared to the level of serine se expression or activity in the absence of interaction, e.g., binding, with a fusion protein described herein.

Pharmaceutical compositions according to the invention can include a fusion protein of the invention, ing modified fusion proteins and other variants, along with a suitable carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits.

Brief Description of the Drawings Figure 1A is a schematic representation of some embodiments of WAP domain ning polypeptide-Fc fusion proteins according to the invention. The WAP domain containing polypeptide can be located at any position within the fusion n.

Variants of these fusion proteins incorporating more than one WAP domain containing ptide are also represented. Figure IB is a photograph of a SDS-PAGE gel showing Elafm-Fcl (lane 1, human IgGl Fc), and SLPI-Fc 1 (lane 2, human IgGl Fc). Figure 1C is a graph showing the tion of neutrophil elastase activity by Elafm-Fc and SLPI-Fc fusion proteins. Figure ID is a raph of a SDS-PAGE gel showing the fusion protein consisting Elafm-Fc-SLPI. Figure IE is a graph showing the tion of neutrophil elastase activity by Elafm-Fc-SLPI. Serum derived AAT is shown as a control for NE inhibition. Figure IF is a graph depicting the serum concentrations over time of recombinant SLPI ed to SLPI-Fc in rats (3 per test protein) dosed with lOmg/kg protein. The half life of c is substantially longer than that of recombinant SLPI.

Figure 2A is a schematic representation of some embodiments of the WAP domain containing polypeptide-cytokine targeting fusion ns of the invention. The WAP domain containing polypeptide can be fused to either the heavy chain, the light chain, or both of an antibody. WAP domain containing polypeptide -cytokine or fusion proteins are also depicted. Figure 2B is a photograph of a SDS-PAGE gel showing the D2E7 antibody (lane 1), the D2E7 antibody with-SLPI fused to heavy chain (lane 2), and the D2E7 antibody with SLPI fused to the light chain (lane 3). Figure 2C is a graph showing the inhibition of neutrophil elastase activity by a D2E7 antibody fused to SLPI on either the heavy chain or the light chain. Serum derived AAT is shown and positive control, whereas the D2E7 antibody alone is shown as a negative control for NE tion.

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

Figure 4 is a schematic representation of some embodiments of the WAP domain containing polypeptide-HSA fusion proteins.

Detailed ption of the Invention The whey acidic n (WAP) domain is motif of approximately 50 amino acids characterized by eight conserved cysteines at defined positions, which form four disulfide bonds (Ranganathan et al 1999 J. Mol. cs Modell. 17, 134-136). The most well characterized function of the WAP domain is serine protease inhibition. Several WAP domain containing proteins are involved in innate immune protection of multiple epithelia; surfaces (Abe et al 1991 J . Clin. Invest. 87(6): 2207-15; Maruyama et al 1994 J Clin Invest. 94(l):368-375; Si-Tahar et al 2000 Gastroenterology 118(6): 1061-71; King et al 2003 Reproductive Biology and Endocrinology 1:1 16). Some of these proteins have been shown to possess antibacterial activities. ary WAP domain containing proteins are secretory yte protease inhibitor (SLPI), Elafin, and Eppin.

The SLPI and Elafin genes are members of the trappin gene family. Proteins d by members of the trappin genes, are characterized by an N-terminal transglutaminase domain substrate and a C-terminal WAP domain (Schalkwijk et al 1999 Biochem. J . 340 77). SLPI and Elafin are inhibitors of neutrophil serine proteases, yet have slightly distinct target proteases. While SLPI and Elafin are both potent inhibitors of neutrophil se, SLPI has also ts Cathepsin G but not nase-3, and Elafin inhibits Proteinase-3, but not Cathepsin G (Eisenberg, et al. 1990 J. Biol. Chem. 265, 7976-7981, Rao et al 1993 Am. J. Respir. Cell Mol. Biol. 8, 612-616, Ying et al 2001 Am.

J. Respir. Cell Mol. Biol. 24, 83-89). In addition, Elafin inhibits endogenous vascular elastase (EVE), a serine protease produced by diseased vascular tissue (Rabinovitch, M. 1999 Am. J. Physiol. Lung Cell. Mol. Physiol. 277, L5-L12; Cowan, et al 1996 J. Clin.

Invest. 97,2452-2468).

During inflammation and injury proteases released from phils generally serve to amplify the inflammatory response, through ation the ECM, tion of chemotactic peptides, MMP activation, and the induction of proteolytic and pro inflammatory ne signaling cascades. SLPI and Elafin represent endogenous regulators of inflammatory signaling that serve to prevent excess inflammation and protect tissues from proteolytic destruction at sites of local inflammation.

In numerous in vitro and in vivo both SLPI and Elafin demonstrated to maintain board anti-inflammatory ties (Doumas et al. 2005. Infect Immun 73, 1271-1274; Williams et al 2006 Clin Sci (Lond) 110, 21-35, Scott et al 201 1 Biochem. Soc. Trans. 39(5) 440; Shaw and Wiedow, 201 1 Biochem. Soc. Trans. 39, 454). While many of the anti-inflammatory activities of these proteins are due to protease inhibition, both SLPI and Elafin possess anti-inflammatory ties that are ndent of direct protease inhibition. For instance both bind bacterial LPS and prevent its binding to CD 14 and downstream signaling by macrophages (Ding et al 1999 Infect. Immun. 67 4485-4489, McMichael et al 2005 Am. J. Respir. Cell Mol. Biol. 32 443-452). Studies using human monocytes exposed to LPS, have shown that SLPI inhibited Toll-like receptor and NF-KB activation and subsequent IL-8 and TNFa production signaling in tes d to LPS (Lentsch et al 1999 Am. J. Pathol. 154, 239-247; t et al 2005 J . Exp. Med. 202, 1659-1668). SLPI deficient mice are significantly more susceptible to LPS induced endotoxin shock and had higher rates of mortality (Nakamura et al 2003 J . Exp. Med. 197, 669-674). rly, mice that overexpressed Elafin displayed reduced pro-inflammatory cytokines, including TNFa, MIP-2 and MCP-1, ed to than wild-type mice following LPS challenge (Sallenave et al 2003 Infect. Immun. 71, 3766-3774).

In addition to their anti-protease and anti-inflammatory activities, SLPI and Elafin possess anti-infective functionalities against a board class of pathogens, including viruses, bacteria, and fungi. Both SLPI and Elafin have demonstrated antibacterial activity against Gram-positive and Gram-negative species. SLPI has been found to be effective against pathogenic species common in the upper airways such as Pseudomonas aeruginosa and lococcus aureus, in addition to lococcus epidermidis and Escherichia coli.

While Elafin also exhibits bactericidal activity against Ps. aeruginosa and S . aureus (Hiemstra, et al 1996 Infect. Immun. 64, 4520-4524; Wiedow et al 1998 Biochem. Biophys.

Res. Commun. 248, 904-909; Simpson et al 2001 Hum. Gene Ther. 12, 1395-1406; Meyer- Hoffert et al. 2003 Exp. Dermatol. 12, 418-425; Simpson et al 1999 FEBS Lett. 452, 309- 313). SLPI has been demonstrated to have fungicidal activity against the pathogenic fungi Aspergillus tus and Candida albicans (Tomee et al 1997 J. Infect. Dis. '6:1'40-747; padhyay et al 2004 Infect. Immun. 72: 1956-1963). SLPI also been shown to have anti-HIV activity (McNeely et al 1995 J. Clin. Investig. 96:456-464; McNeely et al \991Blood 90:1 141-1 149; Hocini et al 2002 Clin. Diagn. Lab. Immunol. 7:515-518; PiUay et al 2001 J. Infect. Dis. 183:653-656).

Based upon reported studies, it has been suggested that recombinant human SLPI may be useful in the treatment of allergic asthma, emphysema, cystic fibrosis, AAT deficiency, COPD, ARDS, arthritis, bacterial, fungal, and viral infections, spinal cord injuries, wound healing, and ischemia/reperfusion injury following cardiac transplantation (Lucey, E.C., Stone, P.J., Ciccolella, D.E., Breuer, R., ensen, T.G., Thompson, R.C., and , G.L. (1990). Recombinant human secretory leukocyte -protease inhibitor: in vitro properties, and amelioration of human neutrophil elastase-induced emphysema and secretory cell asia in the hamster. J Lab Clin Med 115, 224-232; Stolk, J., Rudolphus, A., and Kramps, J.A. (1991). Lipopolysaccharide-induced alveolar wall destruction in the hamster is ted by racheal treatment with r-secretory leukocyte protease inhibitor.

AnnN Y Acad Sci 624, 350-352; Stromatt, S.C. (1993). Secretory leukocyte protease inhibitor in cystic fibrosis. Agents Actions Suppl 42, 103-1 10; Watterberg, K.L., Carmichael, D.F., , J.S., Werner, S., Backstrom, C , and Murphy, S. (1994).

Secretory leukocyte se inhibitor and lung mation in developing bronchopulmonary dysplasia. J Pediatr 125, 264-269; McNeely, T.B., Dealy, M., Dripps, DJ., Orenstein, J.M., Eisenberg, S.P., and Wahl, S.M. . Secretory leukocyte protease inhibitor: a human saliva protein exhibiting anti-human immunodeficiency virus 1 activity in vitro. J Clin Invest 96, 456-464; Fath, M.A., Wu, X., Hileman, R.E., Linhardt, R.J., Kashem, M.A., Nelson, R.M., Wright, CD., and Abraham, W.M. (1998). Interaction of secretory leukocyte protease tor with heparin inhibits proteases ed in . J Biol Chem 273, 13569; Jin, F., Nathan, C.F., Radzioch, D., and Ding, A. (1998).

Lipopolysaccharide-related stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor. Infect Immun 66, 2447-2452; Song, X., Zeng, L., Jin, W., on, J., Mizel, D.E., Lei, K., Billinghurst, R.C., Poole, A.R., and Wahl, S.M. (1999). Secretory leukocyte protease inhibitor suppresses the inflammation and joint damage of bacterial cell wall-induced arthritis. J Exp Med 190, 535- 542; Wright, CD., Havill, A.M., Middleton, S.C., Kashem, M.A., Lee, P.A., , D.J., O'Pviordan, T.G., Bevilacqua, M.P., and Abraham, W.M. (1999). Secretory leukocyte protease inhibitor prevents allergen-induced pulmonary responses in animal models of asthma. J Pharmacol Exp Ther 289, 1007-1014; Ashcroft, G.S., Lei, K., Jin, W., Longenecker, G., Kulkarni, A.B., Greenwell-Wild, T., Hale-Donze, H., McGrady, G., Song, X.Y., and Wahl, S.M. (2000). Secretory leukocyte se inhibitor mediates nonredundant functions necessary for normal wound healing. Nat Med 6, 1147-1 153; Mulligan, M.S., Lentsch, A.B., Lang, M., Guo, R.F., Sarma, V., Wright, CD., Ulich, T.R., and Ward, P.A. (2000). Anti-inflammatory effects of mutant forms of secretory leukocyte protease inhibitor. Am J Pathol 156, 1033-1039; Forteza, R.M., Ahmed, A., Lee, T., and Abraham, W.M. (2001). Secretory leukocyte protease inhibitor, but not alpha-1 protease tor, blocks tryptase-induced bronchoconstriction. Pulm Pharmacol Ther 14, 107-1 10; Pillay, K., Coutsoudis, A., Agadzi-Naqvi, A.K., Kuhn, L., ia, H.M., and Janoff, E.N. (2001). Secretory yte protease inhibitor in vaginal fluids and perinatal human deficiency virus type 1 ission. J Infect Dis 183, 653-656; Feuerstein, G. (2006). mation and : therapeutic effects of adenoviral expression of secretory Leukocyte Protease Inhibitor. Front Biosci 11, 1750-1757; Weldon, S., McGarry, N., Taggart, C.C, and McElvaney, N.G. (2007). The role of secretory leucoprotease inhibitor in the resolution of inflammatory responses. Biochem Soc Trans 35, 273-276; Nishimura, J., Saiga, H., Sato, S., Okuyama, M., Kayama, FL, Kuwata, FL, oto, S., Nishida, T., Sawa, Y., Akira, S., Yoshikai, Y., Yamamoto, M., and Takeda, K. (2008). Potent antimycobacterial activity of mouse secretory leukocyte protease inhibitor. J Immunol 180, 4032-4039; Schneeberger, S., Hautz, T., Wahl, S.M., Brandacher, G., Sucher, R., Steinmassl, O., assl, P., Wright, CD., Obrist, P., Werner, E.R., Mark, W., air, J., Margreiter, R., and Amberger, A. (2008). The effect of secretory leukocyte protease inhibitor (SLPI) on ischemia/reperfusion injury in cardiac lantation. Am J Transplant 8, 773-782; Ghasemlou, N., Bouhy, D., Yang, J., Lopez-Vales, R., Haber, M., Thuraisingam, T., He, G., Radzioch, D., Ding, A., and David, S. . Beneficial effects of ory leukocyte protease inhibitor after spinal cord injury. Brain 133, 126-138; Marino, R., Thuraisingam, T., Camateros, P., ratham, C , Xu, Y.Z., Henri, J., Yang, J., He, G., Ding, A., and Radzioch, D. (201 1). Secretory leukocyte protease inhibitor plays an important role in the regulation of allergic asthma in mice. J Immunol 186, 4433-4442).

Recombinant versions of SLPI and Elafin have be generated the administered to man. In fact recombinant Elafin is currently being evaluated in a human clinical trial to treat the matory component of various types of vascular injuries (ref). The recombinant version of both SLPI and Elafin display very short serum halves (<3hours, Bergenfeldt et al 1990 ScandJClin Lab Invest. 50(7):729-37, WO/201 1/107505). The short half life represents a major limitation to the therapeutic use of these proteins. Thus effective treatment with these version of the protein would require frequent dosing (multiple doses per day).

The fusion ns of the present ion were generated to e therapeutic potential of SLPI and Elafin. To extend the half life of recombinant SLPI and Elafin, Fc and albumin fusion proteins were created. While it was known that fusion of Fc domains or albumin to some proteins, protein s or peptides could extend their halflives {see e.g., Jazayeri, J .A., and Carroll, G.J. . Fc-based cytokines : prospects for engineering or therapeutics. gs 22, 11-26; Huang, C. (2009). or-Fc fusion therapeutics, traps, and MIMETIBODY technology. Curr Opin Biotechnol 20, 692- 699; mann, R.E. (2009). Strategies to extend plasma half-lives of recombinant antibodies. BioDrugs 23, 93-109; Schmidt, S.R. (2009). Fusion-proteins as biopharmaceuticals—applications and challenges. Curr Opin Drug Discov Devel 12, 284- 295), it was n, prior to the studies described herein, whether a Fc domain or albumin fused to SLPI or Elafin, would e their capacity to inhibit neutrophil elastase or have the desired effect of increasing serum half life. The studies described herein demonstrate that the fusion proteins of the present invention are e of potent NE inhibition and display enhanced serum half lives. These fusions proteins of the present invention provide more effective therapeutics over the previous unmodified versions of SLPI or Elafin.

In some embodiments, the WAP domain fusion proteins include SLPI or Elafin polypeptide sequences fused to a cytokine targeting protein.

In some embodiments, the fusion proteins described herein include at least a WAP domain containing polypeptide or an amino acid ce that is derived from a WAP domain containing polypeptide and a cytokine targeting polypeptide or an amino acid sequence that is derived from a cytokine targeting polypeptide. For example, the invention provides WAP domain containing polypeptide or a sequence derived from a WAP domain containing polypeptide fused to a human cytokine receptor or derivative thereof. Another embodiment of the invention provides WAP domain containing polypeptide or a sequence derived from a WAP domain containing polypeptide fused to a ne targeting antibody, e.g., an anti-cytokine antibody, or a sequence derived from of a cytokine targeting antibody, e.g., an anti-cytokine antibody, or sequence d from a fragment of cytokine targeting antibody, e.g. , a fragment of an anti-cytokine antibody. For e, the invention provides a WAP domain containing polypeptide or a sequence d from a WAP domain containing polypeptide fused to a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds any of the following human cytokines: TNFa, IgE, IL-12, IL- 23, IL-6, IL-la, IL- I b, IL-17, IL-13, the p40 subunit of IL-12 and IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

For example, in some ments, the cytokine targeting polypeptide targets TNFa and includes any of the following TNFa-targeting ptide or sequences derived from the following TNFa-targeting polypeptides: Remicade®, Humira®, Simponi®, ®, or Enbrel®.

For e, 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 FcsRI.

For e, in some embodiments, the ne targeting polypeptide s the shared p40 subunit of IL-12 and IL-23 and includes the Stelara® polypeptide or sequences derived from the a® polypeptide.

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

The invention provides a WAP domain containing polypeptide or a sequence derived from a WAP domain containing polypeptide fused to a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds any of the following human cytokine receptors of TNFa, IgE, IL-12, IL-23, IL-6, IL-la, IL- I b, IL-17, IL-13, the p40 subunit of IL-12 and IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32, thereby preventing binding between receptor and cytokine.

In some embodiments, the fusion proteins described herein include at least a SLPI polypeptide or an amino acid sequence that is derived from SLPI and a cytokine targeting ptide or an amino acid sequence that is derived from a cytokine targeting ptide. For example, the invention provides SLPI fused a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds any of the following human cytokines: TNFa, IgE, IL-6, IL-la, IL- I b, IL-12, IL-17, IL-13, IL-23, the p40 subunit of IL-12 and IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

In some embodiments, the fusion proteins described herein include at least an Elafin polypeptide or an amino acid sequence that is derived from Elafin and a cytokine targeting polypeptide or an amino acid sequence that is d from a cytokine targeting polypeptide. For example, the invention provides Elafin fused a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds any of the following human cytokines: TNFa, IgE, IL-6, IL-la, IL- I b, IL-12, IL-17, IL-13, IL-23, the p40 subunit of IL-12 and IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

In some embodiments the cytokine targeting polypeptide binds a cytokine or and ts binding between receptor and cytokine. For example, the present invention includes a serpin fused to a cytokine receptor targeting antibody. For e, the invention provides SLPI fused a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds the receptor of any of the following human nes: TNFa, IgE, IL-6, IL-la, IL- I b, IL-12, IL-17, IL-13, IL-23, the p40 subunit of IL-12 and IL-23, IL- 4, IL-10, IL-2, IL-18, IL-27, or IL-32. For example, the invention provides Elafin fused a cytokine targeting polypeptide in which the cytokine targeting polypeptide binds the receptor of any of the following human cytokines: TNFa, IgE, IL-6, IL-la, IL- I b, IL-12, IL-17, IL-13, IL-23, the p40 subunit of IL-12 and IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

For example, in some embodiments, the cytokine targeting polypeptide targets the IL-6 receptor and es the Actemra® polypeptide or sequences derived from the Actemra® polypeptide. For example, Actemra® the cytokine targeting polypeptide targets the IL-6 receptor and es the tocilizumab polypeptide or sequences derived from the tocilizumab polypeptide.

The ing of inflammatory nes and immune-stimulating agents by protein therapeutics has demonstrated al success in numerous inflammatory 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 biologies, Remicade®, Humira®, i®, Cimiza®, and Enbrel®, and the IL- 12/23 p40 targeting antibody, Stelara®. This is likely to be a common problem of targeting inflammatory cytokines leading to immune suppression in patients. As mentioned above, SLPI and Elafin demonstrate both anti-infective and anti inflammatory activities. Thus, the WAP domain containing polypeptide-cytokine ing polypeptide fusion proteins of this invention can dampen nt cytokine activities while alleviating the risk of infections.

In some embodiments, the fusion ns described herein include at least the following components: a WAP domain containing ptide or an amino acid sequence that is derived from a WAP domain containing, a serpin polypeptide or an amino acid sequence that is derived from a serpin and an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide. For example, the invention provides a WAP domain-containing polypeptide, serpin polypeptide, and human IgGl-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc or IgM-Fc derivatives ly linked together in any functional combination. In some embodiments, the serpin polypeptide is human AAT or derived from AAT. The WAP-Fc-serpin fusion proteins of the invention are ed to have enhanced anti-protease, anti-infective, and anti-inflammatory ties over fusion proteins composed of only a WAP domain containing polypeptide or a serpin ptide.

In some embodiments the fusion proteins described herein include at least a WAP domain containing polypeptide or an amino acid sequence that is derived from a WAP domain containing and a human serum albumin (HSA) polypeptide or an amino acid sequence that is derived from a HSA polypeptide. Further embodiments of invention include WAP domain containing polypeptide-albumin g polypeptide fusion ns, wherein the albumin g polypeptide is responsible for the association of the WAP domain containing polypeptide 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 WAP domain containing polypeptide or an HSA polypeptide. For example, the ion provides a WAP domain containing polypeptide fused to human HSA, or HSA derivatives, or HSA binding peptide or polypeptides.

In some embodiments, the fusion proteins described herein include at least a SLPI polypeptide or an amino acid sequence that is derived from SLPI and a HSA polypeptide or an amino acid sequence that is derived from a HSA polypeptide. For example, the invention es SLPI fused to HSA or a fragment derived from HSA, or an albumin binding polypeptide. In some embodiments, the fusion proteins bed herein include at least a Elafin polypeptide or an amino acid sequence that is derived from Elafin and a HSA polypeptide or an amino acid sequence that is derived from an HSA polypeptide.

For example, the invention provides Elafin fused to HSA or a fragment derived from HSA, or an albumin binding polypeptide.

The fusion proteins and fusion protein derivatives described herein are expected to be useful in treating a y of indications, including, by way of non-limiting example, alpha- 1-antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), ischemia-reperfusion injury, including, e.g., ischemia/reperfusion injury following cardiac transplantation, arthritis, allergic asthma, acute respiratory distress me (ARDS), cystic fibrosis, type I and/or type II diabetes, deficiency, bacterial, fungal, and viral infections, spinal cord injury, wound healing, graft ion, graft versus host disease (GVHD), pulmonary arterial hypertension (PAH), chronic thromboembolic pulmonary hypertension and ischemia/reperfusion injury following cardiac transplantation.

] Unless otherwise d, scientific and cal terms used in connection with the present invention shall have the meanings that are ly understood by those of ordinary skill in the art. Further, unless otherwise required by t, ar terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and ques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation {e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed ing to manufacturer's specifications or as ly accomplished 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 bed in various 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, N.Y. (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 al syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. The term patient includes human and veterinary subjects.

It will be appreciated that administration of therapeutic entities in ance with the ion will be administered with le carriers, buffers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, nce, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly r 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of s molecular weights), sem i 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 ion, provided that the active ient in the formulation is not inactivated by the ation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. "Pharmaceutical excipient development: the need for preclinical guidance." Regul. l Pharmacol. 32(2):210-8 (2000), Wang W.

"Lyophilization and development of solid protein ceuticals." Int. J . Pharm. 203(1- 2): 1-60 (2000), Charman WN s, lipophilic drugs, and oral drug delivery-some emerging concepts." J Pharm Sci. 89(8):967-78 (2000), Powell et al. "Compendium of excipients for parenteral formulations" PDA J Pharm Sci Technol. 52:238-31 1 (1998) and the ons therein for additional information related to formulations, excipients and carriers well known to ceutical chemists.

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

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

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

The fusion ns bed 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 s within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). The terms "physiological " and "biological sample," used interchangeably, herein are intended to include tissues, cells and ical fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the terms "physiological " and "biological sample", therefore, is blood and a fraction or ent of blood including blood serum, blood plasma, or lymph.

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

] A fusion protein of the invention can be used to isolate a particular target using standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. Detection can be facilitated by coupling (i.e., physically linking) the fusion protein to a detectable substance. es of detectable substances include various enzymes, prosthetic , fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ctosidase, or acetylcholinesterase; examples of le prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, scein, fluorescein isothiocyanate, rhodamine, rotriazinylamine fluorescein, dansyl chloride or rythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

A therapeutically effective amount of a fusion protein of the invention relates generally to the amount needed to achieve a eutic objective. As noted above, this may be a binding interaction between the fusion n 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 a fusion protein or fragment thereof invention may be, by way of nonlimiting example, from about 0 .1 mg/kg body weight to about 250 mg/kg body weight.

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

] Where fusion protein fragments are used, the smallest inhibitory fragment that specifically binds to the target is preferred. For example, peptide molecules can be designed that retain the ability to bind the target. Such peptides can be synthesized chemically and/or produced by recombinant DNA logy. (See, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with mentary 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, -inhibitory agent, an anti-inflammatory agent or anti-infective agent. Such molecules are suitably t in combination in amounts that are effective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared, 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 psules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes. ned-release ations can be prepared. Suitable examples of ned-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, -hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and g ethyl-L- glutamate, gradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid mers such as the LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)hydroxybutyric acid.

While rs such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release ns for shorter time periods.

Pharmaceutical compositions The fusion proteins of the ion (also referred to herein as "active compounds"), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical itions suitable for administration. Such compositions typically se the fusion protein and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin.

Liposomes and non-aqueous es 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 tional media or agent is incompatible with the active compound, use f in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

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

Pharmaceutical compositions suitable for injectable use include sterile aqueous ons (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) 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, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of tants. Prevention of the action of microorganisms can be ed by various antibacterial and antifungal agents, for example, parabens, butanol, phenol, ascorbic acid, osal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, ol, sodium chloride in the composition. Prolonged absorption of the able itions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate 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 ns a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, s of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterilefiltered solution thereof.

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

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

For administration by tion, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means.

For transmucosal or transdermal stration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such ants are generally known in the art, and include, for example, for transmucosal administration, ents, bile salts, and fusidic acid tives. ucosal stration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as lly known in the art.

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

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

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and ctic acid. Methods for preparation of such formulations will be nt to those skilled in the art. The materials can also be obtained cially 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 e, as described in U.S. Patent No. 4,522,81 1.

It is especially 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 carrier. The ication for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of duals.

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

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

EXAMPLES e 1: SLPI-Fc and Elafin-Fc Fusion Proteins and Variants Exemplary, but non-limiting examples of SLPI-Fc and Elafin Fc fusion proteins according to the invention include the following sequences, where the SLPI or Elafin polypeptide portion of the fusion protein is shown in bold, the WAP domain is underlined, the IgG-Fc polypeptide portion of the fusion protein is italicized, the Met98Leu (ML) mutation in SLPI is boxed, the Fc mutations Met252Tyr, Ser254Thr, Thr256Glu (YTE) or Met428Leu, Asn434Ser (LS) which enhance FcRn binding are boxed, in bold text, and shaded in grey. While these examples include a hinge sequence and/or a linker sequence, fusion proteins of the invention can be made using any hinge sequence and/or a linker sequence suitable in length and/or flexibility. Alternatively fusion proteins can be made without using a hinge and/or a linker sequence. For example, the polypeptide components can be directly attached.

] An exemplary SLPI~FC fusion protein is the Fc. As shown below, the SLPI polypeptide portion of the fusion protein is shown in bold (SEQ ID NO: 2), the WAP domain is underlined, the hinge region is shown in normal text (SEQ ID NO: 48), and the IgG—Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 7).

SLPI—hFcl (human IgG1 Fc, long Hinge) (SEQ ID NO:16) SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKC CLMLNPPNFCEMDGQCKRDLKCCMGMCGKSCVSPVKAEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK (SEQ ID NO:16) An exemplary SLI’I—Ec fusion protein is the SLPI—hFcZ (human IgG2 Fe, long Hinge). As shown below, the SLPI ptide portion ofthe fusion protein is shown in bold 2O (SEQ ID NO: 2), the WAP domain is underlined, the hinge region is shown in normal text (SEQ ID NO: 49), and the 1gG—Fc ptide portion ofthe fusion protein is italicized (SEQ ID NO: SLPI—hFcZ (human IgG2 FC, long Hinge) (SEQ ID NO:17) SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKC PVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGMCGKSCVSPVKAERKCCVECPPCPAPPVAGPSV PKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO:17) 1001117592 ] An exemplary SLPI-Fc fusion protein is the SLPI-ML—hFcl. As shown below, the SLPI polypeptide portion of the fusion protein is shown in bold (SEQ ID NO: 39), the WAP domain is underlined, the hinge region is shown in normal text (SEQ ID NO: 48), the IgG-Fe polypeptide portion of the fusion protein is ized (SEQ ID NO: 7), and Met98Leu (ML) mutation in SLPI is boxed.

SLPI—ML—hFcl (human IgG1 Fc, Met98Leu) (SEQ ID NO:18) SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKC PVTYGQCLMLNPPNFCEMDGQCKRDLKCCMEICGKSCVSPVKAEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK (SEQ ID NO:18) An exemplary C fusion protein is the SLPI—hFcl—YTE. As shown below, the SLI’I ptide portion of the fusion protein is shown in bold (SEQ ID NO: 39), the WAP domain is underlined, the hinge region is shown in normal text (SEQ ID NO: 48), the lgG—Fe polypeptide portion of the fusion protein is italicized (SEQ ID NO: 40), and the Fe ons Met252Tyr, Ser254Thr, Thr256Glu (YTE) are boxed, in bold text, and shaded in grey.

SLPI—hFcl—YTE (human IgG1 Fc, Met252Tyr, Ser254Thr, Thr256Glu) (SEQ ID NO:19) 2O SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKC PVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGLCGKSCVSPVKAEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDT139]lflmfiPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK (SEQ ID NO:19) An exemplary SLPI-Fc fusion protein is the SLPI-hFcl -LS. As shown below, the SLPI polypeptide portion of the fusion n is shown in bold (SEQ ID NO: 39), the WAP domain is underlined, the hinge region is shown in normal text (SEQ ID NO: 48), the IgG—Fc lOOlll7592 polypeptide portion of the fusion protein is italicized (SEQ ID NO: 41), and Met428Leu, Asn434Ser (LS) which enhance FcRn binding are boxed, in bold text, and shaded in grey.

SLPI—hFcl—LS (human IgG1 Fc, Met428Leu, Asn434Ser) SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKC CLMLNPPNFCEMDGQCKRDLKCCMGLCGKSCVSPVKAEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSWE HEALHEHYTQKSLSLSPGK (SEQ ID NO:20) ] An exemplary Elafm—Fc fusion protein is the Ela‘l‘in—hFcl. As shown below, the Elafin polypeptide portion of the fusion protein is shown in bold (SEQ ID NO: 5), the WAP domain is underlined, the hinge region is shown in normal text (SEQ ID NO: 48), the lgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 41), and the Asn434Ser (LS) which enhance FcRn binding are boxed, in bold text, and shaded in grey.

Elafin—hFcl (human IgG1) VKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQEPVKGPVSTKPGSCPIILIRCAML NPPNRCLKDTDCPGIKKCCEGSCGMACFVPQEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSWEHEELHEHYTQKS LSLSPGK (SEQ ID NO:21) An exemplary 0 fusion protein is the SLPI—hFcl -SLPI. As shown below, the SLPI polypeptide portion of the fusion protein is shown in bold (SEQ ID NO: 39), the WAP domain is underlined, the hinge region is shown in normal text (SEQ ID NO: 48), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 7), and the ASTGS linker is shown in normal text (SEQ ID NO: 50).

SLPI —hFcl — SLPI (human IgG1) lOOlll7592 SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKC PVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGLCGKSCVSPVKAEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKASTGSSGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCP DTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGLCGKSCVSP VKA (SEQ ID NOz22)

[00138] An exemplary Elafin—Fc fusion protein is the Elaiin~hFc1—Elafin. As shown below, the Elafm polypeptide portion ofthe fusion protein is shown in bold (SEQ ID NO: 5), the WAP domain is underlined, the hinge region is shown in normal text (SEQ ID NO: 48), the IgG— Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 7), and the ASTGS linker is shown in normal text (SEQ ID NO: 50). —hFcl—Elafin (human IgG1) AVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQEPVKGPVSTKPGSCPIILIRCAML NPPNRCLKDTDCPGIKKCCEGSCGMACFVPQEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKASTGSAVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQEPVKGPVSTKPG IRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ (SEQ ID NOz23) An exemplary EIafm—Fc fusion protein is the Elafin—hFc1 —SLPI. As shown below, the SLPI polypeptide (SEQ ID NO: 39) n of the fusion protein is shown in bold, with the WAP domain underlined, the Elafin (SEQ ID NO: 5) polypeptide portion of the fusion protein is shown in bold and italics, the WAP domain is underlined, the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 7), the hinge region is shown in normal text (SEQ ID NO: 48) and the ASTGS linker is shown in normal text (SEQ ID NO: 50).

Elafin—hFcl—SLPI (human IgG1) 1001117592 AVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQEPVKGPVSTKPGSCPIILIRCAML NPPNRCLKDTDCPGIKKCCEGSCGMACFVPQEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKASTGSSGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVD RKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGLCGKSCVSPVKA (SEQ ID NO:24) An exemplary SLPI~Fc fusion protein is the SLPI—hFcl—Elafin. As shown below, the SLPI (SEQ ID NO: 39) portion of the fusion protein is shown in hold, with the WAP domain underlined, the Elatin (SEQ ID NO: 5) polypeptide portion of the fusion protein is shown in bold and italics, the WAP domain is ined, and the lgG—Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 7), the hinge region is shown in normal text (SEQ ID NO: 48) and the ASTGS linker is shown in normal text (SEQ ID NO: 50).

SLPI—hFcl—Elafin (human IgG1) SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKC PVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGLCGKSCVSPVKAEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFTLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKASTGSAVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQ EPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ (SEQ ID NO:25) These exemplary SLPl—Fc and Fc fusion proteins were made using the following techniques.

] The genes encoding human SLPI and Elafin were PCR amplified from human spleen cDNA (Zyagen). Specific point ons within the gene encoding SLPI, Elafin or the Fc region were generated by overlapping PCR. The SLPI or Elafin encoding gene was cloned in frame with a gene encoding the hinge region, followed by a CH2 domain, and a CH3 domain of human IgG1, IgGZ, 1gG3, IgG4, or IgM into a mammalian expression vector, containing a 1001117592 ian secretion signal sequence up stream of the SLPI or Elafin gene insertion site. In some cases, these vectors were further modified, wherein the gene encoding a linker sequence and either SLPI or Elafin was cloned in frame to the 3’ end of the CH3 domain, to generate SLPI-Fc-SLPI, Elafin—Fc-Elafin, SLPI—Fc-Elafin, or Fc-SLPI. 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 SLPI-Fc and Elafin-Fc fusion proteins were purified from the expression cell supernatant by n A chromatography. Figure 1B shows a reducing SDS—PAGE gel of the Elafin—Fc (SEQ ID N022l, lane 1) and SLPl—Fc (SEQ ID NOzl6, lane 2) fusion 1001l17592 ns. Figure ID shows a reducing SDS-PAGE gel of the Elafm-Fc-SLPI (SEQ ID NO:24). The ns were visualized by staining with sie blue.

To monitor human Neutrophil se (NE) activity a fluorescent microplate assay was used. Inhibitory activity was measured by a concomitant decrease in the al 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-100. Human NE is used at a final concentration of 5 nM (but can also be used from 1-20 nM). The fluorescent peptide substrate AAVPAMC is used at a final concentration of 100 mM in the assay. The Gemini EM plate reader from Molecular s is used to read the assay kinetics using excitation and emission wavelengths of 370 nm and 440 nm respectively, and a cutoff of 420 nm. The assay is read for 10 min at room temperature scanning every 5 to 10 seconds. The Vmax per second corresponds to the residual NE activity, which is plotted for each concentration of inhibitor.

The intercept with the x-axis indicates the concentration of inhibitor needed to fully inactivate the starting concentration of NE in the assay. Human serum derived AAT (sdAAT) was used as a positive control for NE inhibition in these assays. The Elafm-Fc and c fusion proteins display potent inhibition of NE (Figure 1C). The Elafm-Fc- SLPI fusion protein also was a potent inhibitor of NE (Figure IE). rmore, the c fusion protein displayed a longer serum half life in rats compared to E.coli produced unmodified SLPI (Figure IF), demonstrating that the fusion proteins of the invention have improved pharmacokinetic properties and are a superior eutic format over unmodified versions of SLPI and Elafin, for treating numerous human inflammatory conditions Example 2 : SLPI-TNFa Targeting Molecule Fusion ns The studies presented herein describe several, non-limiting examples of recombinant SLPI derivatives sing human SLPI fused to an anti-TNFa antibody or a derivative of a TNFa receptor. These examples are provided below to further illustrate ent features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not and are not intended to limit the claimed invention.

The fusion proteins below include cytokine targeting polypeptide ces that are from or are derived from (i) the anti-TNFa antibody D2E7 (also known as Adalimumab or Humira®), or (ii) the extracellular domain of Type 2 TNFa Receptor (TNFR2-ECD). The SLPI polypeptide portion of the fusion protein is in bold text, the WAP domain is underlined, the antibody nt s (CHl-hinge-CH2-CH3, or CL) are italicized, and D2E7-VH, D2E7-VK, and TNFR2-ECD are shaded in grey and in bold text.

While these examples include a hinge sequence and/or a linker sequence, fusion proteins of the invention can be made using any hinge sequence and/or a linker sequence le in length and/or ility. Alternatively fusion proteins can be made without using a hinge and/or a linker sequence. For example, the polypeptide components can be directly attached.

An ary NFa Targeting Molecule fusion protein is D2E7-Light Chain-SLPI (G3S)2 Linker. As shown below the SLPI polypeptide portion of the fusion protein is in bold text (SEQ ID NO: 2), the WAP domain is underlined, the antibody constant regions (CHl-hinge-CH2-CH3, or CL) are italicized (SEQ ID NO: 43), and D2E7- VK is shaded in grey and in bold text (SEQ ID NO: 42).

D2E7-Light Chain-SLPI (G3S )2 Linker SPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKA.PKLLIYAASTLQSGVPSR FSGSGSGTDFTLTIS L EDVA YCQRYNRAPY G GTKVE R VAAPS VFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYA CEVTHQGL SSPVTKS FNRGECGGGSGGGSSGKSFKAGVCPPKKSAQCLRYK KPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNP TRRKPGKCPVTYGQCLMLNPPNFCEMD GQCKRDLKCCMGMCGKSCVSPVKA (SEQ ID NO: 26) An exemplary SLPI-TNFa Targeting Molecule fusion protein is D2E7-Light Chain-SLPI ASTGS Linker. As shown below the SLPI polypeptide portion of the fusion protein is in bold text (SEQ ID NO: 2), the WAP domain is underlined, the antibody constant regions (CHl-hinge-CH2-CH3, or CL) are italicized (SEQ ID NO: 43), and D2E7- VK is shaded in grey and in bold text (SEQ ID NO: 42).

D2E7 -Light Chain-SLPI ASTGS Linker SPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSR FSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVE .KRTVAAPS VFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLS SPVTKS FAKGECASTGSSGKSFKAGVCPPKKSAQCLRYKKPE CQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQC KRDLKCCMGMCGKSCVSPVKA (SEQ ID NO: 27) An exemplary SLPI-TNFa Targeting Molecule fusion protein is D2E7- Heavy Chain-SLPI (G S)2 Linker. As shown below the SLPI polypeptide portion of the fusion protein is in bold text (SEQ ID NO: 2), the WAP domain is underlined, the antibody nt s (CHl-hinge-CH2-CH3, or CL) are italicized (SEQ ID NO: 45), and D2E7- VH is shaded in grey and in bold text (SEQ ID NO: 44).

D2E7-Heavy SLPI (G3S )2 Linker EVQLVESGGGLVQPGRSLRLSCAASGF FDDYAMHWVRQAPGKGLEWVSA SGHIDYA DSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVrVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF 5C5 i5 5 5PGiGGGSGGGSSGKSFKAGVCPPKKSAQCLRYKKPECQSD WQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDL KCCMGMCGKSCVSPVKA (SEQ ID NO: 28) An ary SLPI-TNFa Targeting Molecule fusion protein is D2E7- Heavy SLPI ASTGS Linker. As shown below the SLPI polypeptide portion of the fusion protein is in bold text (SEQ ID NO: 2), the WAP domain is underlined, the antibody constant regions (CHl-hinge-CH2-CH3, or CL) are italicized (SEQ ID NO: 45), and D2E7- VH is shaded in grey and in bold text (SEQ ID NO: 44).

-Heavy Chain-SLPI ASTGS Linker STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGKASTGSSGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRC CPDTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGMCGKSCV SPVKA (SEQ ID NO: 29)

[00151] An exemplary SLPI~TNFoc Targeting Molecule fusion protein is TNFRZ—ECD- PI(G38)2 Linker. As shown below, the SLPI polypeptide portion of the fusion protein is in bold text (SEQ ID NO: 2), the WAP domain is underlined, the Fc polypeptide portion is italicized (SEQ ID NO: 47), the TNFR2—ECD is shaded in grey and in bold text (SEQ ID NO: 46), the hinge region is shown in normal text (SEQ ID NO: 48), and the (G3S)2 linker is shown in normal text (SEQ ID NO: 51).

TNFRZ—ECD—FCl-SLPI(G3S)2 Linker LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQL CLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA RPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAV I5 HLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTY RVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKGGGSGGGSSGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKR CCPDTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGMCGKSC VSPVKA (SEQ ID NO: 30) An ary SLPI-TNFOL Targeting Molecule fusion protein is TNFR2—ECD— Fcl —SLPl ASTGS Linker, As shown below, the SLPl polypeptide portion of the fusion n is in bold text (SEQ ID NO: 2), the WAP domain is underlined, the Fc polypeptide portion is italicized (SEQ ID NO: 47), the TNFRZ-ECD is shaded in grey and in bold text (SEQ ID NO: 46), the hinge region is shown in normal text (SEQ ID NO: 48), and the ASTGS linker is shown in normal text (SEQ ID NO: 50).

TNFRZ—ECD—FCl—SLPI ASTGS Linker LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQL WNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVA SDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAV HLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTHTCPPCPAPELLG 1001117592 GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTY RVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HYTQKSLSLSPGKASTGSSGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCP DTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGMCGKSCVSP VKA (SEQ ID NO: 31) l00153t These cxcmplazy SLPi—TNFCL targeting niolocnlc fusion proteins wen: {nadir using the toilowing techniques. 1001542 The genes mending the variabic heavy tVH) and variable kappa (Vii) ms of tho ttttli-TNFCL antihndy, DZEI were generated by gene 8}“1'1ll1'83i5. The DEE? ‘v‘ H gene was cloned in Frame with .21 gone encoding t1 human lgCi] antibody heavy chain wnstant rcgioni ting of a CHI domain, a hinge ttormiin, a CH2 domain, and at CH} domain. into a mammalian expression vector. containing 2-1 mammalian accretion signal sequence up. Stir/inn ofthc VH domain insertion site (DEBT—11C), The DZET—VK gent: was cloned in tram-c with :1 human antibody kappa light chain nt (CL) . into a mammalian expression vector, containing a lltélt‘t‘lmttiittli secretion signal scqucnco up stream of‘thc \K domain insertion site ("DEEY—LC}. The SLPE encoding gone and tho udjucmt 5‘ Einlccr sequence were cioncd in tit-tint: into the 3" end ofcithcr, the Ci l3 domain oftho DEE? heavy chain gcnc l’1C-SLPIL or the CL dut‘nain of tilt: DEE? light Chitin gene (DEE?- LCLSLPE) coding sequences in the. whom: doacribcd r‘i‘iarnrnalian cxprcnaion vectors. The extracellular domain of the ‘l'NFoL Receptor 2 (fi'NFRZ—ECD) was ted by gene synthesin and Clonttct in frame with it gone encoding the hinge region. Followed by 21 CH2 domain and ti CHE domain ofhnmzm lgCil [hit 1 _} into at mammalian expression containing a rnnrtimalizm secretion signal sequence up stream oi’thc TNFRE—ECD innertion sift: The SLPI encoding gene and the nt 5‘ linker sequence won: chnod in frame into the 3" and of the gene encoding, TNF ZZ—ECD-nljcl into a ian expression vector (‘TNF‘RE— BCDthC t wSLPI).

E The [32 E7-HC’-SLPi expression vector was co—transf‘c-ctcd with either the DBE7—LC‘ or the DZE’F—LC-S LPI nion vector into mammalian cells {specifically H [31(293 or {THO coils) to generate the D2E7 antibody with SLPI fused to the C-tcrminns of the heavy chain or to the C—tcrminus of both the heavy chain and light Chain, rcspcctivciy.

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

The recombinant SLPI-TNFa targeting fusion proteins were purified from the expression cell supernatant by protein A chromatography. Figure 2B shows a reducing SDS-PAGE gel of the D2E7 antibody alone (lane 1), the D2E7 antibody with SLPI fused to the light chain (SEQ ID NO: 27 co-transfected with D2E7 heavy chain, lane 2), the D2E7 antibody with SLPI fused to the heavy chain (SEQ ID NO: 29 co-transfected with D2E7 light chain, lane 3). Arrows denote modified (SLPI fused) and unmodified (no SLPI) heavy and light chains. The proteins were ized by staining with sie blue.

The purified SLPI-TNFa targeting molecule fusion proteins were tested for activity by determining their ability to t neutrophil elastase. Human serum derived AAT (sdAAT) was used as a positive control in these assays. (Figure 2C). ve to serum derived AAT, the D2E7-antibody-SLPI fusion proteins show similar inhibition of neutrophil se, indicating that the inhibitory capacity of SLPI has not been compromised by its fusion to an dy. The NE inhibition assays were conducted as described above.

Example 3 -SLPI and AAT-Fc-Elafin The studies presented herein describe several, non-limiting es of recombinant AAT derivatives comprising human AAT fused a WAP domain containing protein. These examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention. The AAT polypeptide portion of the fusion protein is shown in bold text and shaded in grey, the Fc portion is italicized, the SLPI and Elafin portion are in bold text, and the WAP domain containing polypeptide is underlined. 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 ility.

Alternatively fusion proteins can be made without using a hinge and/or a linker sequence.

For example, the polypeptide components can be directly attached.

An exemplary AAT-Fc-SLPI fusion protein is AAT—hFcl-SLPI (human IgG1 Fc).

As shown below, the AAT polypeptide portion of the fusion n is shown in bold text and shaded in grey (SEQ ID NO: 13), the Fc portion is italicized (SEQ ID NO: 7), the SLPI portion is in bold text (SEQ ID NO: 2), and the WAP domain containing polypeptide is underlined (SEQ ID NO: 3), the hinge region is shown in normal text (SEQ ID NO: 48), and the ASTGS linker is shown in normal text (SEQ ID NO: 50).

AAT—hFcl—SLPI (human IgG1 Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAML SLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVD IO KFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKG KWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPD EGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSG LKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLF MGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGSSGKSFKAGV CPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLM LNPPNFCEMDGQCKRDLKCCMGMCGKSCVSPVKA (SEQ ID NO: 32)

[00160] An exemplary AAT-Fc—Elafin fusion protein is AAT—hFcl—Elafin. As shown below, the AAT ptide portion of the fusion protein is shown in bold text and shaded in grey (SEQ ID NO: 13), the Fc portion is italicized (SEQ ID NO: 7), the Elalin portion is in bold text (SEQ ID NO: 5), the WAP domain containing polypeptide is underlined (SEQ ID NO: 6), the hinge region is shown in normal text (SEQ ID NO: 48), and the ASTGS linker is shown in normal text (SEQ ID NO: 50). cl—Elafin (human IgG1 Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAML SLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVD KFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKG KWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPD EGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSG 1001117592 VTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLF MGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGSAVTGVPVKG QDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQEPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKD TDCPGIKKCCEGSCGMACFVPQ(SEQ ID NO: 33) [00161: The genes encoding the SLPI and Elafin were PC‘R amplificd from human splccn CDNA (Zyagcn). These gene; and flanking linker sequcnmr; were ClOfiCd in fiami: into nmmmalian cxpmssiun vectors cuntaining the 361165 encoding AAT and an lg Fc region r'cin a iiun secretion precedes iln": AAT gem}. c exprcsnim‘i vociom wcz‘c li‘unsl‘cclcd inlu mammalian cells (spucificully HEKZQ3 or CHE? CCHS) and grim-’11 for acvcml (lays in 3% CO; in; 3”,?“ C‘. Tin: i‘cmmbiimnl A.~"\T»F~‘c—W.-‘il’ dmnuin fusion prancing were purified From the uxpmssion Cell supernatant: by protein A umgrupl‘iyn A ncur l pH bullbr was used c AgiAb Elulion Bull’cr, Thermo Scientific) [0 clutc lllC .'~\/3‘7l'~i~'i:~\\<'i¢‘il’ domain linden n from the promin A resin. Figure 38 shows a reducing SIDS-PAGE gel Ulitln”: purified Fusion proteins: A‘z’xT—Fc‘Elulu‘i (SEQ ID NO: 33.1:ii'ic l') und.AfXT¥FC—Sll’l[3lflflll}lV():32wlanc 31 I00162E The purified skAT-FC—‘i’v'r’kl’ domain Fusion proteins wens. [0:5th for unlivity by (lolm‘i’nining their abilily to inhibii licuu'npliil clusluse. H lekm scrum dcrivcd AAT (l'sdA Al) was 11:st as 2; positive cuntml in these assays. e 3C), Relative to serum d AAT, 1hr: AA?FC~V~".»\P {arguing molecule fusion proteins display enhanced potency (JFK ES inhibition ofnculmnlni cluvstusc. Nli inhibition 235321355 were conducted as designbed abmzci Example 4 SLPl‘Albumin and EiafinwAibumin {0016-3} The studies; prcscnlcd herein describe several. nonslimiiing cxampics 0i“ recombinant AAT dcrivativcs comprising human SLPI fused un albumin pulypcpridc‘ Thesa- cxumplcs are provided below to further illustrate different features; 0? tin: t invention.

The examples also iliusu'atc useful mnihoclology for practicing the invention These examples do nut and are not ed to limil thc claimed invention. The AAT [)(mifln is; in bold text, the n portion is italicized, and the WAP domain is underlined. While these examples include a linker sequence, fusion proteins of the ion can be made using any linker sequence suitable in length and/or ility. Alternatively fusion proteins can be made without using a linker sequence.

An exemplary SLPI-Albumin fusion protein is SLPI-HSA. As shown below, the SLPI portion is in bold text (SEQ ID NO: 2), the albumin portion is italicized (SEQ ID NO: 14).

SLPI-HSA SGKSFFLAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKP GKCPVTYGQCLMLNPPNFCEMDGQCKPJ)L^ ENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFG DKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEE TFLKKYL YEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASS AKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDL TKVHTECCHGDLLECA DDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDV CKNYAEAKDVFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKCCAAADPHECYAKVFDE FKPL VEEPQNL IKQNCEL FEQLGEYKFQNALLVR YTKKVPQVST PTE VE VSRNL GKVGSKC CKHPEAKRMPCAEDYLSWLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYV PKEFNAE DIC LSEKERQIKKQ TAL VEL VKHKPKATKEQLKAVMDDFAAFVEKCC KADDKETCFAEEGKKLVAASQAAL GL (SEQ I D NO : 3 4 ) An exemplary SLPI-Albumin fusion protein is SLPI-HSA Domain 3. As shown below, the SLPI portion is in bold text (SEQ ID NO: 2), and the albumin portion is italicized (SEQ ID NO: 15).

SLPI-HSA Domain 3 SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKP GKCPVTYGQCIL^NPPNFCE^GQCKI^LKCCMGMCGKSCVSPVKAASTGS LGEYKFQNALL VRYTKKVPQVSTPTL VEVSRNL GKVGSKCCKHPEAKRMPCAEDY LSWLNQLCVLHEKTPVSDR VTKCCTESL VNRRPCFSALEVDETYVPKEFNAE TFTFHADI CTLSEKER QIKKQ TAL VEL VKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKK (SEQ I D NO : 3 5 ) An exemplary SLPI-Albumin fusion protein is Elafin-HSA. As shown below, the Elafm portion is in bold text (SEQ ID NO: 5), the albumin portion is italicized (SEQ ID NO: 14), and the WAP domain is underlined.

SLPI-HSA AVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQEPVKGPVSTKPGSCPIILIRC NRCLKDTDCPGIKKCCEGSCGMACFV^^ VL IAFAQ YL QQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTL FGDKLC A TLRE T YGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYL YEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQK FGERAFKAWAVARLSQRFPKAEFAEVSKL VTDL TKVHTECCHGDLLECADDRADLAKYICE NQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFL GMFL YE YARRHPDYSWLLLRLAKTYE TTLEKCCAAADPHECYAKVFDEFKPL VEEPQNL I KQNCELFEQLGE YKFQNALLVR YTKKVPQ VS TP TL VE VSRNL GKVGSKCCKHPEAKRMPCA EDYLSWLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFH ADICTL SEKERQIKKQ A VEL VKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEE ASQAALGL (SEQ ID NO: 36) An exemplary Elafin-Albumin fusion protein is Elafin-HSA domain 3. As shown below, the Elafm portion is in bold text (SEQ ID NO: 5), the albumin portion is ized (SEQ ID NO: 15), and the WAP domain is underlined (SEQ ID NO: 6).

Elafin-HSA Domain 3 AVTGVPVKGQDTWGRVPFNGQDPVKGQVSVKGQDKVKAQEPVKGP VSTKPGSCPIILIRC AMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQASTGS EEPQNL I FEQLGE YKF QNALLVR YTKKVPQVSTPTL VEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSWLNQLCVLH EKTPVSDR VTKCC TESL VNRRPCFSAL EVDE FNAE TFTFHAD ICTLSEKER QIKK QTAL VEL VKHKPKATKEQLKA AFVEKCCKADDKETCFAEEGKKLVA (SEQ ID NO: 37) The gene encoding human serum albumin (HSA) was PCR amplified from human liver cDNA (Zyagen). A mammalian expression vector was generated, wherein gene encoding HSA or the domain 3 of HSA, was cloned in frame to the 3' end of the SLPI or Elafin encoding gene, containing a mammalian ion signal sequence up stream of SLPI or Elafm.

These expression vectors were transfected into mammalian cells (specifically HEK293 or CHO cells) and grown for several days in 8% C0 2 at 37° C. The recombinant SLPI-HSA and Elafm-HSA fusion proteins were purified from the expression cell supernatant using the phenyl-sepharose.

Other Embodiments While the invention has been described in conjunction with the detailed description thereof, the ing description is intended 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 of the ing claims.

Claims (30)

What is claimed
1. I. An isolated fusion protein comprising at least one human whey acidic protein (WAP) domain-containing polypeptide comprising a human secretory leukocyte proteinase inhibitor (SLPI) polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a human elafin polypeptide comprising the amino acid sequence of SEQ ID NO: 6 operably linked to an immunoglobulin Fc ptide comprising an amino acid sequence that is at least 98% identical to the amino acid ce of SEQ ID NO: 10.
2. 2. An isolated fusion protein comprising at least one human secretory leukocyte proteinase inhibitor (SLPI) polypeptide comprising the amino acid sequence of SEQ ID NO: I, SEQ ID NO: 2 or SEQ ID NO: 39 operably linked to an immunoglobulin Fc polypeptide comprising an amino acid sequence that is at least 98% cal to the amino acid sequence of SEQ ID NO: 10.
3. 3. An ed fusion protein comprising human at least one human Elafin polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 operably linked to an immunoglobulin Fc polypeptide comprising an amino acid sequence that is at least 98% cal to the amino acid sequence of SEQ ID NO: 10.
4. 4. The isolated fusion protein of claim 1, wherein the WAP domain containing ptide and the immunoglobulin Fc polypeptide are operably linked Via a hinge , a linker region, or both a hinge region and linker region.
5. 5. The isolated fusion protein of claim 2, wherein the SLPI polypeptide and the immunoglobulin Fe polypeptide are operably linked via a hinge region, a linker , or both a hinge region and linker region.
6. 6. The isolated fusion protein of claim 3, wherein the Elafm polypeptide and the immunoglobulin Fc polypeptide are operably linked Via a hinge region, a linker region, or both a hinge region and linker region.
7. 7. The ed fusion protein of any one of claims 4-6, wherein the hinge region, the linker region or both the hinge region and the linker region comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51. 1001117809
8. 8. The isolated fusion protein of claim 5, n the peptide sequence ses the amino acid sequence of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51.
9. 9. The isolated fusion protein of claim 6, n the peptide ce comprises the amino acid sequence of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, or SEQ ID NO: 51.
10. 10. The isolated fusion protein of claim 1, wherein the immunoglobulin Fc polypeptide comprises at least one mutation at a position selected from the group ting of: Met252, Ser254, Thr256, Met428, and Asn434.
11. ll. The isolated fusion protein of claim 2, wherein the immunoglobulin Fe polypeptide is modified to enhance FcRn binding.
12. 12. The isolated fusion protein of claim 2, wherein the immunoglobulin Fe polypeptide comprises at least one mutation at a position selected from the group consisting of: Met252, Ser254, Thr256, Met428, and Asn434.
13. 13. The ed fusion protein of claim 2, wherein the immunoglobulin Fe polypeptide comprises at least one of the ing mutations: Tyr, Ser254Thr, Thr256Glu, Met428Leu or Asn434Ser.
14. 14. The isolated fusion protein of claim 3, wherein the immunoglobulin Fe polypeptide is modified to enhance FcRn binding.
15. 15. The isolated fusion protein of claim 3, wherein the immunoglobulin Fe polypeptide comprises at least one mutation at a position selected from the group consisting of: Met252, Ser254, Thr256, Met428, and Asn434.
16. 16. The isolated fusion protein of claim 3, wherein the immunoglobulin Fc polypeptide comprises at least one of the following ons: Tyr, Ser254Thr, Thr256Glu, Met428Leu or Asn434Ser.
17. 17. The isolated fusion protein of claim 2, wherein the human secretory leukocyte proteinase inhibitor (SLPI) polypeptide comprises the amino acid sequence of SEQ ID NO: 3 operably linked to an immunoglobulin Fc polypeptide comprising the amino acid sequence of SEQ ID NO: 10. 1001117809
18. 18. The isolated fusion protein of claim 3, wherein the human Elafm polypeptide ses the amino acid sequence of SEQID NO: 6 operably linked to an immunoglobulin Fc polypeptide comprising the amino acid sequence of SEQ ID NO: 10.
19. 19. The isolated fusion protein of claim 1, wherein the fusion protein comprises a serpin polypeptide comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 38.
20. 20. The isolated fusion protein of claim 2, wherein the fusion protein comprises an AAT polypeptide comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 38.
21. 21. The isolated fusion protein of any one of claim 1, claim 2 or claim 3, wherein the Fe polypeptide is modified to enhance FcRn binding.
22. 22. The isolated fusion protein of any one of claims 1—3, wherein the immunoglobulin Fc polypeptide comprising at least one of the following mutations: Tyr, Ser254Thr, Thr256Glu or Met428Leu and Asn43 4Ser.
23. 23. Use of the fusion protein of any one of claims 1—22 in the manufacture ofa medicament for ng or alleviating a symptom of a disease or disorder associated with aberrant serine se expression or activity in a subject in need thereof.
24. 24. Use of the fusion protein of any one of claims l~22 in the manufacture of a medicament for treating or alleviating a symptom of an inflammatory disease or disorder in a subject in need thereof.
25. 25. Use of the fusion protein of any one of claims 1—22 in the manufacture of a medicament for treating or alleviating a m of an infectious disease or disorder in a subject in need thereof.
26. 26. The use of the fusion protein of any one of claims 23-25, in the manufacture of a medicament for treating or alleviating a symptom of a disease or er ated with aberrant serine protease expression or activity in a subject in need f, wherein the subject is a human.
27. 27. An isolated fusion protein of claim 1, ntially as before bed.
28. 28. An isolated fusion protein of claim 2, substantially as hereinbefore described. 1001117809
29. 29. An isolated fusion n of claim 3, substantially as hereinbefore described.
30. 30. Use of any one of claims 23 to 25 as hereinbefore described.