Isolated fusion protein

Abstract

This invention relates to molecules, particularly polypeptides, more specifically fusion proteins comprising a serpin polypeptide or an amino acid sequence derived from a serpin and a second polypeptide comprising at least one of the following: an Fc polypeptide or an amino acid sequence derived from an Fc polypeptide; a cytokine-targeted polypeptide or a sequence derived from a cytokine-targeted polypeptide; a WAP domain-containing polypeptide or a sequence derived from a WAP domain-containing polypeptide; and an albumin polypeptide or an amino acid sequence derived from a serum albumin polypeptide. This invention also relates to methods of using such molecules in a variety of therapeutic and diagnostic indications, as well as methods of producing such molecules.

Description

/ 88 “ISOLATED FUSION PROTEIN” Related Orders

[001] This application claims the benefit of the Patent Application. US Provisional Order No. 14 / 524,832, filed Monday, October 27, 2014, the contents of which are incorporated herein by reference in their entirety. Sequence Listing Incorporation

[002] The contents of the text file named “INHI002002WO_SeqList.txt”, which was created on October 26, 2015 and is 228 KB in size, are incorporated here by reference in their entirety. Field of Invention

[003] This invention relates to molecules, particularly polypeptides, more particularly fusion proteins that include a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide and a second polypeptide. Additionally, the invention relates to fusion proteins that include a serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide, a second polypeptide and a third polypeptide.Specifically, this invention relates to fusion proteins comprising at least one serpin polypeptide and a second polypeptide, or fusion proteins comprising at least one serpin polypeptide, a second polypeptide, and a third polypeptide, wherein the second and third polypeptides of the fusion proteins of the invention may be at least one of the following: an Fc polypeptide or an amino acid sequence derived from an Fc polypeptide; a cytokine-targeting polypeptide or a sequence derived from a cytokine-targeting polypeptide; a polypeptide containing a WAP domain or a sequence derived from a WAP-containing polypeptide; or an albumin polypeptide or an amino acid sequence derived from a serum albumin polypeptide. This invention also relates to methods of using such. Petition 870210021782, dated 08 / 03 / 2021, p. 14 / 114 / 88 molecules in a variety of therapeutic and diagnostic indications, as well as methods for producing such molecules. Fundamentals of the Invention

[004] Aberrant serine protease activity or a protease inhibitor-to-protease imbalance can lead to protease-mediated tissue destruction and inflammatory responses. Consequently, there is a need for therapeutics and therapies that target aberrant serine protease activity and / or protease inhibitor-to-protease imbalance. Furthermore, enhanced therapeutic effects can be achieved by attenuating aberrant cytokine signaling and serine protease activity. In addition, serpin proteins have demonstrated anti-infective activities, while targeting inflammatory cytokines has been shown to increase the risk of infection. The fusion proteins of this invention have the potential to decrease inflammatory cytokine activity and limit the risk of infection. Summary of the Invention

[005] The fusion proteins described herein include at least one serpin polypeptide or an amino acid sequence derived from a serpin polypeptide (Polypeptide 1) and a second polypeptide (Polypeptide 2). Additionally, the fusion proteins described herein include a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide (Polypeptide 1), a second polypeptide (Polypeptide 2), and a third polypeptide (Polypeptide 3). As used interchangeably herein, the terms “fusion protein” and “fusion polypeptide” refer to a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide operationally linked to at least one second polypeptide or an amino acid sequence derived from at least one second polypeptide. The individual elements of the fusion protein may be linked in Petition 870210021782, dated 08 / 03 / 2021, page 15 / 114 / 88 any of a variety of modes, including, for example, direct attachment, the use of an intermediate or a spacer peptide, the use of a linker region, the use of a hinge region, or the use of both a linker and a hinge region. In some embodiments, the linker region may be included in the hinge region sequence, or alternatively, the hinge region may be included in the linker region sequence. Preferably, the linker region is a peptide sequence. For example, the linker peptide includes zero to 40 amino acids, for example, zero to 35 amino acids, zero to 30 amino acids, zero to 25 amino acids, or zero to 20 amino acids. Preferably, the hinge region is a peptide sequence. For example, the hinge peptide includes zero to 75 amino acids, for example, zero to 70 amino acids, zero to 65 amino acids, or zero to 62 amino acids.In embodiments where the fusion protein includes both a linker region and a hinge region, preferably each of the linker region and the hinge region is a peptide sequence. In these embodiments, the hinge peptide and the linker peptide together include from zero to 90 amino acids, for example, from zero to 85 amino acids, or from zero to 82 amino acids.

[006] In some embodiments, the serpin polypeptide and the second polypeptide may be linked via an intermediate linking protein. In some embodiments, the serpin-based portion and the second polypeptide portion of the fusion protein may be non-covalently linked.

[007] In some embodiments, the fusion proteins according to the invention may have one of the following formulas, in an N-terminal to C-terminal direction or in a C-terminal to N-terminal direction: Polypeptide 1(a) - hinge- Polypeptide 2(b) Polypeptide 1(a) - ligand- Polypeptide 2(b) Polypeptide 1(a) - ligand- hinge- Polypeptide 2(b) Petition 870210021782, dated 08 / 03 / 2021, page 16 / 114 / 88 Polypeptide 1 (a) - hinge- ligand- Polypeptide 2(b) Polypeptide 1(a) - Polypeptide 2(b)- Polypeptide 3(c) Polypeptide 1(a) - hinge- Polypeptide 2(b)- hinge- Polypeptide 3(c) Polypeptide 1(a) - ligand - Polypeptide 2(b)- ligand Polypeptide 3(c) Polypeptide 1(a) - hinge - ligand - Polypeptide 2(b) hinge - ligand - Polypeptide 3(c) Polypeptide 1(a) - ligand hinge - Polypeptide 2(b) - ligand - hinge - 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 integers of at least 1. These embodiments include the above formulations and any variation or combination thereof. For example, the order of polypeptides in the formulas also includes Polypeptide 3(c) - Polypeptide 1(a) - Polypeptide 2(b), Polypeptide 2(b) - Polypeptide 3(c) - Polypeptide 1(a), or any variation or combination thereof.

[008] In some embodiments, the Polypeptide 1 sequence includes a serpin polypeptide. Serpins are a group of proteins with similar structures that were first identified as a set of proteins capable of inhibiting proteases. Serpin proteins suitable for use in the fusion proteins provided herein include, by way of non-limiting example, alpha-1 antitrypsin (AAT), antitrypsin-related protein (SERPIN2), alpha 1-antichymotrypsin (SERPIN3), callistatin (SERPIN4), neutrophil and monocyte elastase inhibitor (SERPINB1), PI-6 (SERPINB6), antithrombin (SERPINC1), plasminogen activator inhibitor 1 (SERPINE1), alpha 2-antiplasmin (SERPINF2), complement inhibitor 1 (SERPING1), and neuroserpin (SERPINI1).

[009] In some embodiments, the Polypeptide 1 sequence includes an alpha-1 antitrypsin (AAT) polypeptide sequence or a Petition 870210021782, dated 08 / 03 / 2021, p. 17 / 114 / 88 amino acid sequence that is derived from AAT. In some embodiments, the Polypeptide 1 sequence includes a portion of the AAT protein. In some embodiments, the Polypeptide 1 sequence includes at least the loop portion of the reactive site of the AAT protein. In some embodiments, the loop portion of the reactive site of the AAT protein includes at least the amino acid sequence: GTEAAGAMFLEAIPMSIPPEVKFNK SEQ ID NO:1).

[0010] In a preferred embodiment, the AAT polypeptide sequence or an amino acid sequence that is derived from AAT is or is derived from a human AAT polypeptide sequence.

[0011] In some embodiments, the fusion protein includes a full-length human AAT polypeptide sequence having the following amino acid sequence: EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI FFSPVSIATA 61 FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL RTLNQPDSQL QLTTGGNGLFL 121 SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT EEAKKQINDY VEKGTQGKIV DLVKELDRDT 181 VFALVNIFF DEKGTEAAGA MFLEAIPMSI 361 PPEVKFNKPF VFLMIEQNTK SPLFMGKVVN PTQK (SEQ ID NO: 2)

[0012] In some embodiments, the fusion protein includes a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0013] In some embodiments, the AAT polypeptide sequence is, or the amino acid sequence derived from the AAT polypeptide is derived from, one or more of the human AAT polypeptide sequences shown in Genbank, accession nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, and / or AAA51547.1. Petition 870210021782, dated 08 / 03 / 2021, page 18 / 114 / 88

[0014] In some embodiments, the fusion proteins contain one or more mutations. For example, the fusion protein contains at least one mutation in a methionine (Met) residue in the serpine portion of the fusion protein. In these Met mutations, the Met residue can be substituted with any amino acid. For example, the Met residue can be substituted with an amino acid with a hydrophobic side chain, such as, for example, leucine (Leu, L). 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 residue can be substituted with a charged residue, such as, for example, glutamate (Glu, E). In some embodiments, the Met mutation is at position 358 of an AAT polypeptide. For example, the Met mutation is Met358Leu (M358L). In some forms, the Met mutation is located at position 351 of an AAT polypeptide.For example, the Met mutation is Met351Glu (M351E). In some embodiments, the Met mutation is at position 351 and position 358 of an AAT polypeptide, for example, the Met mutation is Met351Glu (M351E) and Met358Leu (M358L). For example, the reactive site loop of this variant of the AAT fusion polypeptide has the following sequence: GTEAAGAEFLEAIPLSIPPEVKFNK (SEQ ID NO: 32). In some embodiments, the Met mutation is at position 351 and position 358 of an AAT polypeptide, for example, the Met mutation is Met351Leu (M351L) and Met358Leu (M358L). For example, the reactive site loop of this variant of the AAT fusion polypeptide has the following sequence: GTEAAGALFLEAIPLSIPPEVKFNK (SEQ ID NO: 33).

[0015] In some embodiments, the fusion protein includes a full-length human AAT polypeptide sequence containing a variant modified reactive site loop at M351 and M358, which has the following amino acid sequence: EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI FFSPVSIATA Petition 870210021782, dated 08 / 03 / 2021, page 19 / 114 / 88 FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL RTLNQPDSQL QLTTGNGLFL 121 SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT EEAKKQINDY VEKGTQGKIV DLVKELDRDT 181 VFALVNYIFF KGKWERPFEV KDTEEEDFHV DQVTTVKVPM MKRLGMFNIQ HCKKLSSWVL 241 LMKYLGNATA IFFLPDEGKL QHLENELTHD IITKFLENNED RRSASLHLPK LSITGTYDLK 301 SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA |Ê]fLEAIP|l]SI 361 PPEVKFNKPF VFLMIEQNTK SPLFMGKVVN PTQK (SEQ ID NO: 80)

[0016] In some embodiments, the fusion protein includes a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 80.

[0017] In some embodiments, the second polypeptide (Polypeptide 2) of the serpin fusion protein is an Fc polypeptide or derived from an Fc polypeptide. These embodiments are collectively referred to herein as “serpin-Fc fusion proteins”. The serpin-Fc fusion proteins described herein include at least one serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide and Fc or an amino acid sequence that is derived from an Fc polypeptide. In some embodiments, the serpin-Fc fusion protein includes a single serpin polypeptide. In other embodiments, the serpin-Fc fusion proteins include more than one serpin polypeptide, and these embodiments are collectively referred to herein as “serpin(a') fusion protein”, wherein (a') is an integer of at least 2. In some embodiments, each serpin polypeptide in a serpin(a')-Fc fusion protein includes the same amino acid sequence.In other embodiments, each serpin polypeptide in a serpin(a')-Fc fusion protein includes serpin polypeptides with distinct amino acid sequences. The serpin polypeptides of serpin(a')-Fc fusion proteins can be located at any position within the fusion protein.

[0018] In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein includes at least the amino acid sequence of the loop portion of the AAT protein's reactive site. In some embodiments, Petition 870210021782, dated 08 / 03 / 2021, p. 20 / 114 / 88 the loop portion of the AAT protein's reactive site includes at least the amino acid sequence of SEQ ID NO:1. In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein includes at least the amino acid sequence of a variant of the loop portion of the AAT protein's reactive site. In some embodiments, the variant of the loop portion of the AAT protein's reactive site includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein includes at least the full-length human AAT polypeptide sequence having the amino acid sequence SEQ ID NO: 2. In some embodiments, the serpin polypeptide of the serpin-Fc fusion protein includes at least the full-length human AAT polypeptide sequence having the amino acid sequence SEQ ID NO: 80.In some embodiments, the serpin polypeptide of the Fc-serpin fusion protein includes a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80.

[0019] In some embodiments, the serpin polypeptide of the Fc-serpin fusion protein includes the AAT polypeptide sequence, or the amino acid sequence derived from the AAT polypeptide is derived from one or more of the human AAT polypeptide sequences shown in GenBank, accession nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, and / or AAA51547.1.

[0020] In some embodiments, the Fc polypeptide of the fusion protein is a human Fc polypeptide, for example, a sequence of Petition 870210021782, dated 08 / 03 / 2021, page 21 / 114 / 88 human IgG Fc polypeptides or an amino acid sequence that is derived from a human IgG Fc polypeptide sequence. For example, in some embodiments, the Fc polypeptide is a human IgG1 Fc polypeptide or an amino acid sequence that is derived from a human IgG1 Fc polypeptide sequence. In some embodiments, the Fc polypeptide is a human IgG2 Fc polypeptide or an amino acid sequence 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 either 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 polypeptide is either a human IgM Fc polypeptide or an amino acid sequence that is derived from a human IgM Fc polypeptide sequence.

[0021] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a sequence of human IgG1 Fc polypeptides having the following amino acid sequence: APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 121 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL 181 TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 3)

[0022] In some embodiments, wherein the fusion protein of the invention includes an Fc polypeptide, the fusion protein includes a hinge region coupled to the N-terminal of the Fc polypeptide of the fusion protein, wherein the Fc polypeptide includes a sequence of human IgG1 Fc polypeptides having the following amino acid sequence: DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK Petition 870210021782, dated 08 / 03 / 2021, p. 22 / 114 / 88 121 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 43)

[0023] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG1 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3 or 43.

[0024] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide is mutated or modified to enhance binding to FcRn. In these embodiments, the mutated or modified Fc polypeptide includes the following mutations: Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E) or Met428Leu and Asn434Ser (M428L, N434S) or Met428Val and Asn434Ser (M428V, N434S) using the Kabat numbering system. In some embodiments, the mutated or modified Fc polypeptide includes one or more selected mutations from the group consisting of Met252Tyr (M252Y), Ser254Thr (S256T), Thr256Glu (T256E), Met428Leu (M428L), Met428Val (M428V), Asn434Ser (N434S), and combinations thereof. In some embodiments, the Fc polypeptide portion is mutated or otherwise modified so as to disrupt Fc-mediated dimerization. In these embodiments, the fusion protein is monomeric in nature.

[0025] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide is mutated or modified. In some embodiments, the mutated or modified Fc polypeptide includes one or more mutations at a position selected from M252, T246, M428 and combinations thereof. In some embodiments, the mutated or modified Fc polypeptide includes the following mutations: Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L) using the Kabat numbering system. Petition 870210021782, dated 08 / 03 / 2021, page 23 / 114 / 88

[0026] In some embodiments, where the fusion protein includes a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes mutations at residues M252, T256, and M428, which correspond to residues 22, 26, and 198 of SEQ ID NO: 3 or residues 32, 36, and 208 of SEQ ID NO: 43 shown above, and has the following amino acid sequence, in which the mutated amino acid residues are boxed: APELLGGPSV FLFPPKPKDT l[i]iSR|d]pEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK 61 preeqynsty rvvsvltvlh qdwlngkeyk ckvsnkalpa piektiskak gqprepqvyt 121 lppsrdeltk nqvsltclvk gfypsdiave wesngqpenn ykttppvlds dgsfflyskl 181 TVDKSRWQQG NVFSCSv|l]hE ALHNHYTQKS LSLSPGK (SEQ ID NO: 44)

[0027] In some embodiments, where the fusion protein includes an N-terminal hinge region of a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes mutations at residues M252, T256, and M428, which correspond to residues 22, 26, and 198 of SEQ ID NO: 3 or residues 32, 36, and 208 of SEQ ID NO: 43 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed: DKTHTCPPCP APELLGGPSV FLFPPKPKDT l[I]iSR|d]pEVT CVVVDVSHED PEVKFNWYVD 61 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK 121 gqprepqvyt lppsrdeltk nqvsltclvk gfypsdiave wesngqpenn ykttppvlds 181 DGSFFLYSKL TVDKSRWQQG NVFSCSvQhE ALHNHYTQKS LSLSPGK (SEQ ID NO: 45)

[0028] In some embodiments, where the fusion protein includes a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes a modified human IgG1 Fc polypeptide sequence in which residue G236, which corresponds to residue 6 of SEQ ID NO: 3 or residue 16 of SEQ ID NO: 43 shown above, is deleted and has the following amino acid sequence: APELLGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 61 reeqynstyr vvsvltvlhq dwlngkeykc kvsnkalpap iektiskakg qprepqvytl 121 ppsrdeltkn qvsltclvkg fypsdiavew esngqpenny kttppvldsd gsfflysklt Petition 870210021782, dated 08 / 03 / 2021, p. 24 / 114 / 88 181 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 46)

[0029] In some embodiments, wherein the fusion protein includes a hinge region coupled to the N-terminal of a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes a modified human IgG1 Fc polypeptide sequence in which residue G236, which corresponds to residue 6 of SEQ ID NO: 3 or residue 16 of SEQ ID NO: 43 shown above, is deleted, and the fusion protein includes at least the following amino acid sequence: DKTHTCPPCP APELLGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG 121 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD 181 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 47)

[0030] In some embodiments, where the fusion protein includes a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO: 3 or residues 14 and 15 of SEQ ID NO: 43 shown above, and has the following amino acid sequence, where the mutated amino acid residues are boxed: ape|vA|ggpsv flfppkpkdt lmisrtpevt cvvvdvshed pevkfnwyvd gvevhnaktk PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 121 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL 181 TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 48)

[0031] In some embodiments, where the fusion protein includes a hinge region coupled to the N-terminal of a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO: 3 or residues 14 and 15 of SEQ ID NO: 43 shown above, and the fusion protein includes at least the following amino acid sequence, in which the mutated amino acid residues are boxed: DKTHTCPPCP APEVAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK 121 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS Petition 870210021782, dated 08 / 03 / 2021, p. 25 / 114 / 88 181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 49)

[0032] In some embodiments, wherein the fusion protein includes a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234 and L235, and the fusion protein includes at least the following amino acid sequence, wherein the mutated amino acid residues are boxed: apeVAgpsvf lfppkpkdtl misrtpevtc vvvdvshedp evkfnwyvdg vevhnaktkp REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL 121 PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT 181 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 50)

[0033] In some embodiments, where the fusion protein includes a hinge region coupled to the N-terminal of a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234 and L235, and has the following amino acid sequence, where the mutated amino acid residues are boxed: DKTHTCPPCP APEVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG 121 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD 181 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 51)

[0034] In some embodiments, wherein the fusion protein includes a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428, and the fusion protein includes at least the following amino acid sequence, wherein the mutated amino acid residues are boxed: apeVAgpsvf lfppkpkdtl ^isr^pevtc vvvdvshedp evkfnwyvdg vevhnaktkp REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL 121 PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT 181 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 52)

[0035] In some embodiments, where the fusion protein includes Petition 870210021782, dated 08 / 03 / 2021, p. 26 / 114 / 88 a hinge region coupled to the N-terminal of a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428, and has the following amino acid sequence, where the mutated amino acid residues are boxed: dkthtcppcp apeVagpsvf lfppkpkdtl ^isr^pevtc vvvdvshedp evkfnwyvdg 61 VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG 121 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD 181 GSFFLYSKLT VDKSRWQQGN VFSCSV0HEA LHNHYTQKSL SLSPGK (SEQ ID NO: 53)

[0036] In some embodiments, wherein the fusion protein of the invention includes a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes a modified human IgG1 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53.

[0037] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a sequence of human IgG2 Fc polypeptides having the following amino acid sequence: APPVAGPSVF LFPPKPKDTL 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: 4)

[0038] 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: 4.

[0039] In some embodiments, where the fusion protein includes Petition 870210021782, dated 08 / 03 / 2021, p. 27 / 114 / 88 a modified IgG2 Fc polypeptide, the modified IgG2 Fc polypeptide of the fusion protein includes a modified human IgG2 Fc polypeptide sequence that has the following amino acid sequence, wherein the mutated amino acid residues are boxed: APPVAGPSVF LFPPKPKDTL IiSR | FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT 181 VDKSRWQQGN VFSCSv|l]hEA LHNHYTQKSL SLSPGK (SEQ ID NO: 54)

[0040] In some embodiments, where the fusion protein of the invention includes a modified IgG2 Fc polypeptide, the modified IgG2 Fc polypeptide of the fusion protein includes a modified human IgG2 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 54.

[0041] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a sequence of human IgG3 Fc polypeptides having the following amino acid sequence: APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD GVEVHNAKTK 61 PREEQYNSTF RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKTK GQPREPQVYT 121 LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESSGQPENN YNTTPPMLDS DGSFFLYSKL 181 TVDKSRWQQG NIFSCSVMHE ALHNRFTQKS LSLSPGK (SEQ ID NO: 5)

[0042] 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: 5.

[0043] In some embodiments, where the fusion protein includes a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein includes a sequence of Fc polypeptides Petition 870210021782, dated 08 / 03 / 2021, p. 28 / 114 / 88 concerning a modified human IgG3 that has the following amino acid sequence, in which the mutated amino acid residues are boxed: APELLGGPSV FLFPPKPKDT l[i]iSR|d]pEVT CVVVDVSHED PEVQFKWYVD GVEVHNAKTK preeqynstf rvvsvltvlh qdwlngkeyk ckvsnkalpa piektisktk gqprepqvyt 121 lppsreemtk nqvsltclvk gfypsdiave wessgqpenn ynttppmlds dgsfflyskl 181 TVDKSRWQQG NIFSCSv|l]hE ALHNRFTQKS LSLSPGK (SEQ ID NO: 55)

[0044] In some embodiments, where the fusion protein includes a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein includes a modified human IgG3 Fc polypeptide sequence in which residue G236, which corresponds to residue 6 of SEQ ID NO: 5 shown above, is deleted and has the following amino acid sequence: APELLGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFKWYVDG VEVHNAKTKP reeqynstfr vvsvltvlhq dwlngkeykc kvsnkalpap iektisktkg qprepqvytl 121 ppsreemtkn qvsltclvkg fypsdiavew essgqpenny nttppmldsd gsfflysklt 181 VDKSRWQQGN IFSCSVMHEA LHNRFTQKSL SLSPGK (SEQ ID NO: 56)

[0045] In some embodiments, where the fusion protein includes a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein includes mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO: 5 shown above, and has the following amino acid sequence, where the mutated amino acid residues are boxed: APEVA|GGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVQFKWYVD GVEVHNAKTK preeqynstf RVVSVLTVLH ynttppmlds dgsfflyskl 181 TVDKSRWQQG NIFSCSVMHE ALHNRFTQKS LSLSPGK (SEQ ID NO: 57)

[0046] In some embodiments, where the fusion protein includes a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234 and L235 and has the following amino acid sequence: APe|vA|gPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFKWYVDG VEVHNAKTKP Petition 870210021782, dated 08 / 03 / 2021, p. 29 / 114 / 88 REEQYNSTFR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKTKG QPREPQVYTL 121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESSGQPENNY NTTPPMLDSD GSFFLYSKLT 181 VDKSRWQQGN IFSCSVMHEA LHNRFTQKSL SLSPGK (SEQ ID NO: 58)

[0047] In some embodiments, where the fusion protein includes a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein includes a deletion at residue G236 and mutations at residues L234, L235, M252, T256, and M428 and has the following amino acid sequence: apeVAgpsvf lfppkpkdtl IisrDpevtc vvvdvshedp evqfkwyvdg vevhnaktkp REEQYNSTFR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKTKG QPREPQVYTL 121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESSGQPENNY NTTPPMLDSD GSFFLYSKLT 181 VDKSRWQQGN IFSCSVMHEA LHNRFTQKSL SLSPGK (SEQ ID NO: 59)

[0048] In some embodiments, wherein the fusion protein of the invention includes a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein includes a modified human IgG3 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 55, 56, 57, 58, or 59.

[0049] In some embodiments, the Fc region of human IgG3 is modified at amino acid Asn297 (Kabat numbering) to prevent glycosylation of the antibody, for example, Asn297Ala (N297A). In some embodiments, the Fc region of human IgG3 is modified at amino acid 435 to extend the half-life, for example, Arg435His (R435H). In some embodiments, the Fc region of human IgG3 lacks Lys447 (EU index of Kabat et al. 1991 Sequences of Proteins of Immunological Interest).

[0050] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a sequence of human IgG4 Fc polypeptides having the following amino acid sequence: APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT Petition 870210021782, dated 08 / 03 / 2021, p. 30 / 114 / 88 121 LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL 181 TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 6)

[0051] In some embodiments, wherein the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a hinge region coupled to the N-terminal of the Fc polypeptide of the fusion protein, wherein the Fc polypeptide includes a human IgG4 Fc polypeptide sequence that has the following amino acid sequence: ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 60)

[0052] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgG4 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6 or 60.

[0053] In some embodiments, where the fusion protein includes a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues M252, T256, and M428, which correspond to residues 22, 26, and 19 of SEQ ID NO: 6 or residues 34, 38, and 210 of SEQ ID NO: 60 shown above, and has the following amino acid sequence, in which the mutated amino acid residues are boxed: APEFLGGPSV FLFPPKPKDT l[I]iSR|d]pEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT 121 LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL 181 TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 61)

[0054] In some embodiments, where the fusion protein includes a hinge region coupled to the N-terminal of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the protein Petition 870210021782, dated 08 / 03 / 2021, p. 31 / 114 / 88: The fusion protein includes mutations in residues M252, T256, and M428, which correspond to residues 22, 26, and 197 of SEQ ID NO: 6 or residues 34, 38, and 210 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed: ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTL[i]iSR|d]pE VTCVVVDVSQ EDPEVQFNWY 61 vdgvevhnak tkpreeqfns tyrvvsvltv lhqdwlngke ykckvsnkgl pssiektisk 121 akgqprepqv ytlppsqeem tknqvsltcl vkgfypsdia vewesngqpe nnykttppvl 181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVL HEALHNHYTQ KSLSLSLGK (SEQ id NO: 62)

[0055] In some embodiments, where the fusion protein includes a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes a modified human IgG4 Fc polypeptide sequence in which residue G236, which corresponds to residue 6 of SEQ ID NO: 6 or residue 19 of SEQ ID NO: 60 shown above, is deleted and has the following amino acid sequence: APEFLGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSQEDP EVQFNWYVDG VEVHNAKTKP 61 reeqfnstyr vvsvltvlhq dwlngkeykc kvsnkglpss iektiskakg qprepqvytl 121 ppsqeemtkn qvsltclvkg fypsdiavew esngqpenny kttppvldsd gsfflysrlt 181 VDKSRWQEGN VFSCSVMHEA LHNHYTQKSL SLSLGK (SEQ id NO: 63)

[0056] In some embodiments, wherein the fusion protein includes a hinge region coupled to the N-terminal of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes a modified human IgG4 Fc polypeptide sequence in which residue G236, which corresponds to residue 6 of SEQ ID NO: 6 or residue 19 of SEQ ID NO: 60 shown above, is deleted, and the fusion protein includes at least the following amino acid sequence: ESKYGPPCPS CPAPEFLGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV 61 dgvevhnakt kpreeqfnst yrvvsvltvl hqdwlngkey kckvsnkglp ssiektiska 121 kgqprepqvy tlppsqeemt knqvsltclv kgfypsdiav ewesngqpen nykttppvld 181 SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGK (SEQ id NO: 64)

[0057] In some embodiments, where the fusion protein includes a modified IgG4 Fc polypeptide, the IgG4 Fc polypeptide Petition 870210021782, dated 08 / 03 / 2021, p. 32 / 114 / 88, states that the modified fusion protein includes a mutation at residue L235, which corresponds to residue 5 of SEQ ID NO: 6 or residue 17 of SEQ ID NO: 60 shown above, and has the following amino acid sequence, where the mutated amino acid residue is boxed: apefEggpsv flfppkpkdt lmisrtpevt cvvvdvsqed pevqfnwyvd gvevhnaktk PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT 121 LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL 181 TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 65)

[0058] In some embodiments, where the fusion protein includes a hinge region coupled to the N-terminal of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes a mutation at residue L235, which corresponds to residue 5 of SEQ ID NO: 6 or residue 17 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residue is boxed: eskygppcps cpapefEggp svflfppkpk dtlmisrtpe vtcvvvdvsq edpevqfnwy VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 66)

[0059] In some embodiments, where the fusion protein includes a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO: 6 or residues 16 and 17 of SEQ ID NO: 60 shown above, and has the following amino acid sequence, where the mutated amino acid residues are boxed: APeVAgGPSV flfppkpkdt lmisrtpevt cvvvdvsqed pevqfnwyvd gvevhnaktk PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT 121 LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL 181 TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 67)

[0060] In some embodiments, where the fusion protein includes a hinge region coupled to the N-terminal of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the protein Petition 870210021782, dated 08 / 03 / 2021, p. 33 / 114 / 88: The fusion protein includes mutations in residues L234 and L235, which correspond to residues 4 and 5 of SEQ ID NO: 6 or residues 16 and 17 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed: ESKYGPPCPS cpapeVaggp svflfppkpk dtlmisrtpe vtcvvvdvsq edpevqfnwy VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 68)

[0061] In some embodiments, where the fusion protein includes a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes a mutation at residue S228, which corresponds to residue 10 of SEQ ID NO: 60 shown above, and has the following amino acid sequence, where the mutated amino acid residue is boxed: ESKYGPPCPp] CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 69)

[0062] In some embodiments, where the fusion protein includes a hinge region coupled to the N-terminal of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues S228 and L235, which correspond to residues 10 and 17 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, where the mutated amino acid residues are boxed: ESKYGPPCPp] CPAPEFgGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY 61 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 70)

[0063] In some embodiments, where the fusion protein includes a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues L235, M252, Petition 870210021782, dated 08 / 03 / 2021, p. 34 / 114 / 88 T256 and M428, which correspond to residues 5, 22, 26, and 197 of SEQ ID NO: 6 or residues 17, 34, 38, and 210 of SEQ ID NO: 60 shown above, have the following amino acid sequence, where the mutated amino acid residues are boxed: apefEggpsv flfppkpkdt l^isr^pevt cvvvdvsqed pevqfnwyvd gvevhnaktk 61 PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT 121 LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL 181 TVDKSRWQEG NVFSCSvjLjHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 71)

[0064] In some embodiments, wherein the fusion protein includes an N-terminal hinge region of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues L235, M252, T256, and M428, which correspond to residues 5, 22, 26, and 197 of SEQ ID NO: 6 or residues 17, 34, 38, and 210 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, wherein the mutated amino acid residues are boxed: ESKYGPPCPS CPAPEfEgGP SVFLFPPKPK DTL0ISR0PE VTCVVVDVSQ EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSV[L HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 72)

[0065] In some embodiments, where the fusion protein includes a hinge region coupled to the N-terminal of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes mutations at residues S228, L235, M252, T256, and M428, which correspond to residues 10, 17, 34, 38, and 210 of SEQ ID NO: 60 shown above, and the fusion protein includes at least the following amino acid sequence, in which the mutated amino acid residues are boxed: ESKYGPPCPp] CPAPEfEgGP SVFLFPPKPK DTl[I]iSr|d]pE VTCVVVDVSQ EDPEVQFNWY 61 VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 121 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 181 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSvQ HEALHNHYTQ KSLSLSLGK (SEQ ID NO: 73)

[0066] In some embodiments, where the fusion protein of Petition 870210021782, dated 08 / 03 / 2021, p. 35 / 114 / 88 invention includes a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes a modified human IgG4 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73.

[0067] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a sequence of human IgM Fc polypeptides having the following amino acid sequence: IAELPPKVSV FVPPRDGFFG NPRKSKLICQ ATGFSPRQIQ VSWLREGKQV GSGVTTDQVQ AEAKESGPTT YKVTSTLTIK ESDWLGQSMF TCRVDHRGLT FQQNASSMCV PDQDTAIRVF 121 AIPPSFASIF LTKSTKLTCL VTDLTTYDSV TISWTRQNGE AVKTHTNISE SHPNATFSAV 181 GEASICEDDW NSGERFTCTV THTDLPSPLK QTISRPKG (SEQ ID NO: 7)

[0068] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide of the fusion protein includes a human IgM Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7.

[0069] In some embodiments, the serpin-Fc fusion protein includes at least the amino acid sequence of the reactive site loop portion of the AAT protein operationally linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of the following SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the Fc polypeptide includes an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In some embodiments, the Fc polypeptide includes a Petition 870210021782, dated 08 / 03 / 2021, p. 36 / 114 / 88 amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In some embodiments, the The loop portion of the AAT protein's reactive site includes at least the amino acid sequence SEQ ID NO:1. In some embodiments, the serpine polypeptide and the Fc polypeptide are operationally linked via a linker region, for example, a glycine-serine linker or a glycine-serine-based linker. In some embodiments, the serpine polypeptide and the Fc polypeptide are operationally linked via a hinge region.In some embodiments, the serpin polypeptide and the Fc polypeptide are operationally linked via a linker region and a hinge region. In other embodiments, the serpin polypeptide and the Fc polypeptide are directly attached.

[0070] In some embodiments, the serpin-Fc fusion protein includes at least the amino acid sequence of a variant of the reactive site loop portion of the AAT protein operationally linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of the following SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the Fc polypeptide includes an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.In some embodiments, the Fc polypeptide includes an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59. Petition 870210021782, dated 08 / 03 / 2021, p. 37 / 114 / 88 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In some embodiments, the loop portion variant of the AAT protein's reactive site includes at least the amino acid sequence SEQ ID NO:32 or SEQ ID NO:33. In some embodiments, the serpine polypeptide and the Fc polypeptide are operationally linked via a linker region, for example, a glycine-serine linker or a glycine-serine-based linker. In some embodiments, the serpine polypeptide and the Fc polypeptide are operationally linked via a hinge region. In some embodiments, the serpine polypeptide and the Fc polypeptide are operationally linked via both a linker region and a hinge region. In other embodiments, the serpin polypeptide and the Fc polypeptide are directly attached.

[0071] In some embodiments, the serpin-Fc fusion protein includes at least the full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO: 2 operationally linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of the SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the Fc polypeptide includes an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.In some embodiments, the Fc polypeptide includes an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In some embodiments, The serpin-Fc fusion protein includes a human AAT polypeptide sequence that is at least 50%, 60%... Petition 870210021782, dated 08 / 03 / 2021, p. 38 / 114 / 88 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 operationally linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of the SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the Fc polypeptide includes an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73.In some embodiments, the Fc polypeptide includes an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In some embodiments, The serpin polypeptide and the Fc polypeptide are operationally linked through a linker region, for example, a glycine-serine linker or a glycine-serine-based linker. In some embodiments, the serpin polypeptide and the Fc polypeptide are operationally linked through a hinge region. In some embodiments, the serpin polypeptide and the Fc polypeptide are operationally linked through both a linker region and a hinge region. In other embodiments, the serpin polypeptide and the Fc polypeptide are directly affixed.

[0072] In some embodiments, the serpin-Fc fusion protein includes at least the full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO: 80 operationally linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of the SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, Petition 870210021782, dated 08 / 03 / 2021, p. 39 / 114 / 88 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the serpin-Fc fusion protein includes a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 80 operationally linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of the SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the serpin polypeptide and the Fc polypeptide are operationally linked through a linker region, for example, a glycine-serine linker or a glycine-serine-based linker. In some embodiments, the serpin polypeptide and the Fc polypeptide are operationally linked through a hinge region.In some embodiments, the serpin polypeptide and the Fc polypeptide are operationally linked via a linker region and a hinge region. In other embodiments, the serpin polypeptide and the Fc polypeptide are directly attached.

[0073] In some embodiments of the fusion proteins provided herein, the second polypeptide (Polypeptide 2) of the serpin fusion protein is a cytokine-targeting polypeptide or derived from a cytokine-targeting polypeptide. These embodiments are collectively referred to herein as “serpinacytokine-targeting polypeptide fusion proteins.” The serpinacytokine-targeting polypeptide fusion proteins described herein include at least one serpin polypeptide or an amino acid sequence that is derived from, or derived from, a serpin polypeptide and a cytokine-targeting polypeptide. In some embodiments, the serpin-cytokine-targeting polypeptide fusion protein includes a single serpin polypeptide. In other embodiments, the serpin-cytokine-targeting polypeptide fusion protein Petition 870210021782, dated 08 / 03 / 2021, page 40 / 114 / 88 cytokine includes more than one serpin polypeptide, and these embodiments are collectively referred to herein as “serpin(a')-cytokine targeting polypeptide fusion proteins”, wherein (a') is an integer of at least 2. In some embodiments, each serpin polypeptide in a serpin(a')-cytokine targeting polypeptide fusion protein includes the same amino acid sequence. In other embodiments, each serpin polypeptide of a serpin(a')-cytokine targeting polypeptide fusion protein includes serpin polypeptides with distinct amino acid sequences.

[0074] In some embodiments, the cytokine targeting polypeptide of the serpinacytokine targeting polypeptide fusion protein is a cytokine receptor or derived from a cytokine receptor. In a preferred embodiment, the cytokine targeting polypeptide or an amino acid sequence that is derived from the cytokine receptor is or is derived from a human cytokine receptor sequence. In other embodiments, the cytokine targeting polypeptide is an antibody or an antibody fragment, for example, an anti-cytokine antibody or anti-cytokine antibody fragment. In a preferred embodiment, the cytokine targeting polypeptide or an amino acid sequence that is derived from the antibody or antibody fragment is derived from a chimeric, humanized, or fully human antibody sequence.The term antibody fragment includes a single-chain antibody, a Fab fragment, an F(ab')2 fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody.

[0075] In other embodiments, the cytokine-targeting polypeptide binds to a cytokine receptor and prevents a cytokine from binding to the receptor. In other embodiments, the cytokine-targeting polypeptide is an antibody or an antibody fragment, for example, a Petition 870210021782, dated 08 / 03 / 2021, p. 41 / 114 / 88 anti-cytokine receptor antibody or anti-cytokine receptor antibody fragment.

[0076] In some embodiments, the serpin polypeptide of the serpin-cytocin targeting polypeptide fusion proteins includes at least the amino acid sequence of the loop portion of the AAT protein's reactive site. In some embodiments, the loop portion of the AAT protein's reactive site includes at least the amino acid sequence of SEQ ID NO:1. In some embodiments, the serpin polypeptide of the serpin-cytocin targeting polypeptide fusion proteins includes at least the amino acid sequence of a variant of the loop portion of the AAT protein's reactive site. In some embodiments, the variant of the loop portion of the AAT protein's reactive site includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33.In some embodiments, the serpin polypeptide of the serpina-cytocin bleaching fusion protein includes or is derived from at least the full-length human AAT polypeptide sequence having the amino acid sequence SEQ ID NO: 2. In some embodiments, the serpin polypeptide of the serpina-cytocin bleaching fusion protein includes or is derived from at least the full-length human AAT polypeptide sequence having the amino acid sequence SEQ ID NO: 80. In some embodiments, the serpin polypeptide of the serpina-cytocin bleaching fusion protein includes a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of amino acids with SEQ ID NO: 2 or 32 or 33 or 80.

[0077] In some embodiments, the serpin polypeptide of the serpin-cytokine bleaching fusion protein includes an AAT polypeptide sequence or an amino acid sequence derived from the AAT polypeptide that is or is derived from one or more of the sequences of Petition 870210021782, dated 08 / 03 / 2021, page 42 / 114 / 88 human AAT polypeptides shown in Genbank, accession numbers AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, and / or AAA51547.1.

[0078] The serpin-cytokine targeting polypeptide fusion protein can incorporate a portion of the serpinFc fusion protein. For example, an antibody contains an Fc polypeptide. Therefore, in some embodiments, where the cytokine targeting polypeptide is a cytokine targeting antibody, the serpin-cytokine targeting fusion protein will incorporate a portion of the serpinFc fusion protein. Furthermore, most receptor fusion proteins that are of therapeutic utility are Fc fusion proteins. Thus, in some embodiments, where the serpin-cytokine targeting polypeptide fusion protein is a serpin-cytokine receptor fusion protein, the serpin-cytokine targeting polypeptide fusion protein can incorporate an Fc polypeptide in addition to the serpin portion and the cytokine receptor portion.

[0079] In some embodiments, wherein the serpine-cytokine targeting polypeptide fusion protein includes an Fc polypeptide sequence, the Fc polypeptide sequence includes or is derived from the amino acid sequence of any of the following SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, wherein the serpine-cytokine targeting fusion protein includes an Fc polypeptide sequence, the Fc polypeptide sequence Fc has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of the sequences of Petition 870210021782, dated 08 / 03 / 2021, page. 43 / 114 / 88 amino acids of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the serpin polypeptide and the cytokine-targeting polypeptide are operationally linked via a linker region, for example, a glycine-serine linker or a glycine-serine-based linker. In some embodiments, the serpin polypeptide and the cytokine-targeting polypeptide are operationally linked via a hinge region. In some embodiments, the serpin polypeptide and the cytokine-targeting polypeptide are operationally linked via a linker region and a hinge region. In other embodiments, the serpin polypeptide and the cytokine-targeting polypeptide are directly attached.

[0080] In some embodiments of the fusion proteins provided herein, the second polypeptide (Polypeptide 2) of the serpin fusion protein is a whey acid protein (WAP) domain containing polypeptide, or an amino acid sequence that is derived from a WAP domain containing polypeptide. These embodiments are collectively referred to herein as “serpin-WAP domain fusion proteins.” The serpin-WAP domain fusion proteins described herein include at least one serpin polypeptide or at least one amino acid sequence that is derived from a serpin-containing polypeptide, a WAP domain, or an amino acid sequence that is derived from a WAP domain-containing polypeptide. In some embodiments, the serpin-WAP domain fusion protein includes a single serpin polypeptide. In other embodiments, the serpin-WAP targeting polypeptide fusion protein includes more than one serpin polypeptide.These embodiments are collectively referred to herein as “WAP serpin(a')-domain fusion proteins”, wherein (a') is an integer of at least 2. In some embodiments, serpin polypeptides of the fusion protein. Petition 870210021782, dated 08 / 03 / 2021, page 44 / 114 / 88 serpin(a')-domain WAP include the same amino acid sequence. In other embodiments, serpin polypeptides of the serpin(a')-cytokine targeting polypeptide fusion protein include serpin polypeptides with distinct amino acid sequences.

[0081] These WAP serpine-domain fusion proteins include a WAP domain-containing polypeptide or a polypeptide sequence that is or is derived from a WAP domain-containing polypeptide. The WAP domain is an evolutionarily conserved 50-amino acid sequence motif containing eight cysteines found in a 4-disulfide core arrangement (also called a 4-disulfide core motif). The WAP domain sequence motif is a functional motif characterized by serine protease inhibition activity in a number of proteins.

[0082] Polypeptides containing WAP domains suitable for use in the fusion proteins provided herein include, by way of non-limiting example, secretory leukocyte protease inhibitor (SLPI), Elafin and Epina.

[0083] In some embodiments, the WAP domain-containing polypeptide sequence of the fusion protein includes a secretory leukocyte protease inhibitor (SLPI) polypeptide sequence or an amino acid sequence that is derived from SLPI. These embodiments are referred to herein as “serpina-SLPI-derived fusion proteins”. In some embodiments, the SLPI polypeptide sequence comprises a portion of the SLPI protein, such as, for example, the WAP2 domain or a subportion thereof. In a preferred embodiment, the SLPI polypeptide sequence or an amino acid sequence that is derived from SLPI is or is derived from a human SLPI polypeptide sequence.

[0084] In some embodiments of the serpinaSLPI fusion proteins of the invention, the SLPI sequence or an SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide sequence. Petition 870210021782, dated 08 / 03 / 2021, p. 45 / 114 / 88 of total length which has the following amino acid sequence: MKSSGLFPFL VLLALGTLAP WAVEGSGKSF KAGVCPPKKS AQCLRYKKPE CQSDWQCPGK KRCCPDTCGI KCLDPVDTPN PTRRKPGKCP VTYGQCLMLN PPNFCEMDGQ CKRDLKCCMG 121 MCGKSCVSPV KA (SEQ ID NO:8)

[0085] In some embodiments of the serpinaSLPI fusion protein of the invention, the SLPI sequence or an SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8.

[0086] In some embodiments of the serpinaSLPI fusion protein of the invention, the SLPI sequence or an SLPI-derived sequence of the fusion protein includes a portion of the full-length human SLPI polypeptide sequence, wherein the portion has the following amino acid sequence: SGKSFKAGVC PPKKSAQCLR YKKPECQSDW QCPGKKRCCP DTCGIKCLDP VDTPNPTRRK 61 PGKCPVTYGQ CLMLNPPNFC EMDGQCKRDL KCCMGMCGKS CVSPVKA (SEQ ID NO: 9)

[0087] In some embodiments of the serpinaSLPI fusion protein of the invention, the SLPI sequence or an SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9.

[0088] In some embodiments of the serpinaSLPI fusion protein of the invention, the SLPI sequence or an SLPI-derived sequence of the fusion protein includes the WAP2 domain of the full-length human SLPI polypeptide sequence, wherein the WAP2 domain has the following amino acid sequence: TRRKPGKCPV TYGQCLMLNP PNFCEMDGQC KRDLKCCMGM CGKSCVSPVK A (SEQ ID NO: 10)

[0089] In some embodiments of the serpin fusion protein Petition 870210021782, dated 08 / 03 / 2021, p. 46 / 114 / 88 SLPI of the invention, the SLPI sequence or an SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10.

[0090] In some embodiments of the serpinaSLPI fusion proteins of the invention, the SLPI polypeptide sequence or the amino acid sequence derived from an SLPI polypeptide is, or is derived from, one or more of the human SLPI polypeptide sequences shown in GenBank, accession nos. CAA28187.1, NP_003055.1, EAW75869.1, P03973.2, AAH20708.1, CAB64235.1, CAA28188.1, AAD19661.1, and / or BAG35125.1.

[0091] In some embodiments of the serpinaSLPI fusion proteins of the invention, the SLPI polypeptide sequence or an SLPI-derived sequence of the fusion protein includes a human SLPI polypeptide sequence that is modified at a Methionine (Met) residue. In these Met mutations, the Met residue can be substituted with any amino acid. For example, the Met residue can be substituted with an amino acid with a hydrophobic side chain, such as, for example, leucine (Leu, L) or valine (Val, V). Without wishing to be bound by theory, the Met mutation(s) prevent oxidation and subsequent inactivation of the inhibitory activity of the fusion proteins of the invention. In some embodiments, the Met mutation is at position 98 of an SLPI polypeptide. For example, the modified serpina-SLPI SLPI polypeptide sequence includes M98L or M98V mutations at SEQ ID NO: 8.

[0092] In other embodiments, the WAP domain-containing polypeptide sequence of the fusion protein includes an elafin polypeptide sequence or an amino acid sequence that is derived from elafin. These embodiments are referred to here as “serpin-elafin fusion proteins”. Petition 870210021782, dated 08 / 03 / 2021, page 47 / 114 / 88 In some embodiments, the elafin polypeptide sequence includes a portion of the elafin protein, such as, for example, the WAP domain or a subportion thereof. In a preferred embodiment, the elafin polypeptide sequence or an amino acid sequence that is derived from elafin is or is derived from a human elafin polypeptide sequence.

[0093] In some embodiments of serpinaelafin fusion proteins, the fusion protein includes a full-length human elafin polypeptide sequence that has the following amino acid sequence: 1 MRASSFLIVV VFLIAGTLVL EAAVTGVPVK GQDTVKGRVP FNGQDPVKGQ VSVKGQDKVK 61 AQEPVKGPVS TKPGSCPIIL IRCAMLNPPN RCLKDTDCPG IKKCCEGSCG MACFVPQ (SEQ ID NO: 11)

[0094] In some embodiments of serpinaelafin fusion proteins, the fusion protein includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11.

[0095] In some embodiments of serpinaelafin fusion proteins, the fusion protein includes a portion of the full-length human elafin polypeptide sequence, where the portion has the following amino acid sequence: AVTGVPVKGQ DTVKGRVPFN GQDPVKGQVS VKGQDKVKAQ EPVKGPVSTK PGSCPIILIR 61 CAMLNPPNRC LKDDTCPGIK KCCEGSCGMA CFVPQ (SEQ ID NO: 12)

[0096] In some embodiments of serpinaelafin fusion proteins, the fusion protein includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12.

[0097] In some embodiments of serpinaelaffin fusion proteins, the fusion protein includes the WAP domain of the full-length human elaffin polypeptide sequence, in which the domain of Petition 870210021782, dated 08 / 03 / 2021, p. 48 / 114 / 88 WAP has the following amino acid sequence: VSTKPGSCPI ILIRCAMLNP PNRCLKDTDC PGIKKCCEGS CGMACFVPQ (SEQ ID NO: 13)

[0098] In some embodiments of serpinaelafin fusion proteins, the fusion protein includes a human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13.

[0099] In some embodiments of serpinaelafin fusion proteins, the elafin polypeptide sequence or the amino acid sequence derived from an elafin polypeptide is derived from one or more of the human elafin polypeptide sequences shown in GenBank, accession nos. P19957.3, NP_002629.1, BAA02441.1, EAW75814.1, EAW75813.1, Q8IUB2.1, and / or NP_542181.1.

[00100] In other embodiments, the WAP domain-containing polypeptide sequence of the fusion protein includes an Epin polypeptide sequence or an amino acid sequence that is derived from Epin. These embodiments are referred to herein as “serpin(a')Epin fusion proteins”. In some embodiments, the Epin polypeptide sequence of the serpin-Epin fusion protein includes a portion of the Epin protein, such as, for example, the WAP domain or a subportion thereof. In a preferred embodiment, the Epin polypeptide sequence or an amino acid sequence that is derived from Epin is or is derived from a human Epin polypeptide sequence.

[00101] In some embodiments of serpinaEpina fusion proteins, the Epina polypeptide sequence or amino acid sequence derived from an Epina polypeptide is or is derived from one or more of the human Epina polypeptide sequences shown in GenBank, accession nos. O95925.1, NP_065131.1, AAH44829.2, AAH53369.1, AAG00548.1, AAG00547.1, and / or AAG00546.1. Petition 870210021782, dated 08 / 03 / 2021, page 49 / 114 / 88

[00102] In some embodiments, the serpin polypeptide of the WAP serpin-domain fusion protein includes at least the amino acid sequence of the loop portion of the AAT protein's reactive site. In some embodiments, the loop portion of the AAT protein's reactive site includes at least the amino acid sequence of SEQ ID NO:1. In some embodiments, the serpin polypeptide of the WAP serpin-domain fusion protein includes at least the amino acid sequence of a variant of the loop portion of the AAT protein's reactive site. In some embodiments, the variant of the loop portion of the AAT protein's reactive site includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In some embodiments, the serpin polypeptide of the WAP serpin-domain fusion protein includes at least the full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO:2.In some embodiments, the serpin polypeptide of the serpin-cytokine bleaching fusion protein includes or is derived from at least the full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO: 80. In some embodiments, the serpin polypeptide of the WAP serpin-domain fusion protein includes a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80.

[00103] In some embodiments, the serpin polypeptide of the WAP serpin-domain fusion protein includes the AAT polypeptide sequence, or the amino acid sequence derived from the AAT polypeptide is derived from one or more of the human AAT polypeptide sequences shown in GenBank, accession nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, Petition 870210021782, dated 08 / 03 / 2021, page 50 / 114 / 88 NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, and / or AAA51547.1.

[00104] In some embodiments, the WAP serpinadomain fusion protein may also include an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide. These embodiments are collectively referred to herein as “WAP serpina-Fc-domain fusion proteins.” In these embodiments, no particular order should be interpreted by this terminology. For example, the fusion protein order may be serpina-Fc-WAP domain, serpina-WAP-Fc domain, or any combination of variations thereof. The WAP serpina-Fc-domain fusion proteins described herein include at least one serpina polypeptide or an amino acid sequence that is derived from a serpina-containing polypeptide, a WAP domain or an amino acid sequence that is derived from a WAP-domain-containing polypeptide, and an Fc polypeptide or an amino acid sequence that is derived from an Fc polypeptide.

[00105] In some embodiments, where the WAP serpin-domain fusion protein includes an Fc polypeptide sequence, the Fc polypeptide sequence may have the amino acid sequence of SEQ ID NO: 3-7 and 43-73. In other embodiments, where the WAP serpin-domain fusion protein includes an Fc polypeptide sequence, the Fc polypeptide sequence may be at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 3-7 and 43-73. In some embodiments, the WAP serpin domain fusion protein may also include an albumin polypeptide or an amino acid sequence that is derived from an albumin polypeptide. These embodiments are collectively referred to here as “fusion proteins of Petition 870210021782, dated 08 / 03 / 2021, page 51 / 114 / 88 serpine-albumin-WAP domain”. In these embodiments, no particular order should be interpreted by this terminology. For example, the fusion protein order may be serpine-albumin-WAP domain, serpine-WAP domain-albumin, or any combination of variations thereof. The serpine-albumin-WAP domain fusion proteins described herein include at least one serpine polypeptide or an amino acid sequence derived from a serpine-containing polypeptide, a WAP domain or an amino acid sequence derived from a WAP domain-containing polypeptide, and an albumin polypeptide or an amino acid sequence derived from an albumin polypeptide.

[00106] In some embodiments, where the WAP serpin-domain fusion protein includes an albumin polypeptide sequence, the albumin polypeptide sequence includes the amino acid sequence with SEQ ID NO: 14-15, described herein. In other embodiments, where the WAP serpin-domain fusion protein includes an albumin polypeptide sequence, the albumin polypeptide sequence has at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of the amino acid sequences with SEQ ID NO: 14 or 15.

[00107] In some embodiments, the second polypeptide (Polypeptide 2) of the serpin fusion protein is an albumin polypeptide or is derived from an albumin polypeptide. These embodiments are collectively referred to herein as “serpin(a')albumin fusion proteins”. The serpin-albumin fusion proteins described herein include at least one serpin polypeptide or an amino acid sequence that is derived from a serpin-albumin polypeptide or an amino acid sequence that is derived from an albumin polypeptide. Furthermore, the invention relates to albumin-to-serpin linking polypeptide fusion proteins, wherein albumin is operationally linked to serpin. Petition 870210021782, dated 08 / 03 / 2021, page 52 / 114 / 88 by means of an intermediate linking molecule. Here, serpin is covalently or non-covalently linked to human serum albumin.

[00108] In embodiments where the fusion protein of the invention includes an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein is either human serum albumin (HSA) or an amino acid sequence derived from HSA. In some embodiments, the fusion protein includes an HSA polypeptide sequence that has the following amino acid sequence: DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAEN CDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV MCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPCLD ELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTE CCHGDLLECADDRADLAKYICENQDSISSSKLKECCEKPLLEKSHCIAEVENDEMPADLPSL AADFVESKDVCNKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLLRAKTYETTLEKCCAAADP HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYFQNALLVRYTKKVPQVSTPTLVEVS RNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCF SALEVDETYVPKEFNAETTFFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMD DFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 14)

[00109] In embodiments where the fusion protein of the invention includes an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein includes a human serum albumin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14.

[00110] In embodiments where the fusion protein of the invention includes an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein includes a domain 3 of the human serum albumin polypeptide sequence that has the following amino acid sequence: EEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPE AKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFN Petition 870210021782, dated 08 / 03 / 2021, page 53 / 114 / 88 AETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDK ETCFAEEGKKLVA (SEQ ID NO: 15)

[00111] In embodiments where the fusion protein of the invention includes an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein includes a human serum albumin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 15.

[00112] In some embodiments where the fusion protein of the invention includes an albumin polypeptide sequence, the fusion protein is linked to human serum albumin via an albumin-binding polypeptide intermediate. The albumin-binding polypeptide may be an antibody or an antibody fragment, or derived from an antibody or antibody fragment. In a preferred embodiment, the albumin-binding polypeptide or an amino acid sequence derived from the antibody or antibody fragment is derived from a chimeric, humanized, or fully human antibody sequence. The term antibody includes a single-chain antibody, a Fab fragment, an F(ab')2 fragment, an scFv, an scAb, a dAb, a single-domain heavy chain antibody, and a single-domain light chain antibody. Furthermore, the albumin-binding polypeptide may be an albumin-binding peptide.Another embodiment of the invention is a fusion of albumin-binding polypeptide to serpin, wherein the albumin-binding polypeptide is streptococcal G protein domain 3 or a sequence derived from streptococcal G protein domain 3.

[00113] In some embodiments, the serpin polypeptide of serpin(α')-albumin fusion proteins includes at least the amino acid sequence of the loop portion of the AAT protein's reactive site. In some embodiments, the loop portion of the AAT protein's reactive site includes at least the amino acid sequence of SEQ ID NO:1. In some Petition 870210021782, dated 08 / 03 / 2021, p. 54 / 114 / 88 embodiments, the serpin polypeptide of the serpinaalbumin fusion protein includes at least the amino acid sequence of a variant of the loop portion of the reactive site of the AAT protein. In some embodiments, the variant of the loop portion of the reactive site of the AAT protein includes at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In some embodiments, the serpin polypeptide of the serpina-albumin fusion proteins includes at least the full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO:2. In some embodiments, the serpin polypeptide of the serpina-cytokine targeting fusion protein includes or is derived from at least the full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO:80.In some embodiments, the serpin polypeptide of serpin-albumin fusion proteins includes a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80.

[00114] In some embodiments, the serpin polypeptide of serpin-albumin fusion proteins includes the AAT polypeptide sequence or the amino acid sequence derived from the AAT polypeptide is or is derived from one or more of the human AAT polypeptide sequences shown in GenBank, accession nos. AAB59495.1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1, and / or AAA51547.1.

[00115] In some embodiments, fusion proteins are modified to increase or otherwise inhibit proteolytic cleavage, for example, by mutation of one or more proteolytic cleavage sites. In Petition 870210021782, dated 08 / 03 / 2021, page 55 / 114 / 88 In some embodiments, fusion proteins are modified to alter or otherwise modulate an Fc effector function of the fusion protein, while simultaneously retaining binding and inhibitory function compared to an unaltered fusion protein. Fc effector functions include, but are not limited to, Fc receptor binding, prevention of pro-inflammatory mediator release by Fc receptor binding, phagocytosis, modified antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), modified glycosylation at the Asn297 residue (EU index of Kabat numbering, Kabat et al 1991 Sequences of Proteins of Immunological Interest) of the Fc polypeptide. In some forms, the fusion proteins are mutated or otherwise modified to influence binding to the Fc receptor.In some embodiments, the Fc polypeptide is modified to enhance binding to FcRn. Examples of Fc polypeptide mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E) (numbering by Kabat, Dall'Acqua et al 2006, J. Biol Chem Vol 281(33) 23514-23524), or Met428Leu and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28(2) 157-159). (EU index of Kabat et al 1991 Sequences of Proteins of Immunological Interest) or Met428Val and Asn434Ser (M428V, N434S) using the Kabat numbering system. In some embodiments, the mutated or modified Fc polypeptide includes one or more mutations selected from the group consisting of Met252Tyr (M252Y), Ser254Thr (S256T), Thr256Glu (T256E), Met428Leu (M428L), Met428Val (M428V), Asn434Ser (N434S), and combinations thereof.In some embodiments, the Fc polypeptide portion is mutated or otherwise modified so as to disrupt Fc-mediated dimerization (Ying et al 2012 J. Biol Chem 287(23): 19399-19408). In these embodiments, the fusion protein is monomeric in nature.

[00116] Fusion proteins and their variants are provided here Petition 870210021782, dated 08 / 03 / 2021, page 56 / 114 / 88, exhibit inhibitory activity, for example, by inhibiting a serine protease such as human neutrophil elastase (NE), a chymotrypsin-folded serine protease secreted by neutrophils during an inflammatory response. The fusion proteins provided herein completely or partially reduce, or otherwise modulate, the expression or activity of serine protease by binding to or otherwise interacting with a serine protease, for example, a human serine protease. The reduction or modulation of a serine protease's biological function is complete or partial through interaction between the fusion proteins and the human serine protease protein, polypeptide, and / or peptide.Fusion proteins are considered to completely inhibit serine protease expression or activity when the level of serine protease expression or activity in the presence of the fusion protein is decreased by at least 95%, for example, 96%, 97%, 98%, 99% or 100% compared to the level of serine protease expression or activity in the absence of interaction, for example, binding, with a fusion protein described herein. 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%, for example, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90% compared to the level of serine protease expression or activity in the absence of interaction, for example, binding, with a fusion protein described herein.

[00117] The fusion proteins described herein are useful in a variety of therapeutic, diagnostic, and prophylactic indications. For example, fusion proteins are useful in the treatment of a variety of diseases and disorders in an individual. In some modalities, serpin fusion proteins, including the fusion proteins described herein, are useful in the treatment, symptom relief, improvement, and / or delay of the progression of a disease or disorder in an individual suffering from or identified as having a disease or disorder. Petition 870210021782, dated 08 / 03 / 2021, page 57 / 114 / 88 as being at risk of a disease or disorder selected from alpha-1-antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, lung cancers, ischemia-reperfusion injury, including, for example, ischemia-reperfusion injury after heart transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and / or type II diabetes, bacterial infections, fungal infections, viral infections, pneumonia, sepsis, graft-versus-host disease (GVHD), systemic lupus erythematosus and multiple sclerosis.

[00118] The pharmaceutical compositions according to the invention include a fusion protein of the invention, including modified fusion proteins and other variants, together with a suitable carrier. These pharmaceutical compositions can be included in kits, such as, for example, diagnostic kits. Brief Description of the Drawings

[00119] Figure 1A is a schematic representation of some embodiments of the serpin-Fc fusion proteins according to the invention. The serpin may be located at any position within the fusion protein. Serpin-Fc fusion proteins incorporating more than one serpin polypeptide are also represented. Figure 1B is a photograph of an SDS-PAGE gel showing serum-derived AAT (lane 1), AAT-Fc1 (lane 2, human IgG1 Fc) and IgG1 Fc (lane 3, Met351Glu, Met358Leu mutations within AAT, human IgG1 Fc). Figure 1C is a graph showing the inhibition of neutrophil elastase activity by AAT-Fc fusion proteins. Figure 1D is a photograph of an SDS-PAGE gel showing tetravalent AAT-Fc-AAT, with two AAT polypeptides per Fc polypeptide. Figure 1E is a graph showing the inhibition of neutrophil elastase activity by an AAT-Fc-AAT fusion protein. Petition 870210021782, dated 08 / 03 / 2021, page 58 / 114 / 88 tetravalent. Figure 1F is a graph demonstrating the effect of low pH elution of protein A resin, where the NE inhibition capacity of low pH eluted AAT-Fc fusion protein is dramatically reduced. Figure 1G is a graph showing that the double mutant AAT-EL-Fc (Met351Glu, Met358Leu mutations) is resistant to H2O2 (conc.) inactivation, compared to wild-type AAT and the single mutant AAT-EM-Fc (Met351Glu). Figure 1H is a graph representing serum clearance rates of serum-derived AAT (sdAAT) compared to AAT-Fc in rats dosed with 10 mg / kg of protein (3 rats / test protein). The half-life of AAT-Fc is substantially longer than that of sdAAT.

[00120] Figure 2A is a schematic representation of some embodiments of the serpin-cytokine targeting fusion proteins of the invention. The serpin can be fused to the heavy chain, the light chain, or both of an antibody. The serpin-cytokine receptor fusion proteins are also shown. Figure 2B is a photograph of an SDS-PAGE gel showing the D2E7 antibody (lane 1) and the D2E7 antibody with AAT fused to the heavy chain (lane 2). Figure 2C is a graph showing the inhibition of neutrophil elastase activity by a D2E7 antibody fused to AAT. Serum-derived AAT is shown as a positive control, while the D2E7 antibody alone is shown as a negative control for NE inhibition.

[00121] Figure 3A is a schematic representation of some embodiments of serpin-Fc-WAP fusion proteins. Figure 3B is a photograph of an SDS-PAGE gel showing AAT-Fc-ELAFIN (lane 1) and AAT-Fc-SLPI (lane 2). Figure 3C is a graph showing the inhibition of neutrophil elastase activity by an AAT-Fc-ELAFIN fusion protein and an AAT-Fc-SLPI fusion protein. A serum-derived AAT-Fc and AAT fusion protein are included for comparison.

[00122] Figure 4A is a schematic representation of some Petition 870210021782, dated 08 / 03 / 2021, page 59 / 114 / 88 modalities of AAT-HSA fusion proteins. Figure 4B is a photograph of an SDS-PAGE gel showing an AAT-HSA fusion. Figure 4C is a graph showing the inhibition of neutrophil elastase activity by an AAT-HSA compared to serum-derived AAT. Detailed Description of the Invention

[00123] Human neutrophil elastase (NE) is a chymotrypsin-folded serine protease secreted by neutrophils during inflammation. Aberrant NE activity results in progressive degradation of elastin tissues and slow destruction of alveolar lung structures, leading to emphysema and pulmonary fibrosis (Lungarella et al 2008 Int. J. Biochem Cell Biol 40:1287). Often, the erroneous NE activity is due to an imbalance of the protease with its natural inhibitor, alpha1-antitrypsin (AAT). This imbalance can result from intensified neutrophil infiltration into the lungs, as observed in the lungs of smokers and patients with Cystic Fibrosis (CF) or Acute Respiratory Distress Syndrome (ARDS). Conversely, an AAT deficiency, usually resulting from a point mutation that causes ATT to aggregate and accumulate in the liver, leaves the lungs exposed to uncontrolled NE activity.Individuals with AAT deficiencies are at increased risk of emphysema, COPD, liver disease, and numerous other conditions.

[00124] AAT deficiency affects approximately 100,000 Americans (according to estimates from the Alpha-1 Foundation), and many of those affected die between the ages of 30 and 40. There are currently only a few FDA-approved medications for the treatment of AAT deficiency (Prolastin®, Aralast™, Zemaira®, Glassia™). Each drug is pooled natural AAT derived from human plasma, which appears insufficient to meet anticipated clinical demand. Furthermore, these products have short serum half-lives (T1 / 2 of approximately 5 days) and require weekly high-dose infusions (60 mg / kg body weight). The Petition 870210021782, dated 08 / 03 / 2021, page 60 / 114 / 88 The current market for these drugs is estimated at approximately US$400 million. The market for AAT-type drugs is likely substantially larger, based on the estimate that up to 95% of individuals with AAT deficiencies are undiagnosed, and the fact that these drugs have the potential to be effective therapies for pathologies characterized by enhanced NE activity (e.g., cystic fibrosis (CF), acute respiratory distress syndrome (ARDS), smoking-induced emphysema and / or COPD).

[00125] It has been suggested that AAT has broad-spectrum anti-inflammatory activity (Tilg et al 1993 J Exp Med 178:1629-1636, Libert et al 1996 Immunol 157:5126-5129, Pott et al, Journal of Leukocyte Biology 85 2009, Janciauskiene et al 2007 J. Biol Chem 282(12): 8573-8582, Nita et al 2007 Int J Biochem Cell Biol 39:1165-1176). Recently, evidence shows that AAT may be useful in the treatment of numerous human pathologies, beyond the commonly suggested inflammatory pulmonary conditions. Human AAT has been shown to protect mice from clinical and histopathological signs of experimental autoimmune encephalomyelitis (EAE), suggesting that it could be a potential treatment for autoimmune diseases such as multiple sclerosis or systemic lupus erythematosus (Subramanian et al 2011 Metab Brain Dis 26:107-113). Serum AAT has shown activity in rodent models of Graft-Versus-Host Disease (GVHD) (Tawara et al 2011 Proc. Natl. Acad. Sci.USA 109: 564-569, Marcondes et al 2011 Blood Nov 3;118(18):5031-9), which led to a human clinical trial using AAT to treat individuals with acute steroid-unresponsive GVHD (NCT01523821). Additionally, AAT has been effective in animal models of type I and type II diabetes, attenuating inflammation, protecting islet cells from apoptosis, and enabling durable islet cell allograft (Zhang et al 2007 Diabetes 56:1316-1323, Lewis et al 2005 Proc Natl Acad Sci USA 102:12153-12158, Lewis et al 2008 Proc Natl Acad Sci). Petition 870210021782, dated 08 / 03 / 2021, p. 61 / 114 / 88 EUA 105: 16236-16241, Kalis et al 2010 Islets 2: 185-189). Currently, there are numerous early human clinical trials of type I diabetes using serum-derived AAT products (NCT01183468, NCT01319331, NCT01304537).

[00126] Current serum-derived AAT products undergo extensive purification and testing to ensure the removal of pathogenic viruses; however, the risk of transmission of infectious agents cannot be completely eliminated. Furthermore, serum is limited, which restricts the production capacity of serum-derived AAT. Attempts to address concerns about serum-derived products and production issues have been directed towards the expression of recombinant AAT. However, after 20 years of work, the generation of a therapeutically viable recombinant AAT has not yet reached the market (Karnaukhova et al 2006 Amino Acids 30: 317). Like plasma-derived products, recombinant versions of AAT suffer from short serum half-lives, low production yields, and poor pulmonary distribution.

[00127] The fusion proteins of the present invention have enhanced functionalities compared to the unmodified AAT molecule. The fusion of an AAT polypeptide with a second polypeptide that interacts with the neonatal Fc receptor (FcRn) serves to increase the serum half-life, providing a much-needed dosing benefit for patients. These FcRn-interacting polypeptides of the fusion protein include immunoglobulin (Ig) Fc polypeptides derived from human IgG1, IgG2, IgG3, IgG4, or IgM and derived from human albumin. In some embodiments, the fusion protein incorporates mutations with the AAT moiety that make the molecule more resistant to oxidative inactivation. For example, Met351Glu, Met358Leu (AAT-EL-Fc) demonstrates resistance to oxidative inactivation by H2O2 (Figure 1G). Although AAT is a natural anti-inflammatory protein, some embodiments of the invention Petition 870210021782, dated 08 / 03 / 2021, page 62 / 114 / 88 provides enhanced inflammation attenuation capability through the fusion of an AAT polypeptide and a cytokine-targeting polypeptide. The coupling of dual anti-inflammatory functionalities from AAT and a second polypeptide will provide a more potent therapeutic protein than either polypeptide alone. Additionally, the coupling of the anti-infective activity of AAT will attenuate the risk of infection from most cytokine biological products. Some embodiments provide more potent anti-inflammatory and anti-infective proteins through the fusion of an AAT polypeptide and a polypeptide containing a WAP domain. The fusion proteins of the present invention are expected to be of great therapeutic utility and to be superior to current serum-derived AAT products.

[00128] To prolong the half-life of recombinant AAT, recombinant DNA technology was used to create a fusion of the AAT gene with the Fc domain of human IgG1, IgG2, IgG3, IgG4, IgM, or HSA, such that the expected protein product would be AAT followed by an Fc domain (AATFc (IgG1), AAT-Fc (IgG2), AAT-Fc (IgG3), AAT-Fc (IgG4), AAT-Fc (IgM), or AAT followed by HSA. (2009) BioDrugs 23, 93-109, Schmidt et al. (2009) Curr Opin Drug Discov Devel 12, 284-295), it was not known whether an Fc domain or HSA fused with AAT would allow for adequate folding and maintenance of NE inhibitory activity, or could prolong the half-life of recombinant AAT.The fusion proteins of the present invention are shown to be potent inhibitors of NE, have prolonged serum half-lives, and, in some embodiments, are resistant to oxidation. In other embodiments, the fusion proteins described herein have distinct properties through the incorporation of others. Petition 870210021782, dated 08 / 03 / 2021, page 63 / 114 / 88 functional polypeptides, including cytokine-targeting polypeptides and polypeptides containing the WAP domain.

[00129] Neutrophils, the primary source of neutrophil elastase (NE), frequently undergo an oxidative burst simultaneously with NE secretion. Therefore, it is of great therapeutic utility that the fusion proteins of the present invention are active in an oxidizing environment. Oxidation of AAT within the reactive site loop at Met351 and / or Met358 attenuates the ability of AAT to inhibit neutrophil elastase. As illustrated in Figure 1G, oxidative inactivation can be reduced through the Met351Glu and Met358Leu (M351E / M358L) mutations shown in SEQ ID NO: 32. Furthermore, oxidation of an Fc region at Met252 and Met428 has been shown to reduce FcRn binding and subsequently reduce the serum half-life of the Fc-containing protein. The Met252 and Met428 mutation reduces the oxidation of the Fc region.The present invention describes and optimizes the AAT-Fc fusion protein, which is resistant to oxidation-inactivation within the AAT portion of the fusion protein through mutations in M351 and M358; and oxidative disruption of the FcRn interaction through mutations in M252 and M428, and has a prolonged half-life through the adjusted mutation of M252I, T256D, and M428L.

[00130] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide is mutated or modified to enhance binding to FcRn. In these embodiments, the mutated or modified Fc polypeptide includes the following mutations: Met252Ile, Thr256Asp, and Met428Leu (M252I, T256D, M428L) using the Kabat numbering system.

[00131] In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide is a modified IgG1 Fc polypeptide, and the fusion protein includes at least the amino acid sequence of SEQ ID NO: 53. Petition 870210021782, dated 08 / 03 / 2021, p. 64 / 114 / 88

[00132] In some embodiments where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide is a modified IgG1 Fc polypeptide, and the fusion protein includes at least the amino acid sequence of SEQ ID NO: 73.

[00133] In some embodiments, where the fusion protein of the invention includes an Fc polypeptide, the Fc polypeptide is mutated or modified to reduce binding to Fc-gamma receptors (FcgRs). In some embodiments, reduced FcgR binding can be achieved by Fc glycosylation modification at Asn297. For example, mutation of Asn297Ala (N297A) or Asn297Gln (N297Q). In some embodiments, reduced FcgR binding is achieved by modification of the lower hinge region of Fc. In some embodiments, the Fc polypeptide is derived from human IgG1. In some of these variants, the lower hinge region is modified to mimic that of IgG2, through mutation of Leu234Val and Leu235Ala (L235V / L235A) and deletion of Gly236 (AG236) using the Kabat numbering system: IgG1-wt hinge: DKTHTCPPCPAPELlGgPS (SEQ ID NO: 74) IgG1-VAAG Hinge: DKTHTCPPCPAPE^A-GPS (SEQ ID NO: 75)

[00134] In some of these embodiments, the fusion protein includes at least the amino acid sequence of SEQ ID NO:1. 51.

[00135] In some embodiments, the Fc polypeptide is derived from human IgG4. In some of these embodiments, the lower hinge region is modified by mutation in Leu235Glu (L235E). Furthermore, in embodiments of the present invention where the Fc polypeptide is derived from IgG4, the hinge region is modified by the Ser228Pro (S228P) stabilization mutation using the Kabat numbering system: IgG4-wt hinge: ESKYGPPCPSCPAPEFLGGPS (SEQ ID NO: 76) Petition 870210021782, dated 08 / 03 / 2021, p. 65 / 114 / 88 IgG4-PE Hinge: ESKYGPPCP|CPAPEF|GGPS (SEQ ID NO: 77)

[00136] In some of these embodiments, the fusion protein includes at least the amino acid sequence of SEQ ID NO:1. 70.

[00137] The fusion proteins described herein include at least one serpin polypeptide or an amino acid sequence that is derived from a serpin and a second polypeptide. In some embodiments, for example, the invention provides a serpin polypeptide fused to derivatives of human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or HSA.The serpin fusion described here is expected to be useful in treating a variety of indications, including, but not limited to, alpha-1-antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, lung cancers, ischemia-reperfusion injury, including, for example, ischemia-reperfusion injury after heart transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and / or type II diabetes, bacterial infections, fungal infections, viral infections, pneumonia, sepsis, graft-versus-host disease (GVHD), systemic lupus erythematosus, and multiple sclerosis.

[00138] In some embodiments, the fusion proteins described herein include at least one alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a second polypeptide. For example, the invention provides alpha-1-antitrypsin (AAT) fused to derivatives of IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or human HSA.

[00139] In some embodiments, the serpin-Fc fusion proteins described herein include at least one serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide and a cytokine-targeting polypeptide or an amino acid sequence that is derived from a cytokine-targeting polypeptide. For example, the Petition 870210021782, dated 08 / 03 / 2021, page 66 / 114 / 88 invention provides serpin polypeptide or a sequence derived from a serpin polypeptide fused to a human cytokine receptor or a derivative thereof. Another embodiment of the invention provides serpin polypeptide or a sequence derived from a serpin polypeptide fused to a cytokine targeting antibody, for example, an anti-cytokine antibody, or a sequence derived from a cytokine targeting antibody, for example, an anti-cytokine antibody, or a sequence derived from a fragment of a cytokine targeting antibody, for example, a fragment of an anti-cytokine antibody.For example, the invention provides a serpin polypeptide or a sequence derived from a serpin polypeptide fused to a cytokine-targeting polypeptide wherein the cytokine-targeting polypeptide binds to any of the following human cytokines: TNFα, IgE, IL-12, IL-23, IL-6, IL-1α, IL-ίβ, IL-17, IL-13, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

[00140] For example, in some embodiments, the cytokine-targeting polypeptide targets TNFα and includes any of the following TNFα-targeting polypeptides or sequences derived from the following TNFα-targeting polypeptides: Remicade®, Humira®, Simponi®, Cimiza®, Enbrel®, or ATN-103 and ATN-192.

[00141] For example, in some embodiments, the cytokine-targeting polypeptide targets IgE and includes any of the following IgE-targeting polypeptides or sequences derived from the following IgE-targeting polypeptides: Xolair or FcεRI.

[00142] For example, in some embodiments, the cytokine-targeting polypeptide targets the shared p40 subunit of IL-12 and IL-23 and includes the Stelara® polypeptide or sequences derived from the Stelara® polypeptide.

[00143] For example, the Stelara® cytokine-targeting polypeptide targets IL-13 and includes the CDP7766 polypeptide or sequences Petition 870210021782, dated 08 / 03 / 2021, p. 67 / 114 / 88 derived from the CDP7766 polypeptide.

[00144] In some embodiments, the serpin-Fc fusion proteins described herein include at least one alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and a cytokine-targeting polypeptide or an amino acid sequence that is derived from a cytokine-targeting polypeptide. For example, the invention provides an alpha-1-antitrypsin (AAT) inhibitor fused to a cytokine-targeting polypeptide wherein the cytokine-targeting polypeptide binds to any of the following human cytokines: TNFα, IgE, IL-6, IL-1α, IL-1-1β, IL-12, IL-17, IL-13, IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

[00145] In some embodiments, the cytokine-targeting polypeptide binds to a cytokine receptor and prevents cytokine binding. For example, the present invention includes a serpin fused to a cytokine receptor-targeting antibody. For example, the invention provides an alpha-1-antitrypsin (AAT) inhibitor fused to a cytokine-targeting polypeptide wherein the cytokine-targeting polypeptide binds to the receptor of any of the following human cytokines: TNFα, IgE, IL-6, IL-1α, IL-13, 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.

[00146] For example, in some embodiments, the cytokine-targeting polypeptide targets the IL-6 receptor and includes the Actemra® polypeptide (as described in patent publication EP0628639), or the ALX-0061 polypeptide (as described in WO2010 / 115998), or sequences derived from the Actemra® polypeptide, or the ALX-0061 polypeptide.

[00147] For example, the cytokine-targeting polypeptide Actemra® targets the IL-6 receptor and includes the tocilizumab polypeptide or sequences derived from the tocilizumab polypeptide.

[00148] The targeting of inflammatory cytokines and agents Petition 870210021782, dated 08 / 03 / 2021, page 68 / 114 / 88 Immunostimulant protein therapies have demonstrated clinical success in numerous inflammatory conditions. The most common proteins used as cytokine targeting agents are soluble cytokine receptors and monoclonal antibodies and fragments thereof. A significant disadvantage of targeting cytokines is the increased risk of infection in these patients, as evidenced by the TNFα-targeting biologics Remicade®, Humira®, Simponi®, Cimiza®, and Enbrel®, and the IL-12 / 23 p40-targeting antibody Stelara®. This is likely a common problem with targeting inflammatory cytokines that leads to immune suppression in patients. AAT and other serpin proteins are interesting insofar as they demonstrate both anti-infective and anti-inflammatory activities.Thus, the serpin-cytokine bleaching polypeptide fusion proteins of this invention can attenuate aberrant cytokine activities while alleviating the risk of infections.

[00149] In some embodiments, the WAP serpin-Fcdomain fusion proteins described herein include a serpin polypeptide or an amino acid sequence derived from a serpin-containing polypeptide, a WAP domain or an amino acid sequence derived from a WAP domain-containing polypeptide, and an Fc polypeptide or an amino acid sequence derived from an Fc polypeptide. For example, the invention provides a serpin polypeptide, a WAP domain-containing polypeptide, and derivatives of human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, or IgM-Fc operationally linked in any functional combination. In some embodiments, the WAP domain-containing protein is human SLPI or derived from human SLPI. In other embodiments, the WAP domain-containing protein is human ELAFFIN or derived from human ELAFFIN.In some embodiments, the serpin-Fc fusion proteins described herein include at least one alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from it. Petition 870210021782, dated 08 / 03 / 2021, page 69 / 114 / 88 AAT and an SLPI polypeptide or an amino acid sequence that is derived from an SLPI polypeptide. In some embodiments, the serpin-Fc fusion proteins described herein include at least one AAT polypeptide or an amino acid sequence that is derived from AAT and an ELAFIN polypeptide or an amino acid sequence that is derived from an Elafin polypeptide.

[00150] SLPI and Elafin are proteins containing a WAP domain that exhibit serine protease inhibitory activity. Both proteins have an anti-inflammatory function. In addition, these proteins possess broad anti-infective capabilities against numerous strains of bacteria, viruses, and fungi.

[00151] In some embodiments, the serpin-Fc fusion proteins described herein include at least one serpin polypeptide or an amino acid sequence that is derived from a serpin polypeptide and human serum albumin (HSA), or an amino acid sequence that is derived from an HSA polypeptide. Additional embodiments of the invention include albumin-binding serpin polypeptide fusion proteins, wherein the albumin-binding polypeptide is responsible for the association of serpin and HSA. Thus, the invention includes covalent and non-covalent linkages of the serpin polypeptide and the HSA polypeptide, or sequences derived from the serpin polypeptide or an HSA polypeptide. For example, the invention provides a serpin polypeptide fused with human HSA, or HSA derivatives, or HSA-binding peptide or polypeptides.

[00152] In some embodiments, the serpin-Fc fusion proteins described herein include at least one alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequence that is derived from AAT and an HSA polypeptide or an amino acid sequence that is derived from an HSA polypeptide. For example, the invention provides alpha-1-antitrypsin Petition 870210021782, dated 08 / 03 / 2021, p. 70 / 114 / 88 (AAT) fused to HSA or an HSA-derived fragment, or an albumin-binding polypeptide.

[00153] In some embodiments, the fusion proteins described herein include a serpin polypeptide or an amino acid sequence derived from a serpin-containing polypeptide, HSA or an amino acid sequence derived from an HSA polypeptide, and a WAP domain-containing polypeptide, or an amino acid sequence derived from a WAP domain-containing polypeptide. In some embodiments, the Fc-serpin fusion proteins described herein include at least one alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequence derived from AAT and an HSA polypeptide, or an amino acid sequence derived from an HSA polypeptide and an SLPI polypeptide or an amino acid sequence derived from SLPI.In some embodiments, the serpin-Fc fusion proteins described herein include at least one alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequence derived from AAT and an HSA polypeptide, or an amino acid sequence derived from an HSA polypeptide and an Elafin polypeptide, or an amino acid sequence derived from Elafin.

[00154] The fusion proteins of the present invention can be readily produced in mammalian cell expression systems. For example, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, COS cells, PER.C6® cells, NS0 cells, SP2 / 0 cells, YB2 / 0 cells can be readily used for the expression of the serpin fusion proteins described herein. Importantly, mammalian cell expression systems produce proteins that are generally more ideal for therapeutic use. In contrast to bacterial, insect, or yeast expression systems, mammalian cell expression systems produce proteins with glycosylation patterns that are similar to or the same as those found in natural human proteins. The appropriate glycosylation of Petition 870210021782, dated 08 / 03 / 2021, page 71 / 114 / 88 a protein can greatly influence serum stability, pharmacokinetics, biodistribution, protein folding, and functionality. Therefore, the ability to produce therapeutic proteins in mammalian expression systems has distinct advantages over other systems. Furthermore, most mammalian cell expression systems (e.g., CHO, NS0, PER.C6® cells) can be easily scaled up in commercial manufacturing facilities to produce therapeutic proteins to meet clinical demands. The fusion proteins described herein have enhanced functionalities over the natural form of AAT and can be produced in mammalian expression systems for clinical and commercial supply. Some embodiments of the invention include a purification system that allows the isolation of serpin fusion proteins that retain their ability to inhibit NE.Importantly, the purification process of the present invention can be easily incorporated into current commercial mammalian cell manufacturing processes.

[00155] Unless otherwise defined, the scientific and technical terms used in connection with the present invention shall have the meanings that are normally understood by those skilled in the art. Furthermore, unless required by the context, singular terms shall include plurals and plural terms shall include the singular. Generally, the nomenclatures used in connection with the cell and tissue culture, molecular biology, and oligo- or polynucleotide and protein chemistry and hybridization techniques described herein are those well known and commonly used in the art. Standard techniques for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection) are used. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications or as normally performed in the art or as described herein.The techniques and procedures described above are generally performed in accordance with conventional methods well known in the art. Petition 870210021782, dated 08 / 03 / 2021, pp. 72 / 114 / 88 and as described in various general and more specific references that are cited and discussed throughout this description. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclatures used in connection with the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry laboratory procedures and techniques described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and patient treatment. The term patient includes human and veterinary individuals.

[00156] It will be recognized that the administration of therapeutic entities according to the invention will be administered with suitable carriers, buffers, excipients and other agents that are incorporated into formulations to provide improved transfer, dispensing, tolerance and other similar effects. A variety of suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbocera emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbocera.Any of the above mixtures may be appropriate in treatments and therapies according to the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient. Petition 870210021782, dated 08 / 03 / 2021, pp. 73 / 114 / 88 development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients, and carriers well known to pharmaceutical chemists.

[00157] The therapeutic formulations of the invention, which include a fusion protein of the invention, are used to treat or alleviate a symptom associated with a disease or disorder associated with aberrant serine protease activity in an individual. The present invention also provides methods for treating or alleviating a symptom associated with a disease or disorder associated with aberrant serine protease activity in an individual. A therapeutic regimen is carried out by identifying an individual, for example, a human patient suffering from (or at risk of developing) a disease or disorder associated with aberrant serine protease activity, using conventional methods, including any of a variety of clinical pathologies and / or laboratory procedures. The term patient includes human and veterinary individuals. The term subject includes humans and other mammals.

[00158] The effectiveness of the treatment is determined in association with any known method for diagnosing or treating the particular disease or disorder associated with aberrant serine protease activity. Relief 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.

[00159] Methods for screening fusion proteins that possess the desired specificity include, but are not limited to, assays for Petition 870210021782, dated 08 / 03 / 2021, page 74 / 114 / 88 enzyme-linked immunosorbent assay (ELISA), enzyme assays, flow cytometry and other immunologically mediated techniques known in the art.

[00160] The fusion proteins described herein may be used in methods known in the art relating to the localization and / or quantification of a target such as a serine protease, for example, for use in measuring levels of such targets within appropriate physiological samples, for use in diagnostic methods, for use in protein imaging, and the like. The terms “physiological sample” and “biological sample,” used interchangeably in this document, are intended to include tissues, cells, and biological fluids isolated from an individual, as well as tissues, cells, and fluids present in an individual. Included in the use of the terms “physiological sample” and “biological sample,” therefore, are blood and a fraction or component of blood including blood serum, blood plasma, or lymph.

[00161] In a given embodiment, fusion proteins specific to a given target, or a derivative, fragment, analogue or homolog thereof, containing the target-binding domain, are used as pharmacologically active compounds (referred to hereafter as “Therapeutics”).

[00162] A fusion protein of the invention can be used to isolate a particular target using standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Detection can be facilitated by coupling (i.e., physically linking) the fusion protein to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine Petition 870210021782, dated 08 / 03 / 2021, page 75 / 114 / 88 fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin, and examples of suitable radioactive material include 1251, 1311, 35S or 3H.

[00163] A therapeutically effective amount of a fusion protein of the invention generally refers to the amount required to achieve a therapeutic objective. As noted above, this may be a binding interaction between the fusion protein and its target which, in certain cases, interferes with the target's function. The amount required to be administered will further 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 of the other individual to whom it is administered. Common ranges for the therapeutically effective dosage of a fusion protein or a fragment thereof may be, by way of non-limiting example, from about 0.1 mg / kg body weight to about 250 mg / kg body weight. Common dosing frequencies may vary, for example, from twice daily to once monthly.

[00164] When fusion protein fragments are used, the smallest inhibitory fragment that binds specifically to the target is preferred. For example, peptide molecules that retain the ability to bind to the target can be designed. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation may also contain more than one active compound, as needed for the particular indication to be treated, preferably those with complementary activities that do not adversely affect each other. Alternatively or additionally, the composition may comprise an agent that enhances its function, such as, for example, an agent Petition 870210021782, dated 08 / 03 / 2021, page 76 / 114 / 88 cytotoxic, cytokine, chemotherapeutic agent, growth inhibitor, an anti-inflammatory or anti-infective agent. Such molecules are appropriately present in combination in quantities that are effective for the intended purpose.

[00165] Active ingredients can also be captured in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.

[00166] Formulations to be used for in vivo administration must be sterile. This is easily achieved by filtration through sterile filtration membranes.

[00167] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the fusion protein, the matrices of which are in the form of shaped articles, for example, films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US Pat. 3,773,919), L-glutamic acid and γ-ethyl-L-glutamate copolymers, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.Although polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time. Petition 870210021782, dated 08 / 03 / 2021, p. 77 / 114 / 88 Pharmaceutical compositions

[00168] The fusion proteins of the invention (also referred to herein as “active compounds”), and derivatives, fragments, analogues and homologues thereof, may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the fusion protein and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersing media, coatings, antibacterial and antifungal agents, isotonic and absorption retardant 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 solution, Ringer's solutions, dextrose solution and 5% human serum albumin.Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except to the extent that any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds may also be incorporated into the compositions.

[00169] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, for example, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other solvents. Petition 870210021782, dated 08 / 03 / 2021, page 78 / 114 / 88 synthetic; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

[00170] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (when water-soluble) or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline solution, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to allow easy syringe application. It must be stable under manufacturing and storage conditions and must be protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, and the like) and suitable mixtures thereof.Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions may be... Petition 870210021782, dated 08 / 03 / 2021, pp. 79 / 114 / 88 achieved through the inclusion in the composition of an agent that retards absorption, for example, aluminum monostearate and gelatin.

[00171] Sterile injectable solutions can be prepared by incorporating the active compound in the required quantity into an appropriate solvent with one or a combination of the ingredients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and lyophilization, which yields a powder of the active ingredient plus any desired additional ingredient from a previously filtered sterile solution thereof.

[00172] Oral compositions generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or tablets. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and agitated and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition.Tablets, pills, capsules, lozenges and similar products may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, tragacanth gum or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a slider such as colloidal silicon dioxide; an agent. Petition 870210021782, dated 08 / 03 / 2021, p. 80 / 114 / 88 sweetener such as sucrose or saccharin; or a flavoring agent such as mint, methyl salicylate or orange flavor.

[00173] For administration by inhalation, the compounds are administered in the form of an aerosol from a pressurized container or dispenser containing a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.

[00174] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated in ointments, balms, gels, or creams, as is generally known in the art.

[00175] The compounds can also be prepared in the form of suppositories (for example, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal administration.

[00176] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled-release formulation, including microencapsulated implants and dispensing systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. The methods for preparing such formulations will be evident to those skilled in the art. The materials may also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions may also be used as pharmaceutical carriers. Petition 870210021782, dated 08 / 03 / 2021, page 81 / 114 / 88 acceptable. These can be prepared according to methods known to those skilled in the art, for example, as described in US Patent No. 4,522,811.

[00177] It is especially advantageous to formulate oral or parenteral formulations in unit dosage form for ease of administration and dosage uniformity. The unit dosage form as used herein refers to physically discrete units suitable as unit dosages for the individual to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. The specification for the unit dosage forms of the invention is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the inherent limitations in the technique of compounding such active compound for the treatment of individuals.

[00178] Pharmaceutical compositions may be included in a container, package or dispenser along with instructions for administration.

[00179] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES Example 1: AAT-Fc fusion proteins and variants

[00180] Exemplary, but not limiting, examples of AAT-Fc fusion proteins according to the invention include the following sequences. Although these examples include a hinge sequence and / or a linker sequence, the fusion proteins of the invention can be made using any hinge sequence and / or a linker sequence suitable in length and / or flexibility. Alternatively, the fusion proteins can be made without using a hinge and / or linker sequence. For example, polypeptide components can be directly attached. Petition 870210021782, dated 08 / 03 / 2021, p. 82 / 114 / 88

[00181] An exemplary AAT-Fc fusion protein is AAThFcl (human IgG1 Fc) described herein. As shown below, an AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2) and the IgG-Fc polypeptide portion of the fusion protein is in italics (SEQ ID NO: 3). AAT-hFc1 (Fc of human IgG1) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO:16)

[00182] An exemplary AAT-Fc fusion protein is AAThFc2 (human IgG2 Fc) described herein. As shown below, an AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2) and the IgG-Fc polypeptide portion of the fusion protein is in italics (SEQ ID NO: 4). AAT-hFc2 (Fc of human IgG2) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM ISR TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TFRVVSVL TVVHQDW LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP Petition 870210021782, dated 08 / 03 / 2021, pages 83 / 114 / 88 SDIAVEWESNGQPENNYKTTPPMLDSDGSFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO: 17)

[00183] An exemplary AAT-Fc fusion protein is AATMM-EL-hFcl (human IgG1 Fc, Met351Glu / Met358Leu) described herein. As shown below, an AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 34), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 3), and the Met351Glu mutation is boxed, and the Met358Leu mutation is shaded in gray. AAT-MM-EL-hFc1(human IgG1 Fc, Met351Glu / Met358Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELPRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ lgitkvfsngadlsgvteeaplklskavhkavltidekgteaagaEfleaiplsippevk FNKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK (SEQ ID NO: 18)

[00184] An exemplary AAT-Fc fusion protein is AATMM-EL-hFc2 (human IgG2 Fc, Met351Glu / Met358Leu) described herein. As shown below, an AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 34), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 4), the Met351Glu mutation is boxed, and the Met358Leu mutation is shaded gray. AAT-MM-EL-hFc2(Human IgG2 Fc, Met351Glu / Met358Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKApTHpEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPpSQLQLTTGGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ Petition 870210021782, dated 08 / 03 / 2021, p. 84 / 114 / 88 lgitkvfsngadlsgvteeaplklskavhkavltidekgteaagaEfleaiplsippevk FNKPFVFLMIEQNTKSPLFMGKVVNPTQKERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQD WLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPMLDSDGSFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK (SEQ ID NO: 19)

[00185] An exemplary AAT-Fc fusion protein is AATMM-LL-hFcl (human IgG1 Fc, Met351Glu / Met358Leu) described herein. As shown below, an AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 35), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 3), the Met351Glu mutation is shaded in black, and the Met358Leu mutation is shaded in gray. AAT-MM-LL-hFc1(human IgG1 Fc, Met351Leu / Met358Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AmlslgtkadthdeileglnfNlteipeaqihegeqellrtlnqpdsqlqlttgnglelse GLKLyDKELEDyKKLYHSEAETyNEGDTEEAKKQINDYyEKGTQGKiyDLyKELDRDTyFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVESNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGALFLEAIPLSIPPEyKF NKPFyFLMIEQNTKSPLFMGKVVNPZQKEPKScdkthtcppcpapellggpsvflfppkpk DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK(SEQ ID NO:36)

[00186] An exemplary AAT-Fc fusion protein is AATMM:LL-hFc2 (human IgG2 Fc, Met351Glu / Met358Leu) described herein. As shown below, an AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 35), the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 4), the Met351Glu mutation is shaded in black, and the Met358Leu mutation is shaded in gray. AAT-MM:LL-hFc2 (Human IgG2 Fc, Met351Leu / Met358Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF Petition 870210021782, dated 08 / 03 / 2021, pp. 85 / 114 / 88 AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGALFLEAIPLSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLM ISR TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TFRVVSVL TVVHQDW LNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPMLDSDGSFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK (SEQ ID NO:20)

[00187] An exemplary AAT-Fc fusion protein is AAThFcl-AAT (human IgG1) described herein. As shown below, an AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), the IgG-Fc polypeptide portion of the fusion protein is in italics (SEQ ID NO: 3). AAT-hFcl-AAT (human IgG1 Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKASTGS EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQ LAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLR TLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVE KGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKR LGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSA SLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEWKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:21) Petition 870210021782, of 08 / 03 / 2021, p. 86 / 114 / 88 AAT-EL-Fc-IgG1-DV^G,IDL (AAT: Met351Glu / Met358Leu; Fc IgG1: Leu234Val / Leu235Ala, Gly236 deleted, Met252Ile, Thr256Asp, Met428Leu) EDPQGDAAQKTDTSHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ lgitkvfsngadlsgvteeaplklskavkavltidekgteaaga|e|fleaip|l|sippevk fnkpfvflmieqntxplfmgkvnptqgggggdkppcppcpcppc dtl[i]isr|D|pevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltv LHQDWLNGKEYKCKVSNCALPAPIECTISCAKGQPREPQVYTLPPSRDELTKNQVSLTCLV kgfypsdiavewesngqpennycttppvldsdgsfflyskltvdxrwqqgnvfscsvLh EALHNHYTQKSLSLSPGK (SEQ ID NO: 78) AAT-EL-Fc-IgG4-PE,IDL (AAT: Met351Glu / Met358Leu; Fc IgG4: Ser228Pro Leu235Glu, Met252Ile, Thr256Asp, Met428Leu) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ lgitkvfsngadlsgvteeaplklskavhkavltidekgteaaga|e|fleaip|l|sippevk fnkpfvflmieqntksplfmgkvvnptqkeskygppcp|p|cpapef|e|ggpsvflfppkpkd tlIisrDpevtcvvvdvsqedpevqfnwyvdgvevhnaktkpreeqfnstyrvvsvltvl HQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSV0HE ALHNHYTQKSLSLSLGK (SEQ ID NO: 79)

[00188] These exemplary AAT-Fc fusion proteins were prepared using the following techniques.

[00189] The gene encoding human AAT was amplified by PCR from human liver cDNA (Zyagen). Specific point mutations were generated within the gene encoding AAT or the Fc region by Petition 870210021782, dated 08 / 03 / 2021, page 87 / 114 / 88 PCR overlap. The gene encoding AAT was cloned into a structure with a gene encoding the hinge region, followed by a CH2 domain and a CH3 domain of human IgG1, IgG2, IgG3, IgG4, or IgM in a mammalian expression vector, containing a mammalian secretion signal sequence upstream of the AAT gene insertion site. These expression vectors were transfected into mammalian cells (specifically HEK293 or CHO cells) and cultured for several days in 8% CO2 at 37°C. Recombinant AAT-Fc fusion proteins were purified from cell expression supernatant by protein A chromatography. Importantly, a near-neutral pH buffer (Gentle Ag / Ab Elution Buffer, Thermo Scientific) was used to elute the AAT-Fc fusion protein from the protein A resin.The AAT-Fc fusion protein cannot be eluted from protein A resin using a standard low-pH elution, as AAT's ability to inhibit NE is compromised after low-pH treatment, likely due to low-pH-mediated AAT oligomerization. Figure 1F shows the effects of low-pH elution on AAT's ability to inhibit neutrophil elastase. The AAT-Fc fusion protein can be purified from protein A or from a near-neutral pH elution buffer, using the Alpha-1-Antitrypsin CaptureSelect® affinity matrix (BAC BV).

[00190] Purified AAT-Fc fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase (NE). Figures 1B and 1D show a reducing SDS-PAGE gel of purified serum-derived AAT fusion proteins (sdAAT) and AAT-Fc (Fig. 1B-lane 1: sdAAT, lane 2: AAT-Fc (SEQ ID NO: 16), lane 3: AAT-EL-Fc (SEQ ID NO: 18), Fig 1D AAT-Fc-AAT (SEQ ID NO: 20). The proteins were visualized by Coomassie blue staining.

[00191] A fluorescent microplate assay was used to monitor human neutrophil elastase (NE) activity. The inhibitory activity Petition 870210021782, dated 08 / 03 / 2021, page 88 / 114 / 88 was measured by a concomitant decrease in residual NE activity using the following assay. This assay buffer is composed of 100 mM Tris pH 7.4, 500 mM NaCl, and 0.0005% Triton X-100. Human NE is used at a final concentration of 5 nM (but can also be used from 1 to 20 nM). The fluorescent peptide substrate AAVP-AMC is used at a final concentration of 100 μM in the assay. The Gemini EM plate reader from Molecular Devices is used to read the assay kinetics using excitation and emission wavelengths of 370 nm and 440 nm, respectively, and a cutoff of 420 nm. The assay is read for 10 minutes at room temperature with scans every 5 to 10 seconds. The Vmax per second corresponds to the residual NE activity, which is plotted for each inhibitor concentration. The x-intercept indicates the inhibitor concentration required to completely inactivate the initial NE concentration in the assay.Human serum-derived AAT (sdAAT) was used as a positive control in these assays. AAT-Fc fusion proteins exhibit potent NE inhibitory activity as shown in Figure 1C. The fusion in which two AAT polypeptides are fused with the single Fc polypeptide (AAT-Fc-AAT) shows enhanced potency in both sdAAT and the AAT-Fc fusion protein comprising a single AAT polypeptide (Figure 1E). These findings presented here demonstrate for the first time that AAT can be fused to an Fc region and retain its ability to inhibit NE. Of particular interest, the AAT-Fc-AAT fusion protein was found to be a more potent NE inhibitor.

[00192] Figure 1F demonstrates the resistance of the AAT-EL-Fc fusion protein (M351E, M358L) to inactivation by oxidation. The AAT fusion proteins, AAT-Fc (wt), AAT-EL-Fc (M351E, M358L), and AAT-EM-Fc (M351E), were treated with 33 mM H2O2 and compared to untreated fusion proteins in NE inhibition assays. NE inhibition by AAT-EL-Fc was not affected by oxidation, unlike the other proteins. Petition 870210021782, dated 08 / 03 / 2021, pages 89 / 114 / 88 tested.

[00193] Furthermore, the AAT-Fc fusion protein exhibited a longer serum half-life in rats compared to serum-derived AAT (Figure 1H). In these studies, 10 mg / kg of sdAAT or AAT-Fc was intravenously injected into 3 rats for each assay protein. Serum samples were collected at various time points over a 48-hour period. Serum AAT concentration was determined using an ELISA. These results demonstrate that the fusion proteins of the invention have improved pharmacokinetic properties and are a superior therapeutic format to serum-derived AAT for the treatment of numerous human inflammatory conditions and infectious diseases. Example 2: AATTNFa bleaching molecule fusion proteins

[00194] The studies presented here describe several, non-limiting examples of recombinant AAT derivatives comprising human AAT fused to an anti-TNFα antibody or a derivative of a TNFα receptor. These examples are provided below to better illustrate the different features of the present invention. The examples also illustrate a useful methodology for practicing the invention. These examples are not intended to, and are not intended to, limit the claimed invention.

[00195] The fusion proteins below include cytokine-targeting polypeptide sequences that are or are derived from (i) the anti-TNFα antibody D2E7 (also known as Adalimumab or Humira®), or (ii) the extracellular domain of the TNFα Type 2 Receptor (TNFR2-ECD). The AAT polypeptide portion of the fusion protein is underlined, the antibody constant regions (CH1-hinge-CH2-CH3 or CL) are italicized, and D2E7-VH, D2E7-VK, and TNFR2-ECD are indicated in bold. Although these examples include a hinge sequence and / or a linker sequence, the fusion proteins of the invention can be made Petition 870210021782, dated 08 / 03 / 2021, pp. 90 / 114 / 88 using any hinge sequence and / or a suitable linker sequence in length and / or flexibility. Alternatively, fusion proteins can be made without using a hinge and / or linker sequence.

[00196] An exemplary AAT-TNFα fusion protein is D2E7-Light Chain-AAT (G3S)2Ligand described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VK is indicated in bold (SEQ ID NO: 37), and the antibody constant regions are in italics (SEQ ID NO: 38) D2E7-Light Chain-AAT (G3S)i Binder DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSR FSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGS GGGSEDPQGDAAQKTDTSHHDQDHPT FNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFN LTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGGNGLFLSEGLKLVDKFLEDVKKLYHSEAFT VNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEE EDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENE LTHDIITKFLENEDRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPL KLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKV VNPTQK (SEQ ID NO:22)

[00197] An exemplary AAT-TNFα fusion protein is the D2E7-Light Chain-AAT ASTGS Ligand described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VK is indicated in bold (SEQ ID NO: 37), and the antibody constant regions are in italics (SEQ ID NO: 38) D2E7-Cadeia Leve-AAT ASTGS Ligand DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSR FSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSD EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGECAST GSEDPQGDAAQKTDTSHHDQDHPTFNK ITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTE IPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFSLEGLKLVDKFLEDVKKLYHSEAFTVNF Petition 870210021782, de 08 / 03 / 2021, pág. 91 / 114 / 88 GDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDF HVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTH DIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLS KAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNP TQK (SEQ ID NO:23)

[00198] An exemplary AAT-TNFα fusion protein is the D2E7-Heavy Chain-AAT (G3S)2 Ligand described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VH is indicated in bold (SEQ ID NO: 39), and the antibody constant regions are in italics (SEQ ID NO: 40) D2E7-Heavy Chain-AAT (G3S)2 Binder EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYA DSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSEDPQGDAAQKTDTSHHDQDHPTFNKITPN LAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEA QIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTE EAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIIT KFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVH KAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:24)

[00199] An exemplary AAT-TNFα fusion protein is the D2E7-Heavy Chain-AAT ASTGS Ligand described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VH is indicated in bold (SEQ ID NO: 39), and the antibody constant regions are in italics (SEQ ID NO: 40) D2E7-Heavy Chain-AAT ASTGS Binder Petition 870210021782, dated 08 / 03 / 2021, pages 92 / 114 / 88 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYA DSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGKASTGS EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAE FAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIH EGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAK KQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTT VKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFL ENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAV LTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:25)

[00200] An exemplary AAT-TNFa fusion protein is TNFR2-ECD-Fc1-AAT(G3S)2 Ligand described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), TNFR2-ECD is indicated in bold (SEQ ID NO: 41), and the antibody constant regions are in italics (SEQ ID NO: 42) TNFR2-ECD-Fcy-AAT(G3S)2 Ligand LPAQVAFTPYAPEGSCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTY TQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCR PGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSP TRSMAPGAVHLPQPVSTRSQHTQPTPESTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTH TCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVH NAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPKGGGSGGGSEDPQGDAAQKTDT SHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHD EILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFSLEGLKLVDKFLEDVK KLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWER PFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDE GKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADL Petition 870210021782, de 08 / 03 / 2021, pág. 93 / 114 / 88 SGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNT KSPLFMGKVVNPTQK (SEQ ID NO:26)

[00201] An exemplary AAT-TNFa fusion protein is the TNFR2-ECD-Fc1-AAT ASTGS Ligand described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), TNFR2-ECD is indicated in bold (SEQ ID NO: 41), and the antibody constant regions are in italics (SEQ ID NO: 42). TNFR2-ECD-Fc1-AAT ASTGS Ligand LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTY TQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCR PGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSP TRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVH NAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHH DQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEIL EGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLY HSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFE VKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKL QHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGV TEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSP LFMGKVVNPTQK (SEQ ID NO:27)

[00202] These exemplary AATTNFa targeting molecule fusion proteins were prepared using the following techniques.

[00203] The genes encoding the variable heavy (VH) and variable kappa (VK) regions of the anti-TNFα antibody, D2E7, were generated by gene synthesis. The D2E7-VH gene was cloned into a structure with a gene encoding a constant region of human IgG1 antibody heavy chain, consisting of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain, in a mammalian expression vector containing a mammalian secretion signal sequence. Petition 870210021782, dated 08 / 03 / 2021, page 94 / 114 / 88 upstream of the VH domain insertion site (D2E7-HC). The D2E7-VK gene was cloned into a structure with a constant human antibody kappa light chain (CL) domain, in a mammalian expression vector, containing a mammalian secretion signal sequence upstream of the insertion site in the VK domain (D2E7-LC). The gene encoding AAT and the adjacent 5' linker sequence were cloned into a structure at the 3' end of either the CH3 domain of the D2E7 heavy chain gene (D2E7-HCAAT) or the CL domain of the D2E7 light chain gene (D2E7-LC-AAT) encoding sequences in the mammalian expression vectors described above.The extracellular domain of TNF-α Receptor 2 (TNFR2-ECD) was generated by gene synthesis and cloned into a structure with a gene encoding the hinge region, followed by a CH2 domain and a CH3 domain of human IgG1 (hFc1) in a mammalian expression, containing a mammalian secretion signal sequence upstream of the TNFR2-ECD insertion site. The gene encoding AAT and the adjacent 5' linker sequence were cloned into a structure at the 3' end of the gene encoding TNFR2-ECD-hFc1 in a mammalian expression vector (TNFR2-ECD-hFc1-AAT).

[00204] The D2E7-HC-AAT expression vector was cotransfected with the D2E7-LC or D2E7-LC-AAT expression vector into mammalian cells (specifically HEK293 or CHO cells) to generate the D2E7 antibody with AAT fused to the C-terminal end of the heavy chain or to the C-terminal end of both the heavy and light chains, respectively. The D2E7-LC-AAT was cotransfected with the D2E7-HC expression vector into mammalian cells to generate the D2E7 antibody with AAT fused to the C-terminal end of the light chain. The TNFR2-hFc1-AAT expression vector was transfected into mammalian cells. The transfected cells were cultured for several days in 8% CO2 at 37°C.

[00205] Recombinant AAT-TNFα-targeting fusion proteins were purified from cell supernatant of Petition 870210021782, dated 08 / 03 / 2021, page 95 / 114 / 88 expression by protein A chromatography. A near-neutral pH buffer (Gentle Ag / Ab Elution Buffer, Thermo Scientific) was used to elute the AAT-TNFa bleaching fusion proteins from the protein A resin.

[00206] Figure 2B shows an SDS-PAGE gel of the D2E7 antibody alone (lane 1) and a variant in which AAT is fused with the heavy chain of D2E7 (lane 2). The proteins were visualized by staining with Coomassie blue.

[00207] Purified AATTNFa bleaching molecule fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase. Human serum-derived AAT (sdAAT) was used as a positive control in these assays. The NE inhibitory assay was conducted as described above. Figure 2C demonstrates that, in relation to sdAAT, the AAT-TNFa bleaching molecule fusion protein exhibits similar inhibition of neutrophil elastase, indicating that the inhibitory capacity of AAT was not compromised by its fusion to an antibody. Example 3 AAT-Fc-SLPI and AAT-Fc-Elafina

[00208] The studies presented here describe several, non-limiting examples of recombinant AAT derivatives comprising human AAT fused to a protein containing a WAP domain. These examples are provided below to better illustrate the different features of the present invention. The examples also illustrate a useful methodology for practicing the invention. The AAT polypeptide portion of the fusion protein is underlined, the Fc portion is italicized, and the WAP domain-containing polypeptide is bold. Although these examples include a hinge sequence and / or a linker sequence, the fusion proteins of the invention can be made using any hinge sequence and / or a linker sequence suitable in length and / or flexibility. Petition 870210021782, dated 08 / 03 / 2021, pages 96 / 114 / 88 Alternatively, fusion proteins can be made without using a hinge and / or linker sequence. For example, polypeptide components can be directly attached.

[00209] An exemplary AAT-Fc-SLPI fusion protein is the AAT-hFc1-SLPI (human IgG1 Fc) described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), the Fc portion is italicized (SEQ ID NO: 3), and the WAP domain-containing polypeptide is bold (SEQ ID NO: 9). AAT-hFcl-SLPI (human IgG1 Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKASTGSSGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCP DTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGMCGKSC VSPVKA (SEQ ID NO:28)

[00210] An exemplary AAT-Fc-SLPI fusion protein is AAT-hFc1-SLPI (human IgG1 Fc) described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), the Fc portion is italicized (SEQ ID NO: 3), and the WAP domain-containing polypeptide is bold (SEQ ID NO: 12) AAT-hFcl-Elafin (human IgGl Fc) EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY Petition 870210021782, dated 08 / 03 / 2021, p. 97 / 114 / 88 LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGKASTGSAVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQ EPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ (SEQ ID NO:29)

[00211] The genes encoding SLPI and Elafina were amplified by PCR from human spleen cDNA (Zyagen). These genes were cloned into the mammalian expression vectors of Example 1, in which the SLPI or Elafina gene was inserted into a framework with the AAT-Fc gene. These expression vectors were transfected into mammalian cells (specifically HEK293 or CHO cells) and cultured for several days in 8% CO2 at 37°C. Recombinant AAT-Fc-domain WAP fusion proteins were purified from the cell expression supernatant by protein A chromatography. A near-neutral pH buffer (Gentle Ag / Ab Elution Buffer, Thermo Scientific) was used to elute the AAT-Fc-domain WAP fusion protein from the protein A resin.

[00212] Figure 3B shows an SDS-PAGE gel of AAT-Fc-WAP fusion proteins (lane 1 AAT-Fc-Elafin, lane 2 AAT-Fc-SLPI). The proteins were visualized by Coomassie blue staining. Purified AAT-Fc-WAP domain fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase. NE inhibitory assays were conducted as described above. Human serum-derived AAT (sdAAT) and AAT-Fc fusion protein were used as a positive control in these assays. Compared to sdAAT, the AAT-Fc-WAP fusion proteins exhibit enhanced NE inhibition potency of neutrophil elastase (Figure 3C). Petition 870210021782, dated 08 / 03 / 2021, pages 98 / 114 / 88 Example 4 AAT-Albumin

[00213] The studies presented here describe several non-limiting examples of recombinant AAT derivatives comprising human AAT fused to an albumin polypeptide. These examples are provided below to better illustrate the different features of the present invention. The examples also illustrate a useful methodology for practicing the invention. These examples are not intended to limit the claimed invention. The AAT portion is underlined and the albumin portion is italicized. For example, polypeptide components can be directly affixed.

[00214] An exemplary AAT-Albumin fusion protein is AAT-HSA described here. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), and the albumin polypeptide is in italics (SEQ ID NO: 14) AAT-HSA EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNGFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKF NKPFVFLMIEQNTKSPLFMGKVVNPTQKASTGS DAHKSEVAHRFKDLGEENFKALVLIAFA QYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLERTYGEMAD CCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAF KAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSIS SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYE YARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCEL FEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFHADICTL SEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO:30) Petition 870210021782, dated 08 / 03 / 2021, pages 99 / 114 / 88

[00215] An exemplary AAT-Albumin fusion protein is AAT-HSA Domain 3 described here. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), and the albumin polypeptide is in italics (SEQ ID NO: 15) AAT-HSA Domain 3 EDPQGDAAQKTDTSHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAF AMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSE GLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFA LVNYIFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWLLMKY LGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLPKLSITGTYDLKSVLGQ LGITKVFSNGADLSGVTEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEWKF NKPFVFLMIEQNTKSPLFMGKVNPTQKASTK EQELQNQLQLQLQLQLQL YTKKVPQVS TP TL VEVSRNLGKVGSKCCKHPEAKRMPCAED YLS VVLNQLCVLHEKTP VS DRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTAL AND LVKHKPKATKEQLKAEKCDKVKVKVKFAKKFACKEKFACKEK NO:31)

[00216] The gene encoding human serum albumin (HSA) was amplified by PCR from human liver cDNA (Zyagen). A mammalian expression vector was generated, in which the gene encoding HSA or domain 3 of HSA was cloned into a structure to the 3' end of the gene encoding AAT, containing a mammalian secretion signal sequence upstream of AAT.

[00217] These expression vectors were transfected into mammalian cells (specifically HEK293 or CHO cells) and cultured for several days in 8% CO2 at 37°C. Recombinant AAT-HSA fusion proteins were purified from the expression cell supernatant using the CaptureSelect® Alpha-1-Antitrypsin Affinity Array (BAC BV), where the binding buffer consisted of 20 mM Tris, 150 mM NaCl, pH 7.4 and the elution buffer consisted of 20 mM Tris, 2 mM MgCl2 pH 7.4.

[00218] Figure 4B shows an SDS-PAGE gel of the AAT-HSA fusion protein. The proteins were visualized by staining with blue. Petition 870210021782, dated 08 / 03 / 2021, pages 100 / 114 / 88 Coomassie. Purified AAT-HSA fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase. NE inhibitory assays were conducted as described above. Human serum-derived AAT (sdAAT) was used as a positive control in these assays. Compared to sdAAT, the AAT-HSA fusion protein exhibits similar NE inhibitory potency, demonstrating that fusion with albumin does not attenuate the ability of AAT to inhibit NE (Figure 4C). Other modalities

[00219] Although the invention has been described in conjunction with its detailed description, the preceding description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Petition 870210021782, dated 08 / 03 / 2021, pp. 101 / 114

Claims

8. Isolated fusion protein according to claim 1, characterized in that it is for use in a method of treating ischemia-reperfusion injury, wherein the ischemia-reperfusion injury occurs after heart transplantation.

9. Isolated fusion protein according to claim 1, characterized in that the infection is selected from a bacterial infection, a fungal infection, or a viral infection. Petition 870260068885, dated 12 / 07 / 2026, page 13 / 21 1 / 1 Auxiliary Claims Table (Less Preferred) Petition 870260068885, dated 12 / 07 / 2026, page 14 / 21

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