KR20240005109A - Serpin fusion polypeptides and methods of use thereof - Google Patents

Abstract

The present invention relates to a molecule, in particular a polypeptide, more particularly a serpin and a fusion protein comprising 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 derived from an Fc polypeptide. order; A cytokine targeting polypeptide or a sequence derived from a cytokine targeting polypeptide; A polypeptide comprising a WAP domain or a sequence derived from a polypeptide comprising a WAP; and an amino acid sequence derived from albumin polypeptide or serum albumin polypeptide. The invention also relates to methods of using said molecules for various therapeutic and diagnostic indications, and methods of preparing said molecules.

Description

SERPIN FUSION POLYPEPTIDES AND METHODS OF USE THEREOF}

The present invention relates to molecules, especially polypeptides, more particularly to fusion proteins comprising a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide and a second polypeptide. The present invention also relates to a fusion protein comprising a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide, a second polypeptide, and a third polypeptide. Specifically, the present invention relates to a fusion protein comprising at least one serpin polypeptide, a second polypeptide, or a fusion protein comprising at least one serpin polypeptide, a second polypeptide, and the polypeptide of claim 3, wherein The second and third polypeptides of the fusion protein of the invention may comprise 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 comprising a WAP domain or a sequence derived from a polypeptide comprising a WAP; or an amino acid sequence derived from albumin polypeptide or serum albumin polypeptide. The invention also relates to methods of using the molecules for the various therapeutic and diagnostic indications, and methods of preparing the molecules.

Related applications

This application claims the benefit of U.S. Patent Application No. 14/524,832, filed October 27, 2014, the contents of which are hereby incorporated by reference in their entirety.

Inclusion of Sequence Listing

The content of the text file with the file name "INHI002002WO_SeqList.txt" was created on October 26, 2015, has a size of 228KB, and the content is incorporated by reference into this application in its entirety.

Background of the invention

Aberrant serine protease activity or an imbalance of protease-to-protease inhibitors can cause protease-mediated tissue destruction and inflammatory responses. Accordingly, there is a need for therapeutics and treatments targeting abnormal serine protease activity and/or protease-to-protease inhibitor imbalance. Additionally, improved therapeutic effects can be achieved through reduction of abnormal cytokine signaling and serine protease activity. Additionally, although targeting inflammatory cytokines has been shown to increase the risk of infection, serpin proteins have demonstrated anti-infective activity. The fusion proteins of the invention have the potential to inhibit inflammatory cytokine activity and limit the risk of infection.

Summary of the Invention

The fusion proteins described herein comprise at least a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide (polypeptide 1) and a second polypeptide (polypeptide 2). The fusion proteins described herein also 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 at least a second polypeptide or a serpin operably linked to an amino acid sequence derived from at least a second polypeptide. Refers to an amino acid sequence derived from a polypeptide or serpin polypeptide. The individual components of the fusion protein can be linked in a variety of ways, including, for example, direct attachment, use of a mediator or spacer peptide, use of a linker region, use of a hinge region, or use of a linker and a hinge region. In one embodiment, the linker region can be comprised within the sequence of a hinge region, or alternatively, the hinge region can be comprised within the sequence of a linker region. Preferably, the linker portion is a peptide sequence. For example, the linker peptide includes 0 to 40 amino acids, for example 0 to 35 amino acids, 0 to 30 amino acids, 0 to 25 amino acids, or 0 to 20 amino acids. Preferably, the hinge region is a peptide sequence. For example, the hinge peptide includes 0 to 75 amino acids, for example 0 to 70 amino acids, 0 to 65 amino acids, or 0 to 62 amino acids. In embodiments of a fusion protein comprising both a linker region and a hinge region, preferably each of the linker region and hinge region is a peptide sequence. In this embodiment, the hinge peptide and linker peptide together comprise 0 to 90 amino acids, such as 0 to 85 amino acids or 0 to 82 amino acids.

In one embodiment, the serpip polypeptide and the second polypeptide can be linked via an intermediate binding protein. In one embodiment, the serpin-based portion and the second polypeptide portion of the fusion protein may be non-covalently linked.

In one embodiment, the fusion protein according to the invention may have, in the N-terminus to C-terminus or C-terminus to N-terminus direction, one of the following formulas:

polypeptide 1 _(a) - hinge m - polypeptide 2 _(b)

Polypeptide 1 _(a) - Linker n - Polypeptide 2 _(b)

Polypeptide 1 _(a) - Linker n - Hinge m - Polypeptide 2 _(b)

Polypeptide 1 _(a) - Hinge m - Linker n - Polypeptide 2 _(b)

Polypeptide 1 _(a) - Polypeptide 2 _(b) - Polypeptide 3 _(c)

Polypeptide 1 _(a) - Hinge m - Polypeptide 2 _(b) - Hinge m - Polypeptide 3 _(c)

Polypeptide 1 _(a) - Linker n - Polypeptide 2 _(b) - Linker n - Polypeptide 3 _(c)

Polypeptide 1 _(a) - Hinge m - Linker n - Polypeptide 2 _(b) - Hinge m - Linker n - Polypeptide 3 _(c) Polypeptide 1 _(a) - Linker n - Hinge m - Polypeptide 2 _(b) - Linker n - Hinge m - polypeptide 3 _(c)

where n is an integer from 0 to 20, m is an integer from 1 to 62, and a, b, and c are integers of at least 1. Embodiments of the present invention include the above formulas and any variations or combinations thereof. For example, the order of polypeptides in the formula above can also be polypeptide 3 _(c) - polypeptide 1 _(a) - polypeptide 2 _(b) , polypeptide 2 _(b) - polypeptide 3 _(c) - polypeptide 1 _(a) , or any modification or combination thereof.

In one embodiment, the polypeptide 1 sequence comprises a serpin polypeptide. Serpins are a group of proteins with similar structures that were first identified as a set of proteins that can inhibit proteases. Serpin proteins suitable for use in the fusion proteins provided in the present invention include, but are not limited to, alpha-1 antitrypsin (AAT), antitrypsin-related protein (SERPINA2), Alpha 1-antichymotrypsin (SERPINA3), kallistatin (SERPINA4), monocyte neutrophil elastase inhibitor (SERPINB1), PI-6 (SERPINB6), antithrombin, SERPINC1), plasminogen activator inhibitor 1 (SERPINE1), alpha 2-antiplasmin (SERPINF2), complement 1-inhibitor (SERPING1) and neuroserpin (neuroserpin, SERPINI1), but is not limited thereto.

In one embodiment, the polypeptide 1 sequence comprises an alpha-1 antitrypsin (AAT) polypeptide sequence or an amino acid sequence derived from AAT. In one embodiment, the polypeptide 1 sequence comprises a portion of the AAT protein. In one embodiment, the polypeptide 1 sequence comprises at least a portion of the reactive site loop of the AAT protein. In one embodiment, the reactive site loop portion of the AAT protein comprises at least the amino acid sequence: GTEAAGAMFLEAIPMSIPPEVKFNK (SEQ ID NO:1).

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

In one embodiment, the fusion protein comprises a full-length human AAT polypeptide sequence having the following amino acid sequence:

1 EDPQGDAAQK TDTSHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI FFSPVSIATA

61 FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL RTLNQPDSQL QLTTGGNGLFL

121 SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT EEAKKQINDY VEKGTQGKIV DLVKELDRDT

181 VFALVNYIFF KGKWERPFEV KDTEEEDFHV DQVTTVKVPM MKRLGMFNIQ HCKKLSSWVL

241 LMKYLGNATA IFFLPDEGKL QHLENELTHD IITKFLENED RRSASLHLPK LSITGTYDLK

301 SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA MFLEAIPMSI

361 PPEVKFNKPF VFLMIEQNTK SPLFMGKVVN PTQK (SEQ ID NO: 2)

In one embodiment, the fusion protein has the amino acid sequence of SEQ ID NO:2 and at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% identical human AAT polypeptide sequence.

In one embodiment, the AAT polypeptide sequence, or an amino acid sequence derived from an AAT polypeptide, is described 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.

In one embodiment, the fusion protein includes one or more mutations. For example, the fusion protein includes at least one mutation in a methionine (Met) residue in the serpin portion of the fusion protein. In these Met variations, the Met residue may be substituted with any amino acid. For example, the Met residue can be replaced with an amino acid with a hydrophobic side chain, such as 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 protein of the invention. In one embodiment, the Met residue may be substituted with a charged residue, such as glutamate (Glu, E). In one embodiment, the Met mutation is at position 358 of the AAT polypeptide. For example, the Met mutation is Met358Leu (M358L). In one embodiment, the Met variant is at position 351 of the AAT polypeptide. For example, the Met mutation is Met351Glu (M351E). In one embodiment, the Met variant is at positions 351 and 358 of the AAT polypeptide, for example, the Met variant is Met351Glu (M351E) and Met358Leu (M358L). For example, the reaction site loop of this variant of the fusion AAT polypeptide has the following sequence: GTEAAGA E FLEAIP L SIPPEVKFNK (SEQ ID NO: 32). In one embodiment, the Met variant is at positions 351 and 358 of the AAT polypeptide, for example, the Met variant is Met351Leu (M351L) and Met358Leu (M358L). For example, the reactive site loop of this variant of the fusion AAT polypeptide has the following sequence: GTEAAGA L FLEAIP L SIPPEVKFNK (SEQ ID NO: 33).

In one embodiment, the fusion protein comprises a full-length human AAT polypeptide sequence having the following amino acid sequence and including a variant reactive site loop modified at M351 and M358:

In one embodiment, the fusion protein has the amino acid sequence of SEQ ID NO:80 and at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% identical human AAT polypeptide sequence.

In one embodiment, the second polypeptide (polypeptide 2) of the serpin fusion protein is an Fc polypeptide or is derived from an Fc polypeptide. These embodiments are collectively referred to herein as “serpin-Fc fusion proteins.” In the present invention, the serpin-Fc fusion protein includes at least a serpin polypeptide or an amino acid sequence derived from a serpin and an amino acid sequence derived from an Fc polypeptide or an Fc polypeptide. In one embodiment, the serpin-Fc fusion protein comprises a single serpin polypeptide. In other embodiments, the serpin-Fc fusion protein comprises one or more serpin polypeptides, and such embodiments are referred to herein as "serpin _(a') -Fc fusion protein (serpin _(a') -Fc fusion protein)", where (a') is an integer of at least 2. In one embodiment, each serpin polypeptide in the serpin _(a') -Fc fusion protein comprises a serpin polypeptide having a distinct amino acid sequence. The serpin polypeptide of the serpin _(a') -Fc fusion protein may be present at any position within the fusion protein.

In one embodiment, the serpin polypeptide of the serpin-Fc fusion protein comprises at least the amino acid sequence of the reactive site loop of the AAT protein. In one embodiment, the reactive site loop portion of the AAT protein comprises at least the amino acid sequence of SEQ ID NO:1. In one embodiment, the serpin polypeptide of the serpin-Fc fusion protein comprises a modified amino acid sequence of at least the reactive site loop portion of the AAT protein. In one embodiment, the modification of the reactive site loop portion of the AAT protein comprises at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In one embodiment, the serpin polypeptide of the serpin-Fc fusion protein comprises a full-length human AAT polypeptide sequence having the amino acid sequence of at least SEQ ID NO:2. In one embodiment, the serpin polypeptide of the serpin-Fc fusion protein comprises a full-length human AAT polypeptide sequence having the amino acid sequence of at least SEQ ID NO:80. In one embodiment, the serpin polypeptide of the serpin-Fc fusion protein has at least 50%, 60%, 65%, 70%, 75%, 80% of the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80. , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the human AAT polypeptide sequence.

In one embodiment, the serpin polypeptide of the serpin-Fc fusion protein is described 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.

In one embodiment, the Fc polypeptide of the fusion protein is a human Fc polypeptide, e.g., a human IgG Fc polypeptide sequence or an amino acid sequence derived from a human IgG Fc polypeptide sequence. For example, in one embodiment, the Fc polypeptide is a human IgG1 Fc polypeptide or an amino acid sequence derived from a human IgG1 Fc polypeptide sequence. In one embodiment, the Fc polypeptide is a human IgG2 Fc polypeptide or an amino acid sequence derived from a human IgG2 Fc polypeptide sequence. In one embodiment, the Fc polypeptide is a human IgG3 Fc polypeptide or an amino acid sequence derived from a human IgG3 Fc polypeptide sequence. In one embodiment, the Fc polypeptide is a human IgG4 Fc polypeptide or an amino acid sequence derived from a human IgG4 Fc polypeptide sequence. In one embodiment, the Fc polypeptide is a human IgM Fc polypeptide or an amino acid sequence derived from a human IgM Fc polypeptide sequence.

In one embodiment where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgG1 Fc polypeptide sequence having the following amino acid sequence:

1 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK

61 PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT

121 LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL

181 TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 3)

In one embodiment where the fusion protein of the present invention comprises an Fc polypeptide, the fusion protein comprises a hinge region coupled to the N-terminus of the Fc polypeptide of the fusion protein, and the Fc polypeptide is a human IgG1 having the following amino acid sequence: Contains the Fc polypeptide sequence:

1 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD

61 GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK

121 GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS

181 DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK (SEQ ID NO: 43)

In one embodiment, where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein has the amino acid sequence of SEQ ID NO: 3 or 43 and at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical human IgG1 Fc polypeptide sequence.

In one embodiment where the fusion protein of the present invention includes an Fc polypeptide, the Fc polypeptide is mutated or modified to enhance FcRn binding. In these embodiments the mutated or modified Fc polypeptide comprises the following mutations using the Kobat numbering system: Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E) or Met428Leu and Asn434Ser (M428L, N434S) or Met428Val and Asn434Ser (M428V, N434S). In one embodiment, the mutated or modified Fc polypeptide is a group consisting of Met252Tyr (M252Y), Ser254Thr (S256T), Thr256Glu (T256E), Met428Leu (M428L), Met428Val (M428V), Asn434Ser (N434S), and combinations thereof. Contains one or more mutations selected from. In one embodiment, the Fc polypeptide portion is mutated or otherwise modified to interfere with Fc-mediated dimerization. In this embodiment, the fusion protein is monomeric in nature.

In one embodiment where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide is mutated or modified. In one embodiment, the mutated or modified Fc polypeptide comprises one or more mutations at a position selected from the group consisting of M252, T246, M428, and combinations thereof. In one embodiment, the variant or modified Fc polypeptide comprises the following mutations using the Kobat numbering system: Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L).

In one embodiment where the fusion protein of the invention comprises a modified IgG1 Fc polypeptide, the IgG1 Fc polypeptide of the fusion protein corresponds to residues 22, 26, and 198 of SEQ ID NO: 3 set forth above, or SEQ ID NO: NO: Contains mutations at residues M252, T256, and M428, corresponding to residues 32, 36, and 208 of 43, and has the following amino acid sequence, where the mutated amino acid residues are boxed:

In one embodiment, where the fusion protein of the invention comprises a hinge region connected to the N-terminus of a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein is SEQ ID NO: 22, 26 of SEQ ID NO: 3, as shown above. and mutations at residues M252, T256, and M428, which correspond to residue 198, or which correspond to residues 32, 36, and 208 of SEQ ID NO: 43, and wherein the fusion protein comprises at least the following amino acid sequence, The amino acid residues mutated here are boxed:

In one embodiment where the fusion protein of the invention comprises a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein corresponds to the 6th residue of SEQ ID NO: 3 or SEQ ID NO: 43 as shown above. and has the following amino acid sequence:

1 APELLGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP

61 REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL

121 PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT

181 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 46)

In one embodiment, where the fusion protein of the present invention comprises a hinge region connected to the N-terminus of the modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein has the 6th residue of SEQ ID NO: 3 shown above. or a modified human IgG1 Fc polypeptide sequence with the G236 residue removed, corresponding to or corresponding to residue 16 of SEQ ID NO: 43, and said fusion protein comprising at least the following amino acid sequence:

1 DKTHTCPPCP APELLGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVKFNWYVDG

61 VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG

121 QPREPQVYTL PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD

181 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 47)

In one embodiment where the fusion protein of the invention comprises a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein corresponds to residues 4 and 5 of SEQ ID NO: 3 as shown above or SEQ ID NO: : Contains mutations at residues L234 and L235, corresponding to residues 14 and 15 of 43, and has the following amino acid sequence, where the mutated amino acid residues are boxed:

In one embodiment, where the fusion protein of the present invention comprises a hinge region connected to the N-terminus of a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein has SEQ ID NO: 4 and 5 of SEQ ID NO: 3 shown above. , or corresponding to residues 14 and 15 of SEQ ID NO: 43, and comprising mutations at residues L234 and L235, wherein the fusion protein comprises at least the following amino acid sequence, wherein the mutated amino acid residue is boxed It is displayed as:

In one embodiment where the fusion protein of the invention comprises a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein comprises a deletion of the G236 residue and a mutation of the L234 and L235 residues, and the fusion protein comprises at least It contains the following amino acid sequence, where the mutated residues are boxed:

In one embodiment, where the fusion protein of the invention comprises a hinge region linked to the N-terminus of a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein comprises a deletion of the G236 residue and mutations of the L234 and L235 residues. and has the following amino acid sequence, where the mutated amino acid residues are boxed:

In one embodiment where the fusion protein of the invention comprises a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein comprises a deletion of the G236 residue and mutations of the L234, L235, M252, T256, and M428 residues; , and the fusion protein comprises at least the following amino acid sequence, wherein the mutated amino acid residues are boxed:

In one embodiment, where the fusion protein of the present invention comprises a hinge region connected to the N-terminus of the modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein has a deletion of the G236 residue and L234, L235, M252, T256, and a mutation of residue M428, and has the following amino acid sequence, where the mutated portion is boxed:

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

In one embodiment where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgG2 Fc polypeptide sequence having the following amino acid sequence:

1 APPVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKP

61 REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL

121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLSD GSFFLYSKLT

181 VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK (SEQ ID NO: 4)

In one embodiment where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein is at least 50%, 60%, 65%, 70%, 75%, 80% identical to the amino acid sequence of SEQ ID NO: 4. , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a human IgG2 Fc polypeptide sequence.

In one embodiment where the fusion protein of the invention comprises a modified IgG2 Fc polypeptide, the modified IgG2 Fc polypeptide of the fusion protein comprises a modified human IgG2 Fc polypeptide sequence having the following amino acid sequence, wherein the modified amino acid Residues are boxed:

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

In one embodiment where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgG3 Fc polypeptide sequence having the following amino acid sequence:

1 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)

In one embodiment, where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein is at least 50%, 60%, 65%, 70%, 75%, 80% identical to the amino acid sequence of SEQ ID NO: 5. , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a human IgG3 Fc polypeptide sequence.

In one embodiment where the fusion protein of the invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein comprises a modified human IgG3 Fc polypeptide sequence having the following amino acid sequence, wherein the modified amino acid Residues are boxed:

In one embodiment where the fusion protein of the invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein has a deletion of residue G236, corresponding to the 6th residue of SEQ ID NO: 5 shown above. A modified human IgG3 Fc polypeptide having the following amino acid sequence:

1 APELLGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFKWYVDG VEVHNAKTKP

61 REEQYNSTFR VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKTKG QPREPQVYTL

121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESSGQPENNY NTTPPMLDSD GSFFLYSKLT

181 VDKSRWQQGN IFSCSVMHEA LHNRFTQKSL SLSPGK (SEQ ID NO: 56)

In one embodiment where the fusion protein of the invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein comprises L234 and L235, corresponding to residues 4 and 5 of SEQ ID NO: 5 shown above. contains a mutation in a residue and has the following amino acid sequence, where the mutated amino acid residue is indicated by a box:

In one embodiment where the fusion protein of the invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein comprises a deletion of the G236 residue and a mutation of the L234 and L235 residues, and has the following amino acid sequence: has:

In one embodiment where the fusion protein of the invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein comprises a deletion of the G236 residue and a mutation of the L234, L235, M252, T256, and M428 residues; , and has the following amino acid sequence:

In one embodiment where the fusion protein of the invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein has at least 50% of the amino acid sequence of SEQ ID NO: 55, 56, 57, 58, or 59. , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% matched. Contains a modified human IgG3 Fc polypeptide sequence.

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

In one embodiment where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgG4 Fc polypeptide sequence having the following amino acid sequence:

1 APEFLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD GVEVHNAKTK

61 PREEQFNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKGLPS SIEKTISKAK GQPREPQVYT

121 LPPSQEEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSRL

181 TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK (SEQ ID NO: 6)

In one embodiment where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a hinge region linked to the N-terminus of the Fc polypeptide of the fusion protein, wherein the Fc polypeptide has the following amino acid sequence: Contains an IgG4 Fc polypeptide sequence.

1 ESKYGPPCPS CPAPEFLGGP 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: 60)

In one embodiment, where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein has the amino acid sequence of SEQ ID NO: 6 or 60 and at least 50%, 60%, 65%, 70%, 75%, Includes a human IgG4 Fc polypeptide sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical.

In one embodiment where the fusion protein of the invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises residues 22, 26, and 197 of SEQ ID NO: 6 as indicated above, or SEQ ID NO: : Contains mutations at residues M252, T256, and M428, corresponding to residues 34, 38, and 210 of 60, and has the following amino acid sequence, where the mutated amino acid residues are boxed:

In one embodiment, where the fusion protein of the present invention comprises a hinge region connected to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein is 22, 26 of SEQ ID NO: 6, as shown above. and mutations at residues M252, T256, and M428, corresponding to residue 197 or residues 34, 38, and 210 of SEQ ID NO: 60, wherein the fusion protein comprises at least the following amino acid sequence, wherein: The amino acid residues indicated are boxed:

In one embodiment where the fusion protein of the present invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises the 6th residue of SEQ ID NO: 6 or the 19th residue of SEQ ID NO: 60 as shown above. It comprises a modified human IgG4 Fc polypeptide sequence in which the G236 residue is deleted, corresponding to the residue, and has the following amino acid sequence.

1 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)

In one embodiment, where the fusion protein of the present invention comprises a hinge region connected to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises the 6th residue of SEQ ID NO: 6 shown above, or Comprising a modified human IgG4 Fc polypeptide sequence with the G236 residue deleted, corresponding to residue 19 of SEQ ID NO: 60, said fusion protein comprising at least the following amino acid sequence:

1 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)

In one embodiment where the fusion protein of the present invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises the 5th residue of SEQ ID NO: 6 or the 17th residue of SEQ ID NO: 60 as shown above. It contains a mutation at residue L235, corresponding to the residue, and has the following amino acid sequence, where the mutated amino acid residue is boxed:

In one embodiment, where the fusion protein of the present invention comprises a hinge region connected to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises the 5th residue of SEQ ID NO: 6 shown above, or and a mutation at residue L235, corresponding to residue 17 of SEQ ID NO:60, and the fusion protein comprises at least the following amino acid sequence, wherein the mutated amino acid residue is boxed:

In one embodiment where the fusion protein of the present invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises residues 4 and 5 of SEQ ID NO: 6 or SEQ ID NO: 60 shown above. It contains mutations at residues L234 and L235, corresponding to residues 16 and 17, and has the following amino acid sequence, where the mutated amino acid residues are boxed:

In one embodiment, where the fusion protein of the present invention comprises a hinge region connected to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein is 4 and 5 of SEQ ID NO: 6 shown above. residues or at residues L234 and L235, corresponding to residues 16 and 17 of SEQ ID NO: 60, wherein the fusion protein comprises at least the following amino acid sequence, wherein the mutated amino acid residues are boxed: :

In one embodiment where the fusion protein of the present invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein has a mutation at residue S228, corresponding to the 10th residue of SEQ ID NO: 60 shown above. and has the following amino acid sequence, wherein the mutated amino acid residues are boxed:

In one embodiment, where the fusion protein of the present invention comprises a hinge region connected to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein is 10 and 17 of SEQ ID NO: 60 shown above. Comprising mutations at residues S228 and L235, corresponding to the residues, the fusion protein comprises at least the following amino acid sequence, wherein the mutated amino acid residues are boxed:

In one embodiment where the fusion protein of the invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises residues 5, 22, 26, and 197 of SEQ ID NO: 6 or SEQ ID NO: 6 shown above. ID NO: Contains mutations at residues L235, M252, T256, and M428, corresponding to residues 17, 34, 38, and 210 of 60, and has the following amino acid sequence, wherein the mutated amino acid residues are boxed do:

In one embodiment, where the fusion protein of the present invention comprises a hinge region connected to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein is SEQ ID NO: 5, 22 of SEQ ID NO: 6, as shown above. 26, and 197, or residues 17, 34, 38, and 210 of SEQ ID NO: 60, and mutations in residues L235, M252, T256, and M428, wherein the fusion protein contains at least the following amino acids: Contains the sequence, where the mutated amino acid residues are boxed:

In one embodiment, where the fusion protein of the present invention comprises a hinge region connected to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein is 10, 17 of SEQ ID NO: 60, as indicated above. and mutations at residues S228, L235, M252, T256, and M428, corresponding to residues 34, 38, and 210, and the fusion protein comprises at least the following amino acid sequence, wherein the mutated amino acid residues are boxed: It displays:

In one embodiment where the fusion protein of the invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein is SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69 , 70, 71, 72, or 73 and at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, and a modified human IgG4 Fc polypeptide sequence that is 95%, 96%, 97%, 98% or 99% identical.

In one embodiment where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide of the fusion protein comprises a human IgM Fc polypeptide sequence having the following amino acid sequence:

1 IAELPPKVSV FVPPRDGFFG NPRKSKLICQ ATGFSPRQIQ VSWLREGKQV GSGVTTDQVQ

61 AEAKESGPTT YKVTSTLTIK ESDWLGQSMF TCRVDHRGLT FQQNASSMCV PDQDTAIRVF

121 AIPPSFASIF LTKSTKLTCL VTDLTTYDSV TISWTRQNGE AVKTHTNISE SHPNATFSAV

181 GEASICEDDW NSGERFTCTV THTDLPSPLK QTISRPKG (SEQ ID NO: 7)

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

In one embodiment, the Serpin-Fc fusion protein has at least 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. and the amino acid sequence of the reactive site loop portion of the AAT protein operably linked to the derived Fc polypeptide sequence. In one embodiment, the Fc polypeptide has the following , 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In one embodiment, the Fc polypeptide has the following , 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73, and at least 50%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences. In one embodiment, the reactive site loop portion of the AAT protein comprises at least the amino acid sequence of SEQ ID NO:1. In one embodiment, the serpin polypeptide and the Fc polypeptide are operably linked through a linker moiety, for example, a glycine-serine linker or a glycine-serine based linker. It is done. In one embodiment, the serpin polypeptide and the Fc polypeptide are operably linked through a hinge region. In one embodiment, the serpin polypeptide and Fc polypeptide are operably linked through a linker region and a hinge region. In another embodiment, the serpin polypeptide and Fc polypeptide are directly attached.

In one embodiment, the Serpin-Fc fusion protein has at least 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. It contains the amino acid sequence of the reactive site loop portion of various AAT proteins operably linked to the derived Fc polypeptide sequence. In one embodiment, the Fc polypeptide has the following , 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In one embodiment, the Fc polypeptide has the following , 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73, and at least 50%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences. In one embodiment, the variant of the reactive site loop portion of the AAT protein comprises at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In one embodiment, the serpin polypeptide and the Fc polypeptide are operably linked through a linker moiety, for example, a glycine-serine linker or a glycine-serine based linker. do. In one embodiment, the serpin polypeptide and Fc polypeptide are operably linked through a hinge region. In one embodiment, the serpin polypeptide and Fc polypeptide are operably linked through a linker region and a hinge region. In another embodiment, the serpin polypeptide and Fc polypeptide are directly attached.

In one embodiment, the Serpin-Fc fusion protein has at least 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. It includes a full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO: 2 operably linked to the derived Fc polypeptide sequence. In one embodiment, the Fc polypeptide has the following , 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In one embodiment, the Fc polypeptide has the following , 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73, and at least 50%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences. In one embodiment, the Serpin-Fc fusion protein has 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. and at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence of SEQ ID NO: 2 operably linked to the Fc polypeptide sequence. , 94%, 95%, 96%, 97%, 98% or 99% identical human AAT polypeptide sequence. In one embodiment, the Fc polypeptide has the following , 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In one embodiment, the Fc polypeptide has the following , 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73, and at least 50%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences. In one embodiment, the serpin polypeptide and the Fc polypeptide are operably linked through a linker moiety, for example, a glycine-serine linker or a glycine-serine based linker. In one embodiment, the serpin polypeptide and Fc polypeptide are operably linked through a hinge region. In one embodiment, the serpin polypeptide and Fc polypeptide are operably linked through a linker region and a hinge region. In another embodiment, the serpin polypeptide and Fc polypeptide are directly attached.

In one embodiment, the Serpin-Fc fusion protein has at least 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. It includes a full-length human AAT polypeptide sequence having the amino acid sequence of SEQ ID NO:80, which will be operably linked to the derived Fc polypeptide sequence. In one embodiment, the Serpin-Fc fusion protein has 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. SEQ ID NO:80 amino acid sequence and at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, The 94%, 95%, 96%, 97%, 98% or 99% identity includes the human AAT polypeptide sequence. In one embodiment, the serpin polypeptide and the Fc polypeptide are operably linked through a linker moiety, e.g., a glycine-serine linker or a glycine-serine based linker. . In one embodiment, the serpin polypeptide and Fc polypeptide are operably linked through a hinge region. In one embodiment, the serpin polypeptide and Fc polypeptide are operably linked through a linker region and a hinge region. In another embodiment, the serpin polypeptide and Fc polypeptide are directly attached.

In one embodiment of the fusion protein provided herein, the second polypeptide (polypeptide 2) of the serpin fusion protein is a cytokine targeting polypeptide or is derived from a cytokine targeting polypeptide. These embodiments are collectively referred to herein as “serpin-cytokine targeting polypeptide fusion proteins.” The serpin-cytokine targeting polypeptide fusion protein described herein comprises at least a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide and a cytokine targeting polypeptide or a derivative thereof. In one embodiment, the serpin-cytokine targeting polypeptide fusion protein comprises a single serpin polypeptide. In other embodiments, the serpin-cytokine targeting polypeptide fusion protein comprises one or more serpin polypeptides, and such embodiments are collectively referred to herein as “serpin _(a') -cytokine targeting polypeptide fusion proteins”. refers to where (a') is an integer of at least 2. In one embodiment, each serpin polypeptide of the serpin _(a') -cytokine targeting polypeptide fusion protein comprises the same amino acid sequence. In another embodiment, each serpin polypeptide of the serpin _(a) -cytokine targeting polypeptide fusion protein comprises a serpin polypeptide having a distinct amino acid sequence.

In one embodiment, the cytokine targeting polypeptide of the serpin-cytokine targeting polypeptide fusion protein is a cytokine receptor or is derived from a cytokine receptor. In a preferred embodiment, the cytokine targeting polypeptide or cytokine receptor derived amino acid sequence is or is derived from a human cytokine receptor sequence. In another embodiment, the cytokine targeting polypeptide is an antibody or antibody fragment, eg, an anti-cytokine antibody or anti-cytokine antibody fragment. In a preferred embodiment, the amino acid sequence from the cytokine targeting polypeptide or antibody or antibody fragment is a chimeric, humanized or fully human antibody sequence. The term antibody fragment includes single chain, Fab fragment, F(ab') ₂ fragment, scFv, scAb, dAb, single domain heavy chain antibody, and antibody light chain single domain ( single domain light chain antibody).

In another embodiment, the cytokine targeting polypeptide binds to a cytokine receptor and prevents the cytokine from binding to the receptor. In another embodiment, the cytokine targeting polypeptide is an antibody or antibody fragment, such as an anti-cytokine receptor antibody or an anti-cytokine receptor antibody fragment.

In one embodiment, the serpin polypeptide of the serpin-cytokine targeting polypeptide fusion protein comprises at least the amino acid sequence of the reactive site loop portion of the AAT protein. In one embodiment, the reactive site loop portion of the AAT protein comprises at least the amino acid sequence of SEQ ID NO:1. In one embodiment, the serpin polypeptide of the serpin-cytokine targeting fusion protein comprises the amino acid sequence of at least a variant of the reactive site loop portion of the AAT protein. In one embodiment, the variant of the reactive site loop portion of the AAT protein comprises at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In one embodiment, the serpin polypeptide of the serpin-cytokine targeting fusion protein comprises or is derived from a full-length human AAT polypeptide having the amino acid sequence of at least SEQ ID NO:2. In one embodiment, the serpin polypeptide of the serpin-cytokine targeting fusion protein comprises or is derived from a full-length human AAT polypeptide sequence having the amino acid sequence of at least SEQ ID NO:80. In one embodiment, the serpin polypeptide of the serpin-cytokine targeting fusion protein has the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80 and 50%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the human AAT polypeptide sequence.

In one embodiment, the serpin polypeptide of the serpin-cytokine targeting fusion protein is according to 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 an AAT polypeptide sequence or an amino acid sequence derived from one or more human AAT polypeptide sequences shown in AAA51547.1. Includes.

The serpin-cytokine targeting polypeptide fusion protein may comprise a portion of a serpin-Fc fusion protein. For example, antibodies include Fc polypeptides. Accordingly, in one embodiment where the cytokine targeting polypeptide is a cytokine-targeting antibody, the serpin-cytokine targeting polypeptide fusion protein will comprise a portion of a serpin-Fc fusion protein. Additionally, most receptor fusion proteins used therapeutically are Fc fusion proteins. Accordingly, in one embodiment, wherein the serpin-cytokine targeting polypeptide fusion protein is a serpin-cytokine receptor fusion protein, and wherein the serpin-cytokine targeting polypeptide fusion protein contains an Fc polypeptide in addition to the serpin portion and the cytokine receptor portion. may include.

In one embodiment, where the serpin-cytokine targeting polypeptide fusion protein comprises an Fc polypeptide sequence, the Fc polypeptide sequence is 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 Contains or is derived from any one of 73 amino acid sequences. In one embodiment, the serpin-cytokine targeting fusion protein comprises an Fc polypeptide sequence, wherein the Fc polypeptide sequence is 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 The amino acid sequence of any one of 73 and at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , have 97%, 98% or 99% homology. In one embodiment, the serpin polypeptide and cytokine targeting polypeptide are operable through a linker moiety, for example, a glycine-serine linker or a glycine-serine based linker. connected. In one embodiment, the serpin polypeptide and the cytokine targeting polypeptide are operably linked through a hinge region. In one embodiment, the serpin polypeptide and the cytokine targeting polypeptide are operably linked through a linker region and a hinge region. In another embodiment, the serpin polypeptide and cytokine targeting polypeptide are attached directly.

In one embodiment of the fusion protein provided in the present invention, the second polypeptide (polypeptide 2) of the serpin fusion protein is a polypeptide containing a WAP (whey acidic protein) domain, or is derived from a polypeptide containing a WAP domain. This is the amino acid sequence. These embodiments are collectively referred to herein as “serpin-WAP domain fusion proteins.” The serpin-WAP domain fusion protein comprises at least a serpin polypeptide or at least an amino acid sequence from a serpin, a WAP domain-containing polypeptide, or an amino acid sequence from a WAP domain-containing polypeptide. In one embodiment, the serpin-WAP domain fusion protein comprises a single serpin polypeptide. In another embodiment, the serpin-WAP targeting polypeptide fusion protein comprises one or more serpin polypeptides. These embodiments are collectively referred to herein as "serpin ( _{a ')} -WAP domain fusion proteins", where (a') is an integer of at least 2. In one embodiment, the serpin polypeptides of the serpin _(a') -WAP domain fusion protein comprise identical amino acid sequences. In another embodiment, the serpin polypeptides of the serpin _(a') -cytokine targeting polypeptide fusion protein comprise serpin polypeptides having different amino acid sequences.

This serpin-WAP domain fusion protein includes a polypeptide sequence that is a polypeptide containing a WAP domain or a polypeptide containing a WAP domain, or a polypeptide sequence derived therefrom. The WAP domain is an evolutionarily conserved sequence motif of 50 amino acids containing 8 cysteines found in the characteristic 4-disulfide core arrangement (also called 4-disulfide core motif). The WAP domain sequence motif is a functional motif characterized by serine protease inhibitory activity in a number of proteins.

WAP domain-containing polypeptides suitable for use in the fusion proteins provided herein include, but are not limited to, secretory leukocyte protease inhibitor (SLPI), Elafin, and Eppin. ) includes.

In one embodiment, the WAP domain-comprising polypeptide sequence of the fusion protein comprises a secretory leukocyte protease inhibitor (SLPI) polypeptide sequence or an amino acid sequence derived from SLPI. This embodiment is referred to herein as “serpin-SLPI-derived fusion proteins.” In one embodiment, the SLPI polypeptide sequence comprises a portion of the SLPI protein, such as a WAP2 domain or a subportion thereof. In a preferred embodiment, the SLPI polypeptide sequence or amino acid sequence derived from SLPI is or is derived from a human SLPI polypeptide sequence.

In one embodiment of the serpin-SLPI fusion protein of the invention, the SLPI sequence or SLPI-derived sequence of the fusion protein comprises a full-length human SLPI polypeptide sequence having the following amino acid sequence:

1 MKSSGLFPFL VLLALGTLAP WAVEGSGKSF KAGVCPPKKS AQCLRYKKPE CQSDWQCPGK

61 KRCCPDTCGI KCLDPVDTPN PTRRKPGKCP VTYGQCLMLN PPNFCEMDGQ CKRDLKCCMG

121 MCGKSCVSPV KA (SEQ ID NO:8)

In one embodiment of the serpin-SLPI fusion protein of the invention, the SLPI sequence or SLPI-derived sequence of the fusion protein is at least 50%, 60%, 65%, 70%, 75% identical to the amino acid sequence of SEQ ID NO: 8. %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical human SLPI polypeptide sequence.

In one embodiment of the serpin-SLPI fusion protein of the invention, the SLPI sequence or SLPI-derived sequence of the fusion protein comprises a portion of a full-length human SLPI polypeptide sequence having the following amino acid sequence:

1 SGKSFKAGVC PPKKSAQCLR YKKPECQSDW QCPGKKRCCP DTCGIKCLDP VDTPNPTRRK

61 PGKCPVTYGQ CLMLNPPNFC EMDGQCKRDL KCCMGMCGKS CVSPVKA (SEQ ID NO:　9)

In one embodiment of the serpin-SLPI fusion protein of the invention, the SLPI sequence or SLPI-derived sequence of the fusion protein is at least 50%, 60%, 65%, 70%, 75% identical to the amino acid sequence of SEQ ID NO: 9. %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical human SLPI polypeptide sequence.

In one embodiment of the serpin-SLPI fusion protein of the invention, the SLPI sequence or SLPI-derived sequence of the fusion protein comprises the WAP2 domain of a full-length human SLPI polypeptide sequence wherein the WAP2 domain has the following amino acid sequence:

1 TRRKPGKCPV TYGQCLMLNP PPNFCEMDGQC KRDLKCCMGM CGKSCVSPVK A (SEQ ID NO:　10)

In one embodiment of the serpin-SLPI fusion protein of the invention, the SLPI sequence or SLPI-derived sequence of the fusion protein comprises at least 50%, 60%, 65%, 70%, 75% of the amino acid sequence of SEQ ID NO: 10. %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical human SLPI polypeptide sequence.

In one embodiment of the serpin-SLPI fusion protein of the invention, the SLPI polypeptide sequence or the amino acid sequence derived from the SLPI polypeptide is described in GenBank Accession Nos. One or more human SLPI polypeptide sequences represented by or derived from CAA28187.1, NP_003055.1, EAW75869.1, P03973.2, AAH20708.1, CAB64235.1, CAA28188.1, AAD19661.1, and/or BAG35125.1 do.

In one embodiment of the serpin-SLPI fusion protein of the present invention, the SLPI polypeptide sequence or SLPI-derived sequence of the fusion protein comprises a human SLPI polypeptide sequence with a modified methionine (Methoine, Met) residue. In these Met variations, the Met residue may be substituted with any amino acid. For example, a Met residue can be substituted with an amino acid with a hydrophobic side chain, such as 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 protein of the invention. In one embodiment, the Met variant is at position 98 of the SLPI polypeptide. For example, the modified SLPI polypeptide sequence of Serpin-SLPI includes the M98L or M98V mutation in SEQ ID NO:8.

In another embodiment, the WAP domain-comprising polypeptide sequence of the fusion protein comprises an elafin polypeptide sequence or an amino acid sequence derived from elafin. This embodiment is referred to herein as “serpin-elafin fusion proteins.” In one embodiment, the elafin polypeptide sequence comprises a portion of an elafin protein, such as a WAP domain or a sub-portion thereof. In a preferred embodiment, the elafin polypeptide sequence or amino acid sequence derived from elafin is or is derived from a human elafin polypeptide sequence.

In one embodiment of the serpin-elafin fusion protein, the fusion protein comprises a full-length human elafin polypeptide sequence having the following amino acid sequence:

1 MRASSFLIVV VFLIAGTLVL EAAVTGVPVK GQDTVKGRVP FNGQDPVKGQ VSVKGQDKVK

61 AQEPVKGPVS TKPGSCPIIL IRCAMLNPPN RCLKDTDCPG IKKCCEGSCG MACFVPQ (SEQ ID NO: 11)

In one embodiment of the serpin-elapin fusion protein, the fusion protein comprises at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% of the amino acid sequence of SEQ ID NO: 11. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the human elaffin polypeptide sequence.

In one embodiment of the serpin-elafin fusion protein, the fusion protein comprises a portion of a full-length human elaffin polypeptide sequence, wherein the portion has the following amino acid sequence:

1 AVTGVPVKGQ DTVKGRVPFN GQDPVKGQVS VKGQDKVKAQ EPVKGPVSTK PGSCPIILIR

61 CAMLNPPNRC LKDTDCPGIK KCCEGSCGMA CFVPQ (SEQ ID NO:　12)

In one embodiment of the serpin-elapin fusion protein, the fusion protein comprises at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% of the amino acid sequence of SEQ ID NO: 12. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical human elaffin polypeptide sequence.

In one embodiment of the serpin-elafin fusion protein, the fusion protein comprises a WAP domain of a full-length human elaffin polypeptide sequence, wherein the WAP domain has the following amino acid sequence:

1 VSTKPGSCPI ILIRCAMLNP PNRCLKDTDC PGIKKCCEGS CGMACFVPQ (SEQ ID NO:　13)

In one embodiment of the serpin-elapin fusion protein, the fusion protein comprises at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% of the amino acid sequence of SEQ ID NO: 13. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the human elaffin polypeptide sequence.

In one embodiment of the serpin-elafin fusion protein, the elafin polypeptide sequence or the amino acid sequence derived from the elaffin polypeptide is described in GenBank Accession Nos. It is derived from one or more human elaffin polypeptide sequences designated as P19957.3, NP_002629.1, BAA02441.1, EAW75814.1, EAW75813.1, Q8IUB2.1, and/or NP_542181.1.

In another embodiment, the WAP domain-comprising polypeptide sequence of the fusion protein comprises an Eppin polypeptide sequence or an amino acid sequence derived from Eppin. This embodiment is referred to herein as “serpin ( _{a ')} -Eppin fusion proteins.” In one embodiment, the epin polypeptide sequence of the serpin-epin fusion protein comprises a portion of the epin protein, such as a WAP domain or a sub-portion thereof. In a preferred embodiment, the epin polypeptide sequence or amino acid sequence derived from epin is a human epin polypeptide sequence or is derived therefrom.

In one embodiment of the serpin-epin fusion protein, the serpin polypeptide sequence or the amino acid sequence derived from the serpin polypeptide is described in GenBank Accession Nos. One or more human epin polypeptide sequences shown in O95925.1, NP_065131.1, AAH44829.2, AAH53369.1, AAG00548.1, AAG00547.1, and/or AAG00546.1.

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

In one embodiment, the serpin polypeptide of the serpin-WAP domain fusion protein is described 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, an AAT polypeptide sequence, or an amino acid sequence derived from an AAT polypeptide derived therefrom. do.

In one embodiment, the serpin-WAP domain fusion protein may comprise an Fc polypeptide or an amino acid sequence derived from an Fc polypeptide. These embodiments are collectively referred to herein as “serpin-Fc-WAP domain fusion proteins.” In this embodiment, no particular order is dictated by the terms. For example, the sequence of the fusion protein may be Serpin-Fc-WAP domain, Serpin-WAP domain-Fc, or any modified combination thereof. The serpin-Fc-WAP domain fusion protein disclosed in the present invention includes at least a serpin polypeptide or an amino acid sequence derived from a serpin, a WAP domain-containing polypeptide or an amino acid sequence derived from a WAP domain-containing polypeptide, and an Fc polypeptide or an Fc Contains an amino acid sequence derived from a polypeptide.

In one embodiment, where the serpin-WAP domain fusion protein comprises an Fc polypeptide sequence, the Fc polypeptide sequence may comprise the amino acid sequences of SEQ ID NO: 3-7 and 43-73. In another embodiment, where the serpin-WAP domain fusion protein comprises an Fc polypeptide sequence, the Fc polypeptide sequence is SEQ ID NOs. 3-7 and 43-73 and at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% , may have 96%, 97%, 98% or 99% homology. In one embodiment, the serpin-WAP domain fusion protein may also comprise an albumin polypeptide, or an amino acid sequence derived from an albumin polypeptide. These embodiments are collectively referred to herein as “serpin--albumin-WAP domain fusion proteins.” In this embodiment, no particular order is dictated by the terms. For example, the sequence of the fusion protein may be serpin-albumin-WAP domain, serpin-WAP domain-albumin, or any modified combination thereof. The serpin-albumin-WAP domain fusion protein disclosed in the present invention comprises at least a serpin polypeptide or an amino acid sequence derived from a serpin, a WAP domain-containing polypeptide, or an amino acid sequence derived from a WAP domain-containing polypeptide, an albumin polypeptide, or an albumin Contains an amino acid sequence derived from a polypeptide.

In one embodiment, where the serpin-WAP domain fusion protein comprises an albumin polypeptide sequence, the albumin polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 14-15, disclosed herein. In another embodiment, where the serpin-WAP domain fusion protein comprises an albumin polypeptide sequence, the albumin polypeptide sequence is at least 50%, 60%, 65%, 70% identical to any of the amino acid sequences of SEQ ID NO: 14 or 15. , 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In one embodiment, the second polypeptide (polypeptide 2) of the serpin fusion protein is or is derived from an albumin polypeptide. These embodiments are collectively referred to herein as “serpin ( _{a ')} -albumin fusion proteins.” The serpin-albumin fusion protein disclosed in the present invention includes at least a serpin polypeptide or an amino acid sequence derived from a serpin and an amino acid sequence derived from an albumin polypeptide or an albumin polypeptide. The present invention also relates to a serpin albumin binding polypeptide fusion protein, wherein the albumin is operably linked to the serpin through an intermediate binding molecule. In the present invention, the serpin is non-covalently or covalently bound to human serum albumin.

In one embodiment where the fusion protein of the present invention includes an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein is a human serum albumin (HSA) polypeptide or an amino acid sequence derived from HSA. In one embodiment, the fusion protein comprises an HSA polypeptide sequence having the following amino acid sequence:

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFG ERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQ VSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 14)

In one embodiment where the fusion protein of the present invention comprises an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein is at least 50%, 60%, 65%, 70%, 75% of the amino acid sequence of SEQ ID NO: 14. 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical human serum albumin polypeptide sequence.

In one embodiment where the fusion protein of the invention comprises an albumin polypeptide sequence, the albumin polypeptide sequence of the fusion protein comprises domain 3 of the human serum albumin polypeptide sequence, which has the following amino acid sequence:

EEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA (SEQ ID NO : 15)

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

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

In one embodiment, the serpin polypeptide of the serpin _(a')- albumin fusion protein comprises at least the amino acid sequence of the reactive site loop portion of the AAT protein. In one embodiment, the reactive site loop portion of the AAT protein comprises at least the amino acid sequence of SEQ ID NO:1. In one embodiment, the serpin polypeptide of the serpin-albumin fusion protein comprises the amino acid sequence of at least a variant of the reactive site loop portion of the AAT protein. In one embodiment, the variant of the reactive site loop portion of the AAT protein comprises at least the amino acid sequence of SEQ ID NO:32 or SEQ ID NO:33. In one embodiment, the serpin polypeptide of the serpin-albumin fusion protein comprises a full-length human AAT polypeptide sequence having the amino acid sequence of at least SEQ ID NO:2. In one embodiment, the serpin polypeptide of the serpin-cytokine targeting fusion protein comprises or is derived from a full-length human AAT polypeptide sequence having the amino acid sequence of at least SEQ ID NO:80. In one embodiment, the serpin polypeptide of the serpin-albumin fusion protein has the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80 and at least 50%, 60%, 65%, 70%, 75%, 80%, Contains 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical human AAT polypeptide sequence.

In one embodiment, the serpin polypeptide of the serpin-albumin fusion protein is described 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, or an AAT polypeptide sequence derived therefrom, or an amino acid sequence derived from an AAT polypeptide. .

In one embodiment, the fusion protein is modified to increase or inhibit proteolytic cleavage, for example, by mutation of one or more proteolytic cleavage sites. In one embodiment, the fusion protein is modified to alter or modulate the Fc effector function of the fusion protein while simultaneously maintaining binding and inhibitory functions compared to an unmodified fusion protein. Fc effector functions include, but are not limited to, Fc receptor binding, prevention of release of proinflammatory mediators by binding to receptors, phagocytosis, and transformation. Modified antibody-dependent cell-mediated cytotoxicity (ADCC), modified complement-dependent cytotoxicity (CDC), glycosylation of Asn297 residue of modified Fc polypeptide (modified glycosylation) (EU index of Kabat numbers, Kabat et al 1991 Sequences of Proteins of Immunological Interest ). In one embodiment, the fusion protein is mutated or modified to affect Fc receptor binding. In one embodiment, the Fc polypeptide is modified to enhance FcRn binding. Examples of Fc polypeptide mutations that enhance binding to FcRn include Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E) (Kabat number, 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 using the Kabat numbering system. These are t428Val and Asn434Ser (M428V, N434S). In one embodiment, the mutated or modified Fc polypeptide is a group consisting of Met252Tyr (M252Y), Ser254Thr (S256T), Thr256Glu (T256E), Met428Leu (M428L), Met428Val (M428V), Asn434Ser (N434S), and combinations thereof. Contains one or more mutations selected from. In one embodiment, the Fc polypeptide portion is mutated or modified to interfere with Fc-mediated dimerization (Ying et al 2012 J. Biol Chem 287 (23): 19399-19408). In this embodiment, the fusion protein is monomeric in nature.

The fusion proteins and variants thereof disclosed in the present invention have inhibitory activities, such as human neutrophil elastase (NE), chymotrypsin-fold serine protease and Inhibits serine proteases, such as . The fusion proteins provided herein bind or interact with serine proteases, such as human serine proteases, to completely or partially reduce or regulate serine protease expression or activity. Reduction or modulation of the biological function of the serine protease is complete or partial by the interaction of the fusion protein with the human serine protease protein, polypeptide and/or peptide. The level of serine protease expression or activity in the presence of the fusion protein is at least 95% compared to the level of serine protease expression or activity in the absence of interaction, such as binding to the fusion protein of the present invention, e.g. When reduced by 96%, 97%, 98%, 99% or 100%, the fusion protein is believed to completely inhibit serine protease expression or activity. The level of serine protease expression or activity in the presence of the fusion protein is less than 95% compared to the level of serine protease expression or activity in the absence of interaction, such as binding to the fusion protein of the present invention, for example. When reduced by 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85% or 90%, the fusion protein partially inhibits serine protease expression or activity. It is believed that it does.

The fusion proteins described herein are useful for a variety of therapeutic, diagnostic and prophylactic indications. For example, the fusion proteins are useful for treating various diseases and disorders in a subject. In one embodiment, the serpin fusion proteins comprising the fusion proteins described herein may be used to treat alpha-1-antitrypsin (AAT) deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute Acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, cancers of the lung, for example, ischemia/reperfusion injury after heart transplantation ischemia-reperfusion injury, including following cardiac transplantation, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis (ankylosing spondylitis), Crohn's disease, psoriasis, type I and/or type II diabetes, bacterial infection, fungal infection, viral infection, pneumonia ), a disease or disorder selected from sepsis, graft versus host disease (GVHD), wound healing, systemic lupus erythematosus, and multiple sclerosis It is useful for treating, relieving, ameliorating symptoms and/or delaying the progression of a disease or disorder in an individual suffering from or confirmed to have said disease.

The pharmaceutical composition according to the present invention comprises a fusion protein of the present invention, including modified fusion proteins and other variants, and an acceptable carrier. The pharmaceutical composition of the present invention may include a kit, for example, a diagnostic kit.

1A is a schematic diagram showing one embodiment of a Serpin-Fc fusion protein according to the present invention. Serpin-Fc fusion proteins comprising one or more serpin polypeptides are also shown. Figure 1B is a photograph of an SDS-PAGE gel showing serum-derived AAT (lane 1), AAT-Fc1 (lane 2, human IgG1 Fc) and AAT-EL-Fc1 (lane 3, Met351Glu, Met358Leu mutation in AAT, human IgG1 Fc). am. Figure 1c is a graph showing inhibition of neutrophil elastase activity by AAT-Fc fusion protein. 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 inhibition of neutrophil elastase activity by a tetravalent AAT-Fc-AAT fusion protein. Figure 1f is a graph demonstrating the low elution effect from Protein A resin, and the NE inhibition capacity of the AAT-Fc fusion protein eluted at low pH was drastically reduced. Figure 1g is a graph showing the double mutant, resistance of AAT-EL-Fc (Met351Glu, Met358Leu mutant) to H ₂ O ₂ inactivation (conc.) compared to wild-type AAT and single mutant AAT-EM-Fc ( It is shown in comparison with Met351Glu). Figure 1H is a graph depicting the serum clearance rate of serum derived AAT (sdAAT) compared to AAT-Fc in rats administered 10 mg/kg protein (3 rats/test protein). The half-life of AAT-Fc is generally longer than that of sdAAT.
Figure 2a is a schematic diagram showing one embodiment of the serpin-cytokine targeting fusion protein of the present invention. The serpin may be fused to the heavy chain, light chain, or both heavy and light chains of the antibody. Serpin-cytokine receptor fusion proteins are also depicted. Figure 2B is a photograph of an SDS-PAGE gel showing the D2E7 antibody (lane 1) and the D2E7 antibody with AAT-fused heavy chain (lane 2). Figure 2c is a graph showing inhibition of neutrophil elastase activity by D2E7 antibody fused with AAT. Serum-derived AAT is shown as a positive control, and D2E7 antibody alone is shown as a negative control for NE inhibition.
3a is a schematic representing one embodiment of a serpin-Fc-WAP fusion protein. 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 inhibition of neutrophil elastase activity by AAT-Fc-ELAFIN fusion protein and AAT-Fc-SLPI fusion protein. AAT-FC fusion protein and serum-derived AAT are included for comparison.
Figure 4A is a schematic representing one embodiment of an AAT-HSA fusion protein. Figure 4b is a SDSPAGE gel image showing AAT-HSA fusion. Figure 4c is a graph showing the inhibition of neutrophil elastase activity by AAT-HSA compared with serum-derived AAT.

Human neutrophil elastase (NE) is a chymotrypsin-fold serine protease secreted by neutrophils during inflammation. Abnormal activity of NE causes progressive degradation of elastin tissue and slow destruction of the lung-like structure of the lung, causing emphysema and pulmonary fibrosis (Lungarella et al 2008 Int. J. Biochem Cell Biol 40:1287). Often, misunderstood NE activity is due to an imbalance between proteases and its natural inhibitor, alpha-antitrypsin (AAT). This imbalance may be the result of increased neutrophil infiltration into the lungs, as observed in the lungs of smokers and patients with Cystic Fibrosis (CF) or Acute Respiratory Distress Syndrome (ARDS). there is. Conversely, deficiency of AAT, usually as a result of point mutations that cause aggregation and accumulation of AAT in the liver, leaves the lungs exposed to uninhibited NE activity. Individuals deficient in AAT have an increased risk of emphysema, COPD, liver disease, and many other conditions.

AAT deficiency affects approximately 100,000 Americans (according to estimates by the Alpha-1 Foundation), with many sufferers dying in their 30s and 40s. Currently, only a few FDA-approved drugs exist to treat AAT deficiency (Prolastin®, Aralast™, Zemaira®, Glassia™). Each drug is a natural AAT derived from human plasma, which appears to be insufficient to meet expected clinical needs. Additionally, these drugs have a short serum half-life (T _1/2 of about 5 days) and require weekly administration in high doses (60 mg/kg of body weight). The current market for such drugs is estimated at approximately $400 million. The market for AAT-like drugs is driven by speculation that as many as 95% of individuals with AAT-deficiency are undiagnosed and that these drugs may be used to treat pathologies characterized by enhanced NE activity (e.g., cystic cysts) in non-AAT-deficient individuals. Based on the fact that it has the potential to be effective in the treatment of chronic fibrosis (CF), acute respiratory distress syndrome (ARDS), smoking-induced emphysema and/or COPD), it is expected to grow substantially.

AAT has been thought to have broad-spectrum anti-inflammatory activity (Tilg et al 1993 J Exp Med 178:1629-1636, Libert et al 1996 Immunol 157:5126-5129, Pott et al , Journal of Leukocyte Biology 85 2009, Janciauskiene et al 2007 J. Biol Chem 282 (12): 8573-8582, Nita et al 2007 Int J Biochem Cell Biol 39:1165-1176). Recently, evidence has presented that AAT may be useful in treating a variety of human pathologies beyond the inflammatory lung diseases for which it is commonly recommended. Human AAT has been shown to protect mice from clinical and histopathological signs of experimental autoimmune encephalomyelitis (EAE), autoimmune diseases such as multiple sclerosis or systemic lupus erythematosus (SLE). It appears to have therapeutic potential (Subramanian et al 2011 Metab Brain Dis 26:107-113). Serum AAT has been shown to be active in a rodent model 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 Steroid Non-responsive Acute GVHD (NCT01523821). In addition, AAT was effective in reducing inflammation in type I and type II diabetes animal models, preventing islet cell apoptosis, and providing islet cell allograft durability (Zhang et al 2007 Diabetes 56: 1316-1323, Lewis et al 2005 Proc Natl Acad Sci USA 102:12153-12158, Lewis et al 2008 Proc Natl Acad Sci USA 105:16236-16241, Kalis et al 2010 Islet 2:185-189). Currently, many early human clinical trials are being attempted for type I diabetes using serum-derived AAT substances (NCT01183468, NCT01319331, NCT01304537).

Currently, serum-derived AAT materials are undergoing extensive purification and testing to ensure removal of pathogenic viruses, but the risk of transmission of infectious agents cannot be completely eliminated. Additionally, serum is limited, and this limitation limits the ability to produce serum-derived AAT. We aimed to express recombinant AAT in an attempt to solve problems with serum-derived materials and production issues. However, after 20 years of research, the production of therapeutically viable recombinants has not yet reached the market (Karnaukhova et al 2006 Amino Acids 30: 317). Like the plasma-derived products, the recombinant version of AAT has a short serum half-life, low production yield, and poor pulmonary distribution.

The fusion proteins of the invention have improved functionality compared to unmodified AAT molecules. Fusion of an AAT polypeptide and a second polypeptide that interacts with the neonatal Fc receptor (FcRn) increases serum half-life, providing a much-needed dosing benefit to patients. The FcRn interacting polypeptides of the fusion proteins include immunoglobulin (Ig) Fc polypeptides derived from human IgG1, IgG2, IgG3, IgG4 or IgM and human albumin derivatives. In one embodiment, the fusion protein comprises a mutation in the AAT portion that renders the molecule more resistant to oxidative inactivation. For example, Met351Glu, Met358Leu (AAT-EL-Fc) is resistant to inactivation by _{HO oxidation} (Figure 1g). Although AAT is a natural anti-inflammatory protein, one embodiment of the invention provides enhanced inflammation reduction capabilities through the fusion of an AAT polypeptide and a cytokine targeting polypeptide. Combining the dual anti-inflammatory functions of AAT and a second polypeptide will provide a more potent therapeutic protein than either polypeptide on its own. Additionally, the combination of AAT's anti-infective activity will mitigate the infection risk of most cytokine-targeted biologics. One embodiment provides a more potent anti-inflammatory and anti-infective protein through the fusion of an AAT-polypeptide and a polypeptide comprising a WAP domain. The fusion protein of the present invention is expected to have excellent therapeutic utility and is expected to be superior to conventional serum-derived AAT products.

In order to extend the half-life of recombinant AAT, recombinant DNA technology was used to create an AAT gene fused to the Fc domain of human gG1, IgG2, IgG3, IgG4, IgM or HSA, and the protein product was produced with AAT followed by an Fc domain (AAT- Fc (IgG1), AAT-Fc (IgG2), AAT-Fc (IgG3), AAT-Fc (IgG4), AAT-Fc (IgM)), HSA are expected to come. It is known that fusion of the Fc domain of HSA with other proteins, protein domains or peptides can increase the half-life ( see eg , Jazayeri et al. BioDrugs 22 , 11-26, Huang et al. (2009) Curr Opin Biotechnol 20 , 692-699, Kontermann et al. (2009) BioDrugs 23 , 93-109, Schmidt et al. (2009) Curr Opin Drug Discov Devel 12 , 284-295), AAT fused to the Fc domain or HSA ensures proper folding and NE It is not known whether this will maintain the inhibitory activity or increase the half-life of recombinant AAT. The fusion proteins of the invention have been shown to be potent inhibitors of NE, have a prolonged serum half-life, and, in one embodiment, are resistant to oxidation. In other embodiments, the fusion proteins described herein have different properties by fusion with other functional polypeptides, including a cytokine targeting polypeptide and a WAP domain containing polypeptide.

Neutrophils, the main source of neutrophil elastase (NE), often undergo an oxidative burst concurrent with the secretion of NE. Therefore, the fusion protein of the present invention, which shows activation in an oxidizing environment, is very useful therapeutically. Oxidation of AAT at Met351 and or Met358 within the reactive site loop weakens the ability of AAT to inhibit neutrophil elastase. As shown in Figure 1g, oxidative inactivation can be reduced through mutations in Met351Glu and Met358Leu (M351E/M358L) shown in SEQ ID NO:32. Additionally, it has been shown that Met252 and Met428dml oxidation at the Fc region reduces FcRn binding and subsequently reduces the serum half-life of Fc-containing proteins. Mutations in Met252 and Met428 reduce oxidation of the Fc region. The present invention discloses an optimized AAT-Fc fusion protein, comprising oxidation-inactivation through mutations of M351 and M358 in the AAT portion of the fusion protein; and resistance to oxidative destruction of the FcRn interaction through mutations in M252 and M428, and an extended half-life through a set of mutations in M252I, T256D, and M428L.

In one embodiment where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide is mutated or modified to enhance FcRn binding. In this embodiment the mutated or modified Fc polypeptide comprises the following mutations (using the Kabat numbering system): Met252Ile, Thr256Asp and Met428Leu (M252I, T256D, M428L).

In one embodiment where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide is a modified IgG1 Fc polypeptide, and the fusion protein comprises at least the amino acid sequence of SEQ ID NO:53.

In one embodiment where the fusion protein of the invention comprises an Fc polypeptide, the Fc polypeptide is a modified IgG1 Fc polypeptide, and the fusion protein comprises at least the amino acid sequence of SEQ ID NO:73.

In one embodiment where the fusion protein of the present invention includes an Fc polypeptide, the Fc polypeptide is modified or mutated to reduce binding to Fc-gamma receptors (FcgRs). In one embodiment, reduced FcgRs binding can be achieved by modification of Fc glycosylation at Asn297. For example, mutations in Asn297Ala (N297A) or Asn297Gln (N297Q). In one embodiment, reduced FcgRs binding is achieved by modification of the lower hinge region of the Fc. In one embodiment, the Fc polypeptide is derived from human IgG1. In one embodiment, the lower hinge region is modified to mimic that of IgG2 through mutations of Leu234Val and Leu235Ala (L235V/L235A) and deletion of Gly236 (ΔG236) using the Kabat numbering system:

In one embodiment, the fusion protein comprises at least the amino acid sequence of SEQ ID NO:51.

In one embodiment, the Fc polypeptide is from human IgG4. In one embodiment, the lower hinge region is modified by mutation of Leu235Glu (L235E). Additionally, in embodiments of the invention in which the Fc polypeptide is derived from human IgG4, the hinge region is modified through a stabilizing mutation at Ser228Pro (S228P) in the Kabat numbering system:

In one embodiment, the fusion protein comprises at least the amino acid sequence of SEQ ID NO:70.

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

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

In one embodiment, the fusion proteins described herein comprise at least a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide and a cytokine targeting polypeptide or an amino acid sequence derived from a cytokine targeting polypeptide. For example, the present invention provides sequences from serpin polypeptides or serpin polypeptides fused to human cytokine receptors or derivatives thereof. Another embodiment of the invention is a cytokine targeting antibody, e.g., an anti-cytokine antibody, or a sequence derived from a cytokine targeting antibody, e.g., an anti-cytokine antibody, or a fragment of a cytokine targeting antibody. Provided are sequences, for example, serpin polypeptides or sequences derived from serpin polypeptides fused to fragments of anti-cytokine antibodies. For example, the present 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 the following human cytokines: TNFa, IgE, IL-12, IL-23, IL-6, IL-1a, IL-1b, IL-17, IL-13, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

For example, in one embodiment, the cytokine targeting polypeptide targets TNFa and comprises the following TNFa-targeting polypeptides or sequences derived from the following TNFa-targeting polypeptides: Remicade®, Humira®, Simponi®, Cimiza®, Enbrel ® or ATN-103 and ATN-192.

For example, in one embodiment, the cytokine targeting polypeptide targets IgE and comprises the following IgE-targeting polypeptide or sequence from an IgE-targeting polypeptide: Xolair or FcεRI.

For example, a cytokine targeting polypeptide targets the shared subunit p40 of IL-12 and IL-23 and includes a Stelara® polypeptide or a sequence derived from a Stelara® polypeptide.

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

In one embodiment, the fusion protein described herein comprises at least an alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequence derived from an AAT and a cytokine targeting polypeptide or an amino acid sequence derived from a cytokine targeting polypeptide. For example, the present invention provides an alpha-1-antitrypsin inhibitor (AAT) fused to a cytokine targeting polypeptide, wherein the cytokine targeting polypeptide binds the following human cytokines: TNFa, IgE, IL-6. , IL-1a, IL-1b, IL-12, IL-17, IL-13, IL-23, IL-4, IL-10, IL-2, IL-18, IL-27, or IL-32.

In one embodiment, the cytokine targeting polypeptide binds to the cytokine receptor and prevents binding of the cytokine. For example, the invention includes serpins fused with cytokine receptor targeting antibodies. For example, the present invention provides an alpha-1-antitrypsin inhibitor (AAT) fused to a cytokine targeting polypeptide, wherein the cytokine targeting polypeptide binds to the receptors of the following human cytokines: TNFa, IgE, IL. -6, IL-1a, IL-1b, IL-12, IL-17, IL-13, IL-23, IL-12 and IL-23 subunits p40, IL-4, IL-10, IL-2 , IL-18, IL-27, or IL-32.

For example, in one embodiment, the cytokine targeting polypeptide targets the IL-6 receptor and is an Actemra® polypeptide (described in Patent Publication No. EP0628639), or ALX-0061 polypeptide (described in WO2010/115998), or Actemra ® polypeptide or a sequence derived from the ALX-0061 polypeptide.

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

Targeting inflammatory cytokines and immune-enhancing agents by protein therapy has shown clinical success in many inflammatory diseases. The most common proteins used as cytokine targeting agents are soluble cytokine receptors and monoclonal antibodies and fragments thereof. An important problem with cytokine targeting is the increased risk of infection in patients, which is due to the use of TNFα targeting biologics, Remicade®, Humira®, Simponi®, Cimiza®, and Enbrel®, and IL-12/23 p40 targeting. Antibody, verified by Stelara®. This may be a common problem in targeting inflammatory cytokines that cause immunosuppression in patients. AAT and other serpin proteins are interesting in that both show anti-infectious and anti-inflammatory activities. Therefore, the serpin-cytokine targeting polypeptide fusion protein of the present invention can inhibit abnormal cytokine activity while alleviating the risk of infection.

In one embodiment, the fusion protein described herein comprises a serpin polypeptide or an amino acid sequence from a serpin, an amino acid sequence from a WAP domain-containing polypeptide or a WAP domain-containing polypeptide, and an Fc polypeptide or an amino acid sequence from an Fc polypeptide. do. For example, the invention provides serpin polypeptides, WAP domain-containing polypeptides and human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc or IgM-Fc derivatives operably linked to each other in any functional combination. do. In one embodiment, the WAP domain containing protein is human SLPI or is derived from human SLPI. In another embodiment, the WAP domain containing protein is human ELAFIN or is derived from human elafin. In one embodiment, the fusion protein described herein comprises at least an alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequence derived from AAT and an SLPI polypeptide or an amino acid sequence derived from SLPI. In one embodiment, the fusion protein described herein comprises at least an AAT polypeptide or an amino acid sequence derived from AAT and an elafin polypeptide or an amino acid sequence derived from elafin.

SPLI and Elafin are proteins containing a WAP domain that exhibit serine protease inhibitory activity. The two proteins have anti-inflammatory functions. Additionally, these proteins have broad anti-infective abilities against many species of bacteria, viruses and fungi.

In one embodiment, the fusion protein described herein comprises at least a serpin polypeptide or an amino acid sequence derived from a serpin and a human serum albumin (HSA) polypeptide or an amino acid sequence derived from HSA. A further embodiment of the invention includes a serpin-albumin binding polypeptide fusion protein, wherein the albumin binding polypeptide participates in the association of a serpin and HSA. Accordingly, the present invention includes both covalent and non-covalent combinations of serpin polypeptides and HSA polypeptides or sequences derived from serpin or HSA polypeptides. For example, the invention provides serpin polypeptides fused with human HSA or HSA derivatives, or HSA binding peptides or polypeptides.

In one embodiment, the fusion protein described herein comprises at least an 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. For example, the invention provides alpha-1-antitrypsin (AAT) polypeptides fused with HSA or HSA-derived fragments, or albumin binding polypeptides.

In one embodiment, the fusion protein described herein comprises a serpin polypeptide or an amino acid sequence derived from a serpin, an HSA polypeptide or an amino acid sequence derived from an HSA, and an amino acid sequence derived from a WAP domain-containing polypeptide or a WAP domain-containing polypeptide. In one embodiment, the fusion protein described herein comprises at least an alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequence from AAT and an HSA polypeptide or an amino acid sequence from an HSA polypeptide, and an amino acid sequence from a SLPI polypeptide or SLPI. In another embodiment, the fusion protein described herein comprises at least an 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. Includes sequence.

The fusion proteins of the invention can be easily produced in mammalian cell expression systems. For example, Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) 293 cells, COS cells, PER.C6®, NS0 cells, SP2/0, YB2/0 are used according to the present invention. It can be easily used for the expression of serpin fusion proteins described in . Importantly, mammalian cell expression systems generally produce more optimal proteins for therapeutic use. Unlike bacterial, insect, or yeast-based expression systems, mammalian cell expression systems produce proteins in a glycosylated form, which is similar or identical to that found in native human proteins. Proper glycosylation of proteins can greatly affect serum stability, pharmacokinetics, biodistribution, protein folding, and functionality. Therefore, the ability to produce therapeutic proteins in a mammalian expression system has clear advantages over other systems. Additionally, most mammalian cell expression systems (e.g., CHO, NS0, PER.C6® cells) can be easily scaled up to commercial manufacturing facilities to produce therapeutic proteins for clinical needs. The fusion proteins described herein have improved functionality compared to native AAT and can be produced in mammalian expression systems for clinical and commercial supply. One embodiment of the invention includes a purification system that allows for the isolation of serpin fusion proteins that retain NE inhibitory ability. Importantly, the purification process of the present invention can be readily incorporated into current commercial mammalian cell-based production processes.

Unless otherwise defined, scientific and technical terms used in connection with the present invention will have obvious meanings to those skilled in the art. Additionally, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In general, the nomenclature utilized in connection with cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein, and the techniques thereof are common and obvious in the art. . Standard techniques for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (e.g., electroporation, lipofection) were used. Enzyme reactions and purification techniques were performed according to the manufacturer's instructions, methods commonly performed in the art, or methods described in the present invention. The foregoing techniques and methods are generally performed according to conventional methods obvious to those skilled in the art and as described in the various general and more specific references cited and discussed herein. For example, Sambrook et al . See Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The nomenclature and laboratory procedures and techniques used in connection with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are commonly used and well known in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient care. The term patient includes human and livestock subjects.

Administration of the therapeutic entity according to the present invention may be administered in conjunction with acceptable carriers, buffers, excipients, and other agents included in the formulation to provide improved delivery, transfer, transport, tolerance, etc. You will understand. A number of suitable formulations can be found in formulas known to every pharmaceutical chemist: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), especially Chapter 87 by Blaug, Seymour, in. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vehicles (such as Lipofectin™), DNA conjugates. conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbawax emulsions (water-in-oil) (polyethylene glycols of various molecular weights) (polyethylene glycols), semi-solid gels, and semi-solid mixtures containing carbowax. The foregoing mixtures will be suitable for treatment and treatment according to the present invention, wherein the active ingredients contained in the formulation are not inactivated by the formulation and the formulation is physiologically compatible and appropriate for the route of administration. Also, Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32 (2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203 (1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts.” J Pharm Sci. 89 (8):967-78 (2000), Powell et al . “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the literature cited therein for additional information on formulations, excipients and carriers that should be self-evident to pharmaceutical chemists.

Pharmaceutical formulations of the invention comprising the fusion protein of the invention are used to treat or alleviate symptoms associated with a disease or condition associated with abnormal serine protease activity in a subject. Additionally, the present invention provides a method of treating or alleviating symptoms associated with abnormal serine protease activity and related diseases or disorders in a subject. Therapeutic regimens are performed by identifying an individual, e.g., a human patient suffering from a disease or disorder associated with abnormal serine protease activity, using methods that include a variety of clinical and/or laboratory procedures. The term patient includes humans and livestock. The term subject includes humans and other mammals.

The effectiveness of treatment is determined by known methods for diagnosing or treating specific diseases or disorders associated with abnormal serine protease activity. Alleviation of one or more symptoms of a disease or disorder associated with abnormal serine protease activity indicates that the fusion protein provides clinical benefit.

Methods for screening fusion proteins with desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA), enzyme assay, flow cytometry, and other immunological techniques common in the art. No.

The fusion protein described in the present invention can be used by conventional methods in the art related to localization and/or quantification of targets such as serine proteases, for example, measuring the level of said target in an appropriate physiological sample, diagnostic methods, It can be used for protein imaging, etc. The terms "physiological sample" and "biological sample", used interchangeably herein, refer to tissues, cells and biological fluids isolated from an individual, and tissues present within an individual, Includes cells and body fluids. Accordingly, the use of the terms “physiological sample” and “biological sample” includes blood and fractions or components of blood, including serum, plasma, or lymph fluid.

In a provided embodiment, the fusion protein specific for the target, or a derivative, fragment, analog or homolog thereof containing a target-binding domain, is used as a pharmacologically active compound (hereinafter referred to as “Therapeutics”). It is used.

The fusion proteins of the present invention can be used to isolate specific targets using standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Detection can be facilitated by association (i.e., physical connection) of the fusion protein with 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 substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, Contains dansyl chloride or phycoerythrin; Examples of suitable luminescent materials include luminal; Examples of bioluminescent substances include luciferase, luciferin and aequorin, and suitable radioactive substances include ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

The therapeutically effective amount of the fusion protein of the present invention generally refers to the amount necessary to achieve the therapeutic purpose. As mentioned above, this may be a binding interaction of the fusion protein with its target protein, which in certain cases interferes with the function of the target. Additionally, the amount required for administration will depend on the binding affinity of the fusion protein for a particular target and will depend on the rate at which the administered fusion protein disappears from the free volume of the recipient. A general range for a therapeutically effective dosage of the fusion protein or fragment thereof of the present invention may be, but is not limited to, about 0.1 mg/kg body weight to about 250 mg/kg body weight. Typical frequency of administration would be, for example, twice a day to once a month.

When fusion protein fragments are used, the smallest inhibitor fragment that specifically binds to the target is preferred. For example, a peptide molecule can be designed that retains the ability to bind to the target. Such peptides can be synthesized chemically and/or produced by recombinant DNA techniques (see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993) ). Additionally, the formulation may contain more than one active compound as needed for the specific indication to be treated, preferably with complementary activities that do not exert opposing effects on each other. Alternatively, the composition may also contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapy agents, growth-inhibitory agents. agent), anti-inflammatory agent, or anti-infective agent. These molecules exist in appropriate combinations and in amounts sufficiently effective for the purpose.

Additionally, the active ingredient may be used in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or macroemulsions, e.g. coacervation. ) technology or interfacial polymerization technology, such as hydroxymethylcellulose or gelatin-microcapsules and poly (methyl methacrylate) microcapsules. - (methylmethacrylate) microcapsules), respectively, can be put into.

Formulations used for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filtration membranes.

Sustained-release preparations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the fusion protein, the matrices being in the form of molded articles, for example, films or microcapsules. Examples of sustained-release mattresses include polyesters, hydrogels, such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol). vinylalcohol))), polylactides (U.S. Pat. No. 3,773,919), copolymer of L-glutamic acid and γ ethyl-L-glutamate, 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 Contains -(-)-3-hydroxybutyric acid (poly-D-(-)-3-hydroxybutyric acid). Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release copper molecules for more than 100 days. On the other hand, hydrogels release proteins over a shorter period of time.

Pharmaceutical compositions

The fusion proteins of the invention (also referred to as “active compounds or active compounds”), and their derivatives, fragments, analogs and homologs, may be included in pharmaceutical compositions suitable for administration. Such compositions include, among other things, a fusion protein and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” used in the present invention includes all solvents, dispersions, coatings, antibiotics and antifungal agents, isotonic agents and absorption delay agents, and the like, suitable for pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, which is incorporated herein by reference as a standard reference work in the art. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional vehicle or agent is contemplated for use in the composition, except in cases where it is incompatible with the active compound.

The pharmaceutical composition of the present invention can be formulated to suit the desired administration route. Examples of routes of administration include parenteral administration, e.g. intravenous, intradermal, subcutaneous, oral (e.g. inhalation), transdermal (i.e. , topical, transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal or subcutaneous use may contain the following ingredients: water for injection, saline, fixed oils, polyethylene glycols, glycerine, propylene glycol or sterile diluents such as other synthetic solvents; Antibiotics such as benzyl alcohol or methyl parabens; Antioxidants such as ascorbic acid or sodium bisulfate; Chelating agents such as EDTA (ethylenediaminetetraacetic acid); Buffering agents such as acetate, citrate or phosphate, 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. Parenteral preparations may be enclosed in ampoules, disposable syringes or infusion bottles made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (soluble in water) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid so that it can be easily injected. It must be stable under the conditions of manufacture and storage and must be prevented from contamination by microorganisms such as bacteria and fungi. The carrier may be, for example, a solution or dispersion medium containing water, ethanol, polyols (e.g., glycol, propylene glycol and polyethylene glycol liquids, etc.) and appropriate mixtures thereof. Adequate fluidity can be maintained, for example, by coatings such as lecithin, by maintaining the required particle size during dispersion and by the use of surfactants. The activity of microorganisms can be inhibited by various antibiotics and antifungal agents, and examples of antibiotics and antifungal agents include parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it will be desirable to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by including agents in the composition that delay absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount and one or a combination of ingredients enumerated above in an appropriate solvent, if necessary, followed by filter sterilization. In general, dispersions can be prepared by incorporating the active compound into a sterile vehicle, which can contain the basic dispersion medium and any other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze-drying to obtain powders of the active ingredient and other additional desired ingredients from the sterile-filtered solution.

Oral compositions generally include an inert diluent or edible carrier. The substances are contained in gelatin capsules or compressed into tablets. For oral administration of therapeutic agents, the active compounds may be included together with excipients and may be used in the form of tablets, troches or capsules. Additionally, the oral composition can be prepared as a mouthwash using a liquid carrier, and the compound contained in the liquid carrier is applied orally, moved in the mouth, and spit out or swallowed. Pharmaceutically compatible binders and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, troches and the like may contain the following ingredients, or compounds of similar nature: microcrystalline cellulose, gum tragacanth or gelatin. Same binder; Additives such as starch or lactose, disintegrating agents such as alginic acid, Primogel, or corn starch; Lubricants such as Magnesium Stearate or Sterotes; Glidants such as colloidal silicon dioxide, sweeteners such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For inhalation administration, the compound is delivered in the form of an aerosol spray from a compressed container or dispenser containing a suitable propellant, for example carbon dioxide, or from a nebulizer.

Additionally, systemic administration may be administered via mucosal or transdermal methods. For mucosal or transdermal administration, penetrants suitable for penetrating the intestinal wall are used in the formulation. Such penetrants are well known in the art and include, for example, detergents for mucosal administration, bile salts and fusidic acid derivatives. Mucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are prepared into ointments, salves, gels or creams commonly known in the art.

Additionally, the compounds may be prepared in the form of suppositories (e.g., conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for delivery to the rectum.

In one embodiment, the active compound is prepared with a carrier that can protect the compound from rapid elimination from the body, such as controlled release formulations including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions can also be used as pharmaceutically acceptable carriers. This is a method that is obvious to those skilled in the art, for example, the U.S. method. Patent No. It can be prepared according to the method described in No. 4,522,811.

It is particularly desirable to formulate oral or parenteral compositions in dosage unit forms for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; It means each unit containing a pre-quantified active compound calculated to produce the desired therapeutic effect together with the pharmaceutical carrier. The specification for the unit dosage form of the invention is influenced and directly dependent on the unique properties of the active compound and the particular therapeutic effect to be achieved and the limitations inherent in synthesizing the active compound for the treatment of a subject.

The pharmaceutical composition may be contained in a container, pack, or dispenser with instructions for administration.

The present invention will be further illustrated in the following examples, which do not limit the scope of the invention as set forth in the claims.

Example

Example 1: AAT-Fc fusion proteins and variants

Typical, but non-limiting examples of AAT-Fc fusion proteins according to the invention include the following sequences. Although these examples include hinge sequences and/or linker sequences, fusion proteins of the invention can be made using any hinge sequence and/or linker sequence of appropriate length and/or flexibility. Alternatively, fusion proteins may be made without hinge sequences and/or linker sequences. For example, polypeptide constructs can be attached directly.

An example of an AAT-Fc fusion protein is AAT-hFc1 (human IgG1 Fc) described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2) and the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 3).

AAT-hFc1 (human IgG1 Fc)

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK (SEQ ID NO:16)

An example of an AAT-Fc fusion protein is AAT-hFc2 (human IgG2 Fc) described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2) and the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 4).

AAT-hFc2 (human IgG2 Fc)

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17)

An example of an AAT-Fc fusion protein is AAT-MM-EL-hFc1 (human IgG1 Fc, Met351Glu/Met358Leu) described herein. As shown below, the 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. is indicated, and the Met358Leu mutation is shaded in gray.

AAT-MM-EL-hFc1 (human IgG1 Fc, Met351Glu/Met358Leu)

An example of an AAT-Fc fusion protein is AAT-MM-EL-hFc2 (human IgG2 Fc, Met351Glu/Met358Leu) described herein. As shown below, the 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), and the Met351Glu mutation is It is indicated by a box, and the Met358Leu mutation is shaded in gray.

AAT-MM-EL-hFc2 (human IgG2 Fc, Met351Glu/Met358Leu)

An example of an AAT-Fc fusion protein is AAT-MM-LL-hFc1 (human IgG1 Fc, Met351Leu/Met358Leu) described herein. As shown below, the 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), and the Met351Leu mutation is It is shaded in black, and the Met358Leu mutation is shaded in gray.

AAT-MM-LL-hFc1 (human IgG1 Fc, Met351Leu/Met358Leu)

An example of an AAT-Fc fusion protein is AAT-MM-LL-hFc2 (human IgG2 Fc, Met351Leu/Met358Leu) described herein. As shown below, the 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), and the Met351Leu mutation is It is indicated with black shading, and the Met358Leu mutation is shaded with gray.

AAT-MM:LL-hFc2 (human IgG2 Fc, Met351Leu/Met358Leu)

An example of an AAT-Fc fusion protein is AAT-hFc1-AAT (human IgG1) described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2) and the IgG-Fc polypeptide portion of the fusion protein is italicized (SEQ ID NO: 3).

AAT-hFc1-AAT (human IgG1)

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQKEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:21)

AAT-EL-Fc-IgG1-DV,ΔG,IDL (AAT: Met351Glu/Met358Leu; Fc IgG1: Leu234Val/Leu235Ala, Gly236 deletion, Met252Ile, Thr256Asp, Met428Leu)

AAT-EL-Fc-IgG4-PE, IDL (AAT: Met351Glu/Met358Leu; Fc IgG4: Ser228Pro Leu235Glu, Met252Ile, Thr256Asp, Met428Leu)

This exemplary AAT-Fc fusion protein was made using the following techniques.

The gene encoding human AAT was PCR amplified from human liver cDNA. Specific point mutations within the AAT or Fc region were generated through overlapping PCR. The AAT encoding gene was cloned into a mammalian expression vector in the frame containing the gene encoding the hinge region, followed by the CH2 and CH3 domains of human IgG1, IgG2, IgG3, IgG4, or IgM, and the vector was used to insert the AAT gene. Contains a mammalian secretion signal sequence upstream of the branch. These expression vectors were transfected into mammalian cells (particularly HEK293 or CHO cells) and grown for several days at 8% CO ₂ and 37°C. Recombinant AAT-Fc fusion protein was purified via protein A chromatography from supernatants of expressing cells. Importantly, a near-neutral pH buffer (Gentle Ag/Ab Elution Buffer, Thermo Scientific) was used to elute the AAT-Fc fusion protein from Protein A resin. The AAT-Fc fusion protein cannot be extracted using standard low pH elution on protein A resin, possibly because the ability of AAT to inhibit NE may be impaired following low pH treatment. This is likely due to oligomerization of the mediating AAT. Figure 1F shows the effect of low pH elution on the ability of AAT to inhibit neutrophil elastase. The AAT-Fc fusion protein can be purified by CaptureSelect® Alpha-1 Antitrypsin affinity matrix (BAC BV) using Protein A and a nearly neutral elution buffer.

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

A fluorescence microplate assay was used to monitor human neutrophil elastase (NE) activity. Inhibitory activity was measured through concomitant reduction of residual NE activity using the following assay. The assay buffer consisted of 100mM Tris pH 7.4, 500mM NaCl, and 0.0005% Triton X-100. Human NE was used at a final concentration of 5 nM (but can also be used at 1-20 nM). The fluorescent peptide substrate AAVP-AMC was used in the assay at a final concentration of 100 μM. A Gemini EM plate reader from Molecular Devices was used to read the analytical kinetics using excitation and emission wavelengths of 370 nm, 440 nm, and 420 nm, respectively, and a cutoff wavelength of 420 nm. Analysis was read every 5 to 10 seconds for 10 minutes at room temperature. Vmax per second corresponds to the residual NE activity, which is plotted for each concentration of inhibitor. The x-axis intercept represents the concentration of inhibitor required to completely inactivate the starting concentration of NE in the assay. Human serum derived AAT (sdAAT) was used as a positive control in these tests. As shown in Figure 1c, the AAT-Fc fusion protein exhibits potent NE inhibitory activity. The fusion (AAT-Fc-AAT), in which two AAT polypeptides are fused to one Fc polypeptide, shows enhanced ability compared to the AAT-Fc fusion protein containing sdAAT and one AAT (FIG. 1e). These findings disclosed herein demonstrate for the first time that AAT can be fused to the Fc region while retaining the ability to inhibit NE. What is more interesting is that AAT-Fc-AAT is a more potent NE inhibitor.

Figure 1f shows the resistance of AAT-EL-Fc (M351E, M358L) fusion proteins to inactivation by oxidation. AAT fusion proteins, AAT-Fc (wild type), AAT-EL-Fc (M351E, M358L), and AAT-EM-Fc (M351E) were treated with 33mM H ₂ O ₂ and then compared with untreated fusion proteins for NE inhibition. were compared by test. NE inhibition by AAT-EL-Fc was not impaired, contrary to other proteins tested.

Additionally, the AAT-Fc fusion protein showed a longer serum half-life than serum-derived AAT in rats (Figure 1h). In this study, 3 rats for each test protein were injected intravenously (I.V.) with sdAAT or AAT-Fc at a concentration of 10 mg/kg. Serum samples were collected at various time points over 48 hours. Serum AAT concentrations were measured using ELISA. These findings demonstrate that the fusion proteins of the invention have improved pharmacokinetic properties and are a superior therapeutic form over serum-derived AAT for treating a large number of human inflammatory and infectious diseases.

Example 2: AAT-TNFa Targeting Molecular Fusion Proteins

The studies described herein disclose several non-limiting examples of recombinant AAT derivatives, including human AAT fused with anti-TNFα or derivatives of the TNFα receptor. These embodiments disclosed below are provided to illustrate the distinguishing features of the invention. The above embodiments also illustrate useful methods for carrying out the invention. These examples are not intended to limit or limit the invention as claimed.

The following fusion proteins are derived from (i) the anti-TNFα antibody D2E7 (also known as Adalimumab or Humira®), or (ii) the extracellular domain of the type 2 TNFα-receptor (TNFR2-ECD). Includes cytokine targeting polypeptide sequences produced or derived therefrom. The AAT polypeptide portion of the fusion protein is underlined, the antibody constant region (CH1-hinge-CH2-CH3, or r CL) is in italics, and D2E7-VH, D2E7-VK, and TNFR2-ECD are in bold. displayed. Although this example includes a hinge sequence and/or linker sequence, the fusion proteins of the invention may be made using a hinge sequence and/or linker sequence of appropriate length and/or flexibility. Alternatively, fusion proteins may be made without hinge and/or linker sequences.

One example of an AAT-TNFα fusion protein is the D2E7-Light Chain-AAT (G ₃ S) ₂ linker described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), ID2E7-VK is in bold (SEQ ID NO: 37), and the antibody constant region is in italics. (SEQ ID NO: 38).

D2E7-light chain-AAT (G ₃ S) ₂ Linker (D2E7-Light Chain-AAT (G ₃ S) ₂ Linker)

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGSGGGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:22)

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

D2E7-Light Chain-AAT ASTGS Linker

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:23)

An example of an AAT-TNFα fusion protein is the D2E7-heavy chain-AAT (G ₃ S) ₂ linker described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VH is in bold (SEQ ID NO: 39), and the antibody constant region is in italics. (SEQ ID NO: 40).

D2E7-Heavy Chain-AAT (G ₃ S) ₂ Linker (D2E7-Heavy Chain-AAT (G ₃ S) ₂ Linker)

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:24)

An example of an AAT-TNFα fusion protein is the D2E7-heavy chain-AAT ASTGS linker described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VK is in bold (SEQ ID NO: 39), and the antibody constant region is in italics. (SEQ ID NO: 40).

D2E7-Heavy Chain-AAT ASTGS Linker

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:25)

An example of an AAT-TNFα fusion protein is the NFR2-ECD-Fc1-AAT (G ₃ S) ₂ linker described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), TNFR2-ECD is in bold (SEQ ID NO: 41), and the antibody constant region is in italics. (SEQ ID NO: 42).

TNFR2-ECD-Fc1-AAT (G ₃ S) ₂ Linker (TNFR2-ECD-Fc1-AAT (G ₃ S) ₂ Linker)

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQP TPEPSTAPSTSFLLPMGSPSPPAEGSTGD EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGSGGGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTE EEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:26)

An example of an AAT-TNFα fusion protein is the TNFR2-ECD-Fc1-AAT ASTGS linker described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2), TNFR2-ECD is in bold (SEQ ID NO: 41), and the antibody constant region is in italics. (SEQ ID NO: 42).

TNFR2-ECD-Fc1-AAT ASTGS Linker (TNFR2-ECD-Fc1-AAT ASTGS Linker)

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQP TPEPSTAPSTSFLLPMGSPSPPAEGSTGD EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO:27)

The AAT-TNFα target molecule fusion protein of the above example was created using the following techniques.

Genes encoding the variable heavy (VH) and variable kappa (VK) regions of the anti-TNFα antibody, D2E7, were formed through gene synthesis. The D2E7-VH gene is a mammalian expression vector containing a mammalian secretion signal sequence upstream of the VH domain insertion point in frame with a human IgG1 antibody heavy chain constant region consisting of a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. was cloned (D2E7-HC). The D2E7-VK gene was cloned into a mammalian expression vector containing the mammalian secretion signal sequence upstream of the VK domain insertion point in frame with the human antibody kappa light chain constant (CL) domain (D2E7-LC). ). The 5' linker sequence adjacent to the gene encoding AAT is a sequence encoding the CH3 domain of the D2E7 heavy chain gene (D2E7-HC-AAT) or the (D2E7-LC-AAT) CL domain of the D2E7 light chain gene of the mammalian expression vector disclosed above. It was cloned within frame at the 3' end of . The extracellular domain of TNFα receptor 2 (TNFR2-ECD) was formed through gene synthesis, in frame with the gene encoding the hinge region, followed by the CH2 domain of human IgG1 (hFc1), followed by the CH3 domain, resulting in TNFR2- It was cloned into a mammalian expression vector containing a mammalian secretion signal sequence upstream of the ECD insertion point. The 5' linker sequence adjacent to the gene encoding AAT was cloned into a mammalian expression vector in frame at the 3' end of the gene encoding TNFR2-ECD-hFc1 (TNFR2-ECD-hFc1-AAT).

To generate a D2E7 antibody with AA fused to the C-terminus of the light chain or to the C-termini of the heavy and light chains, respectively, the D2E7-HC-AAT expression vector is used in combination with the D2E7-LC or D2E7-LC-AAT expression vector in a mammalian form. Cells (especially HEK293 or CHO cells) were co-transfected. D2E7-LC-AAT was co-transfected into mammalian cells with the D2E7-H C expression vector to form a D2E7 antibody in which AAT was fused to the C-terminus of the light chain. Mammalian cells were transfected with the TNFR2-hFc1-AAT expression vector. Transfected cells were grown under 8% CO ₂ and 37°C for several days.

The recombinant AAT-TNFα fusion protein was purified from the supernatant of expressing cells by protein A chromatography. A nearly neutral pH buffer (Gentle Ag/Ab Elution Buffer, Thermo Scientific) was used to elute the protein A resin.

Figure 2b shows an SDS-PAGE gel of D2E7 alone (lane 1) and a variant with AAT fused to the heavy chain of D2E7 (lane 2). Proteins were visualized via Coomassie blue.

Purified AAT-TNFα targeting molecule fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase (NE). Human serum derived AAT (sdAAT) was used as a positive control in these tests. NE inhibition assay was performed as described above. Figure 2c demonstrates that the AAT-TNFα target molecule fusion protein showed similar neutrophil elastase inhibition to sdAAT, indicating that the inhibitory ability of AAT was not impaired even by the antibody fusion.

Example 3 AAT-Fc-SLPI and AAT-Fc-Elafin

The studies described herein disclose several non-limiting examples of recombinant AAT derivatives, including human AAT fused to a protein containing a WAP domain. These embodiments disclosed below are provided to illustrate the distinguishing features of the invention. The above embodiments also illustrate useful methods for carrying out the invention. The AAT polypeptide portion of the fusion protein is underlined, the Fc portion is italicized, and the polypeptide containing the WAP domain is bolded. Although this example includes a hinge sequence and/or linker sequence, the fusion proteins of the invention can be made using any hinge sequence and/or linker sequence of appropriate length and/or flexibility. Alternatively, fusion proteins may be made without hinge and/or linker sequences. For example, the polypeptide component can be attached directly.

An example of an 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 polypeptide containing the WAP domain is in bold. indicated (SEQ ID NO: 9).

AAT-hFc1-SLPI (human IgG1 Fc)

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGS SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGMCGKSCVSPVKA (SEQ ID NO:28)

An example of an 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 polypeptide containing the WAP domain is in bold. indicated (SEQ ID NO: 12).

AAT-hFc1-Elafin (human IgG1 Fc)

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGS AVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQEPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ (SEQ ID NO:29)

Genes encoding SLPI and elafin were PCR amplified from human spleen cDNA (Zyagen). These genes were cloned into the mammalian expression vector of Example 1, where the SLPI or Elafin gene was inserted into frame with the AAT-Fc gene. These expression vectors were transfected into mammalian cells (particularly HEK293 or CHO cells) and grown for several days at 8% CO ₂ and 37°C. The recombinant AAT-Fc-WAP domain fusion protein was purified from the supernatant of expressing cells by protein A chromatography. A near-neutral pH buffer (Gentle Ag/Ab Elution Buffer, Thermo Scientific) was used to elute the AAT-Fc-WAP domain fusion protein from the protein A resin.

Figure 3B shows an SDS-PAGE gel of AAT-Fc-WAP fusion protein (lane 1 AAT-Fc-elafin, lane 2 AAT-Fc-SLPI). Proteins were visualized by staining with Coomassie blue. The activity of purified AAT-Fc-WAP fusion proteins was determined to determine their ability to inhibit neutrophil elastase (NE). NE inhibition tests were performed as described above. Human serum derived AAT (sdAAT) and AAT-Fc fusion protein were used as positive controls in these assays. Compared to sdAAT, the AAT-Fc-WAP target molecule fusion protein exhibits enhanced NE inhibition of neutrophil elastase (Figure 3c).

Example 4 4. AAT-albumin

The studies described herein disclose several non-limiting examples of recombinant AAT derivatives, including human AAT fused to albumin polypeptide. These embodiments disclosed below are provided to illustrate the distinguishing features of the invention. The above embodiments also illustrate useful methods for carrying out the invention. These examples are not intended to limit or limit the invention as claimed. The AAT portion is underlined and the albumin portion is in italics. For example, the polypeptide can be attached directly.

One example AAT-albumin fusion protein is AAT-HSA described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2) and the albumin polypeptide portion is italicized (SEQ ID NO: 14).

AAT-HSA

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK ASTGS ( SEQ ID NO:30)

One example AAT-albumin fusion protein is AAT-HSA domain 3 described herein. As shown below, the AAT polypeptide portion of the fusion protein is underlined (SEQ ID NO: 2) and the albumin polypeptide portion is italicized (SEQ ID NO: 15).

AAT-HSA domain 3

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQ VTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQKASTGSEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA (SEQ ID NO:31)

The gene encoding human serum albumin (HSA) was PCR amplified from human liver cDNA (Zyagen). A mammalian expression vector carrying the mammalian secretion signal sequence upstream of AAT was formed, in which the gene encoding HSA or domain 3 of HSA was cloned in frame at the 3' end of the gene encoding AAT.

These expression vectors were transfected into mammalian cells (particularly HEK293 or CHO cells) and grown for several days at 8% CO ₂ and 37°C. Recombinant AAT-HSA fusion protein was purified from the supernatant of expressing cells using antitrypsin affinity matrix (BAC BV), where the binding buffer consisted of 20mM Tris, 150mM NaCl, pH 7.4, and the elution buffer consisted of 20mM Tris. , 2M MgCl ₂ pH 7.4.

Figure 4B shows an SDS-PAGE gel of AAT-HSA fusion protein visualized by staining with Coomassie blue. The activity of purified AAT-HSA fusion proteins was determined to determine their ability to inhibit neutrophil elastase. NE inhibition assay was performed as described above. Human serum derived AAT (sdAAT) was used as a positive control in these tests. Compared with sdAAT, the AAT-HS fusion protein exhibits similar strong NE inhibition, demonstrating that fusion with albumin did not weaken the NE inhibition ability of AAT (Figure 4c).

other real solar

Although the present invention has been described with a detailed description thereof, the above description is intended to illustrate only and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

SEQUENCE LISTING <110> Inhibrx LP Eckelman, Brendan P. Timmer, John C. Deveraux, Quinn <120> SERPIN FUSION POLYPEPTIDES AND METHODS OF USE THEREOF <130> INHI-002/002WO <150> US 14/524,832 <151> 2014-10-27 <160>80 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> reactive site loop subsequence <400> 1 Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser 1 5 10 15 Ile Pro Pro Glu Val Lys Phe Asn Lys 20 25 <210> 2 <211> 390 <212> PRT <213> Homo sapiens <400> 2 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350 Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn 385 390 <210> 3 <211> 217 <212> PRT <213> Homo sapiens <400> 3 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 4 <211> 216 <212> PRT <213> Homo sapiens <400> 4 Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 180 185 190 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 5 <211> 217 <212> PRT <213> Homo sapiens <400> 5 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Asn Thr Thr Pro Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 6 <211> 217 <212> PRT <213> Homo sapiens <400> 6 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 7 <211> 218 <212> PRT <213> Homo sapiens <400> 7 Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg Asp 1 5 10 15 Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala Thr 20 25 30 Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly Lys 35 40 45 Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala Lys 50 55 60 Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile Lys 65 70 75 80 Glu Ser Asp Trp Leu Gly Gln Ser Met Phe Thr Cys Arg Val Asp His 85 90 95 Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro Asp 100 105 110 Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser Phe Ala Ser 115 120 125 Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp Leu 130 135 140 Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln Asn Gly Glu 145 150 155 160 Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala Thr 165 170 175 Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn Ser 180 185 190 Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser Pro 195 200 205 Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly 210 215 <210> 8 <211> 132 <212> PRT <213> Homo sapiens <400> 8 Met Lys Ser Ser Gly Leu Phe Pro Phe Leu Val Leu Leu Ala Leu Gly 1 5 10 15 Thr Leu Ala Pro Trp Ala Val Glu Gly Ser Gly Lys Ser Phe Lys Ala 20 25 30 Gly Val Cys Pro Pro Lys Lys Ser Ala Gln Cys Leu Arg Tyr Lys Lys 35 40 45 Pro Glu Cys Gln Ser Asp Trp Gln Cys Pro Gly Lys Lys Arg Cys Cys 50 55 60 Pro Asp Thr Cys Gly Ile Lys Cys Leu Asp Pro Val Asp Thr Pro Asn 65 70 75 80 Pro Thr Arg Arg Lys Pro Gly Lys Cys Pro Val Thr Tyr Gly Gln Cys 85 90 95 Leu Met Leu Asn Pro Pro Asn Phe Cys Glu Met Asp Gly Gln Cys Lys 100 105 110 Arg Asp Leu Lys Cys Cys Met Gly Met Cys Gly Lys Ser Cys Val Ser 115 120 125 Pro Val Lys Ala 130 <210> 9 <211> 107 <212> PRT <213> Homo sapiens <400> 9 Ser Gly Lys Ser Phe Lys Ala Gly Val Cys Pro Pro Lys Lys Ser Ala 1 5 10 15 Gln Cys Leu Arg Tyr Lys Lys Pro Glu Cys Gln Ser Asp Trp Gln Cys 20 25 30 Pro Gly Lys Lys Arg Cys Cys Pro Asp Thr Cys Gly Ile Lys Cys Leu 35 40 45 Asp Pro Val Asp Thr Pro Asn Pro Thr Arg Arg Lys Pro Gly Lys Cys 50 55 60 Pro Val Thr Tyr Gly Gln Cys Leu Met Leu Asn Pro Pro Asn Phe Cys 65 70 75 80 Glu Met Asp Gly Gln Cys Lys Arg Asp Leu Lys Cys Cys Met Gly Met 85 90 95 Cys Gly Lys Ser Cys Val Ser Pro Val Lys Ala 100 105 <210> 10 <211> 51 <212> PRT <213> Homo sapiens <400> 10 Thr Arg Arg Lys Pro Gly Lys Cys Pro Val Thr Tyr Gly Gln Cys Leu 1 5 10 15 Met Leu Asn Pro Pro Asn Phe Cys Glu Met Asp Gly Gln Cys Lys Arg 20 25 30 Asp Leu Lys Cys Cys Met Gly Met Cys Gly Lys Ser Cys Val Ser Pro 35 40 45 Val Lys Ala 50 <210> 11 <211> 117 <212> PRT <213> Homo sapiens <400> 11 Met Arg Ala Ser Ser Phe Leu Ile Val Val Val Phe Leu Ile Ala Gly 1 5 10 15 Thr Leu Val Leu Glu Ala Ala Val Thr Gly Val Pro Val Lys Gly Gln 20 25 30 Asp Thr Val Lys Gly Arg Val Pro Phe Asn Gly Gln Asp Pro Val Lys 35 40 45 Gly Gln Val Ser Val Lys Gly Gln Asp Lys Val Lys Ala Gln Glu Pro 50 55 60 Val Lys Gly Pro Val Ser Thr Lys Pro Gly Ser Cys Pro Ile Ile Leu 65 70 75 80 Ile Arg Cys Ala Met Leu Asn Pro Pro Asn Arg Cys Leu Lys Asp Thr 85 90 95 Asp Cys Pro Gly Ile Lys Lys Cys Cys Glu Gly Ser Cys Gly Met Ala 100 105 110 Cys Phe Val Pro Gln 115 <210> 12 <211> 95 <212> PRT <213> Homo sapiens <400> 12 Ala Val Thr Gly Val Pro Val Lys Gly Gln Asp Thr Val Lys Gly Arg 1 5 10 15 Val Pro Phe Asn Gly Gln Asp Pro Val Lys Gly Gln Val Ser Val Lys 20 25 30 Gly Gln Asp Lys Val Lys Ala Gln Glu Pro Val Lys Gly Pro Val Ser 35 40 45 Thr Lys Pro Gly Ser Cys Pro Ile Ile Leu Ile Arg Cys Ala Met Leu 50 55 60 Asn Pro Pro Asn Arg Cys Leu Lys Asp Thr Asp Cys Pro Gly Ile Lys 65 70 75 80 Lys Cys Cys Glu Gly Ser Cys Gly Met Ala Cys Phe Val Pro Gln 85 90 95 <210> 13 <211> 48 <212> PRT <213> Homo sapiens <400> 13 Val Ser Thr Lys Pro Gly Ser Cys Pro Ile Ile Leu Ile Arg Cys Ala 1 5 10 15 Met Leu Asn Pro Pro Asn Arg Cys Leu Lys Asp Thr Asp Cys Pro Gly 20 25 30 Ile Lys Lys Cys Cys Glu Gly Ser Cys Gly Met Ala Cys Phe Val Pro 35 40 45 <210> 14 <211> 585 <212> PRT <213> Homo sapiens <400> 14 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 <210> 15 <211> 196 <212> PRT <213> Homo sapiens <400> 15 Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 1 5 10 15 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 20 25 30 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 35 40 45 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 50 55 60 Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 65 70 75 80 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 85 90 95 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val 100 105 110 Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe 115 120 125 His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 130 135 140 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 145 150 155 160 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys 165 170 175 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 180 185 190 Lys Leu Val Ala 195 <210> 16 <211> 626 <212> PRT <213> Artificial Sequence <220> <223> AAT-hFc1 fusion protein <400> 16 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350 Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Glu Pro Lys Ser Cys Asp 385 390 395 400 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 405 410 415 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 420 425 430 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 435 440 445 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 450 455 460 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 465 470 475 480 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 485 490 495 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 500 505 510 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 515 520 525 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 530 535 540 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 545 550 555 560 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 565 570 575 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 580 585 590 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 595 600 605 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 610 615 620 Gly Lys 625 <210> 17 <211> 622 <212> PRT <213> Artificial Sequence <220> <223> AAT-hFc2 fusion protein <400> 17 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350 Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Glu Arg Lys Cys Cys Val 385 390 395 400 Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe 405 410 415 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 420 425 430 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 435 440 445 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 450 455 460 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val 465 470 475 480 Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 485 490 495 Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 500 505 510 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 515 520 525 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 530 535 540 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 545 550 555 560 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp 565 570 575 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 580 585 590 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 595 600 605 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 610 615 620 <210> 18 <211> 626 <212> PRT <213> Artificial Sequence <220> <223> AAT-MM-EL-hFc1 fusion protein <400> 18 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Glu Phe 340 345 350 Leu Glu Ala Ile Pro Leu Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Glu Pro Lys Ser Cys Asp 385 390 395 400 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 405 410 415 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 420 425 430 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 435 440 445 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 450 455 460 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 465 470 475 480 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 485 490 495 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 500 505 510 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 515 520 525 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 530 535 540 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 545 550 555 560 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 565 570 575 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 580 585 590 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 595 600 605 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 610 615 620 Gly Lys 625 <210> 19 <211> 622 <212> PRT <213> Artificial Sequence <220> <223> AAT-MM-EL-hFc2 fusion Protein <400> 19 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Glu Phe 340 345 350 Leu Glu Ala Ile Pro Leu Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Glu Arg Lys Cys Cys Val 385 390 395 400 Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe 405 410 415 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 420 425 430 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 435 440 445 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 450 455 460 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val 465 470 475 480 Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 485 490 495 Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 500 505 510 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 515 520 525 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 530 535 540 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 545 550 555 560 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp 565 570 575 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 580 585 590 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 595 600 605 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 610 615 620 <210> 20 <211> 622 <212> PRT <213> Artificial Sequence <220> <223> AAT-MM:LL-hFc2 fusion protein <400> 20 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Leu Phe 340 345 350 Leu Glu Ala Ile Pro Leu Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Glu Arg Lys Cys Cys Val 385 390 395 400 Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe 405 410 415 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 420 425 430 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 435 440 445 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 450 455 460 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val 465 470 475 480 Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 485 490 495 Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 500 505 510 Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 515 520 525 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 530 535 540 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 545 550 555 560 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp 565 570 575 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 580 585 590 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 595 600 605 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 610 615 620 <210> 21 <211> 1025 <212> PRT <213> Artificial Sequence <220> <223> AAT-hFc1-AAT fusion protein <400> 21 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350 Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Glu Pro Lys Ser Cys Asp 385 390 395 400 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 405 410 415 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 420 425 430 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 435 440 445 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 450 455 460 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 465 470 475 480 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 485 490 495 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 500 505 510 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 515 520 525 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 530 535 540 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 545 550 555 560 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 565 570 575 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 580 585 590 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 595 600 605 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 610 615 620 Gly Lys Ala Ser Thr Gly Ser Glu Asp Pro Gln Gly Asp Ala Ala Gln 625 630 635 640 Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn Lys 645 650 655 Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln Leu 660 665 670 Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser Ile 675 680 685 Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr His 690 695 700 Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro Glu 705 710 715 720 Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn Gln 725 730 735 Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu Ser 740 745 750 Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys Leu 755 760 765 Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu Ala 770 775 780 Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys Ile 785 790 795 800 Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu Val 805 810 815 Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val Lys 820 825 830 Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val Lys 835 840 845 Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys Lys 850 855 860 Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala Thr 865 870 875 880 Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu Asn 885 890 895 Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp Arg 900 905 910 Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr Tyr 915 920 925 Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe Ser 930 935 940 Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys Leu 945 950 955 960 Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly Thr 965 970 975 Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile Pro 980 985 990 Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu Gln 995 1000 1005 Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 1010 1015 1020 Gln Lys 1025 <210> 22 <211> 616 <212> PRT <213> Artificial Sequence <220> <223> D2E7-Light Chain-AAT (G3S)2 Linker <400> 22 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Gly Gly Gly Ser Gly Gly Gly Ser Glu Asp 210 215 220 Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His Asp Gln 225 230 235 240 Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala 245 250 255 Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile 260 265 270 Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu 275 280 285 Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe 290 295 300 Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu 305 310 315 320 Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr 325 330 335 Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe 340 345 350 Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn 355 360 365 Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu 370 375 380 Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg 385 390 395 400 Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp 405 410 415 Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val 420 425 430 Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met 435 440 445 Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu Leu Met 450 455 460 Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly 465 470 475 480 Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys 485 490 495 Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys 500 505 510 Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu 515 520 525 Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr 530 535 540 Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu 545 550 555 560 Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu 565 570 575 Ala Ile Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe 580 585 590 Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe Met Gly 595 600 605 Lys Val Val Asn Pro Thr Gln Lys 610 615 <210> 23 <211> 613 <212> PRT <213> Artificial Sequence <220> <223> D2E7-Light Chain-AAT ASTGS Linker <400> 23 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Ala Ser Thr Gly Ser Glu Asp Pro Gln Gly 210 215 220 Asp Ala Ala Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro 225 230 235 240 Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu 245 250 255 Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser 260 265 270 Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys 275 280 285 Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr 290 295 300 Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg 305 310 315 320 Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly 325 330 335 Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp 340 345 350 Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp 355 360 365 Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr 370 375 380 Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val 385 390 395 400 Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro 405 410 415 Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val 420 425 430 Thr Thr Val Lys Val Pro Met Lys Arg Leu Gly Met Phe Asn Ile 435 440 445 Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu 450 455 460 Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln 465 470 475 480 His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu 485 490 495 Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile 500 505 510 Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr 515 520 525 Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala 530 535 540 Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp 545 550 555 560 Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro 565 570 575 Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu 580 585 590 Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val 595 600 605 Asn Pro Thr Gln Lys 610 <210> 24 <211> 853 <212> PRT <213> Artificial Sequence <220> <223> D2E7-Heavy Chain-AAT (G3S)2 Linker <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Glu Asp Pro Gln Gly 450 455 460 Asp Ala Ala Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro 465 470 475 480 Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu 485 490 495 Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser 500 505 510 Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys 515 520 525 Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr 530 535 540 Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg 545 550 555 560 Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly 565 570 575 Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp 580 585 590 Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp 595 600 605 Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr 610 615 620 Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val 625 630 635 640 Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro 645 650 655 Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val 660 665 670 Thr Thr Val Lys Val Pro Met Lys Arg Leu Gly Met Phe Asn Ile 675 680 685 Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu 690 695 700 Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln 705 710 715 720 His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu 725 730 735 Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile 740 745 750 Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr 755 760 765 Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala 770 775 780 Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp 785 790 795 800 Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro 805 810 815 Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu 820 825 830 Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val 835 840 845 Asn Pro Thr Gln Lys 850 <210> 25 <211> 850 <212> PRT <213> Artificial Sequence <220> <223> D2E7-Heavy Chain-AAT ASTGS Linker <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Ala Ser Thr Gly Ser Glu Asp Pro Gln Gly Asp Ala Ala 450 455 460 Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn 465 470 475 480 Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln 485 490 495 Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser 500 505 510 Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr 515 520 525 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro 530 535 540 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn 545 550 555 560 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu 565 570 575 Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 580 585 590 Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu 595 600 605 Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys 610 615 620 Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu 625 630 635 640 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val 645 650 655 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val 660 665 670 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys 675 680 685 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala 690 695 700 Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu 705 710 715 720 Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp 725 730 735 Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr 740 745 750 Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe 755 760 765 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys 770 775 780 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly 785 790 795 800 Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile 805 810 815 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 820 825 830 Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 835 840 845 Gln Lys 850 <210> 26 <211> 869 <212> PRT <213> Artificial Sequence <220> <223> TNFR2-ECD-Fc1-AAT(G3S)2 Linker <400> 26 Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys 100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro 130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 165 170 175 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 180 185 190 His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 195 200 205 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly 210 215 220 Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Gln Val Lys Phe Asn Trp Tyr Val Asp Gly Val Gln Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Gln Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asn Trp Leu Asp Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys Gly Gly Gly Ser Gly Gly Gly Ser Glu Asp Pro Gln Gly 465 470 475 480 Asp Ala Ala Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro 485 490 495 Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu 500 505 510 Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser 515 520 525 Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys 530 535 540 Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr 545 550 555 560 Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg 565 570 575 Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly 580 585 590 Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp 595 600 605 Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp 610 615 620 Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr 625 630 635 640 Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val 645 650 655 Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro 660 665 670 Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val 675 680 685 Thr Thr Val Lys Val Pro Met Lys Arg Leu Gly Met Phe Asn Ile 690 695 700 Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu 705 710 715 720 Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln 725 730 735 His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu 740 745 750 Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile 755 760 765 Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr 770 775 780 Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala 785 790 795 800 Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp 805 810 815 Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro 820 825 830 Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu 835 840 845 Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val 850 855 860 Asn Pro Thr Gln Lys 865 <210> 27 <211> 866 <212> PRT <213> Artificial Sequence <220> <223> TNFR2-ECD-Fc1-AAT ASTGS Linker <400> 27 Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys 100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro 130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 165 170 175 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 180 185 190 His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 195 200 205 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly 210 215 220 Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 275 280 285 Glu Asp Pro Gln Val Lys Phe Asn Trp Tyr Val Asp Gly Val Gln Val 290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Gln Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asn Trp Leu Asp Gly 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 450 455 460 Pro Gly Lys Ala Ser Thr Gly Ser Glu Asp Pro Gln Gly Asp Ala Ala 465 470 475 480 Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn 485 490 495 Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln 500 505 510 Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser 515 520 525 Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr 530 535 540 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro 545 550 555 560 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn 565 570 575 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu 580 585 590 Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 595 600 605 Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu 610 615 620 Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys 625 630 635 640 Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu 645 650 655 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val 660 665 670 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val 675 680 685 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys 690 695 700 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala 705 710 715 720 Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu 725 730 735 Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp 740 745 750 Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr 755 760 765 Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe 770 775 780 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys 785 790 795 800 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly 805 810 815 Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile 820 825 830 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 835 840 845 Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 850 855 860 Gln Lys 865 <210> 28 <211> 738 <212> PRT <213> Artificial Sequence <220> <223>AAT-hFc1-SLPI <400> 28 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350 Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Glu Pro Lys Ser Cys Asp 385 390 395 400 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 405 410 415 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 420 425 430 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 435 440 445 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 450 455 460 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 465 470 475 480 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 485 490 495 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 500 505 510 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 515 520 525 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 530 535 540 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 545 550 555 560 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 565 570 575 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 580 585 590 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 595 600 605 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 610 615 620 Gly Lys Ala Ser Thr Gly Ser Ser Gly Lys Ser Phe Lys Ala Gly Val 625 630 635 640 Cys Pro Pro Lys Lys Ser Ala Gln Cys Leu Arg Tyr Lys Lys Pro Glu 645 650 655 Cys Gln Ser Asp Trp Gln Cys Pro Gly Lys Lys Arg Cys Cys Pro Asp 660 665 670 Thr Cys Gly Ile Lys Cys Leu Asp Pro Val Asp Thr Pro Asn Pro Thr 675 680 685 Arg Arg Lys Pro Gly Lys Cys Pro Val Thr Tyr Gly Gln Cys Leu Met 690 695 700 Leu Asn Pro Pro Asn Phe Cys Glu Met Asp Gly Gln Cys Lys Arg Asp 705 710 715 720 Leu Lys Cys Cys Met Gly Met Cys Gly Lys Ser Cys Val Ser Pro Val 725 730 735 Lys Ala <210> 29 <211> 726 <212> PRT <213> Artificial Sequence <220> <223>AAT-hFc1-Elafin <400> 29 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350 Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Glu Pro Lys Ser Cys Asp 385 390 395 400 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 405 410 415 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 420 425 430 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 435 440 445 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 450 455 460 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 465 470 475 480 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 485 490 495 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 500 505 510 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 515 520 525 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 530 535 540 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 545 550 555 560 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 565 570 575 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 580 585 590 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 595 600 605 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 610 615 620 Gly Lys Ala Ser Thr Gly Ser Ala Val Thr Gly Val Pro Val Lys Gly 625 630 635 640 Gln Asp Thr Val Lys Gly Arg Val Pro Phe Asn Gly Gln Asp Pro Val 645 650 655 Lys Gly Gln Val Ser Val Lys Gly Gln Asp Lys Val Lys Ala Gln Glu 660 665 670 Pro Val Lys Gly Pro Val Ser Thr Lys Pro Gly Ser Cys Pro Ile Ile 675 680 685 Leu Ile Arg Cys Ala Met Leu Asn Pro Pro Asn Arg Cys Leu Lys Asp 690 695 700 Thr Asp Cys Pro Gly Ile Lys Lys Cys Cys Glu Gly Ser Cys Gly Met 705 710 715 720 Ala Cys Phe Val Pro Gln 725 <210> 30 <211> 984 <212> PRT <213> Artificial Sequence <220> <223> AAT-HSA <400>30 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350 Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Ala Ser Thr Gly Ser Asp 385 390 395 400 Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu 405 410 415 Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln 420 425 430 Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe 435 440 445 Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys Ser 450 455 460 Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg 465 470 475 480 Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu 485 490 495 Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro 500 505 510 Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His Asp 515 520 525 Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg 530 535 540 His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr 545 550 555 560 Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys 565 570 575 Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser 580 585 590 Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg 595 600 605 Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys 610 615 620 Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val 625 630 635 640 His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg 645 650 655 Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser 660 665 670 Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys 675 680 685 Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser Leu 690 695 700 Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu 705 710 715 720 Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg 725 730 735 His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr 740 745 750 Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys 755 760 765 Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln 770 775 780 Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu Tyr 785 790 795 800 Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln 805 810 815 Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys Val 820 825 830 Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala 835 840 845 Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu 850 855 860 Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu 865 870 875 880 Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr 885 890 895 Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile 900 905 910 Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala Leu 915 920 925 Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys 930 935 940 Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala 945 950 955 960 Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala 965 970 975 Ala Ser Gln Ala Ala Leu Gly Leu 980 <210> 31 <211> 595 <212> PRT <213> Artificial Sequence <220> <223> AAT-HSA Domain 3 <400> 31 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350 Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Ala Ser Thr Gly Ser Glu 385 390 395 400 Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu 405 410 415 Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys 420 425 430 Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu 435 440 445 Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met 450 455 460 Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val 465 470 475 480 Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr 485 490 495 Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp 500 505 510 Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His 515 520 525 Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln 530 535 540 Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu 545 550 555 560 Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys 565 570 575 Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys 580 585 590 Leu Val Ala 595 <210> 32 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> reactive site loop subsequence variant <400> 32 Gly Thr Glu Ala Ala Gly Ala Glu Phe Leu Glu Ala Ile Pro Leu Ser 1 5 10 15 Ile Pro Pro Glu Val Lys Phe Asn Lys 20 25 <210> 33 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> reactive site loop subsequence variant <400> 33 Gly Thr Glu Ala Ala Gly Ala Leu Phe Leu Glu Ala Ile Pro Leu Ser 1 5 10 15 Ile Pro Pro Glu Val Lys Phe Asn Lys 20 25 <210> 34 <211> 394 <212> PRT <213> Artificial Sequence <220> <223> AAT-MM-EL peptide <400> 34 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Glu Phe 340 345 350 Leu Glu Ala Ile Pro Leu Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys 385 390 <210> 35 <211> 394 <212> PRT <213> Artificial Sequence <220> <223> AAT-MM-LL peptide <400> 35 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Leu Phe 340 345 350 Leu Glu Ala Ile Pro Leu Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys 385 390 <210> 36 <211> 626 <212> PRT <213> Artificial Sequence <220> <223> AAT-MM-LL-hFc1 fusion protein <400> 36 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Leu Phe 340 345 350 Leu Glu Ala Ile Pro Leu Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Glu Pro Lys Ser Cys Asp 385 390 395 400 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 405 410 415 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 420 425 430 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 435 440 445 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 450 455 460 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 465 470 475 480 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 485 490 495 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 500 505 510 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 515 520 525 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 530 535 540 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 545 550 555 560 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 565 570 575 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 580 585 590 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 595 600 605 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 610 615 620 Gly Lys 625 <210> 37 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> D2E7-VK light chain <400> 37 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 38 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> antibody constant region <400> 38 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 100 105 <210> 39 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> D2E7-VH heavy chain <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu 115 <210> 40 <211> 335 <212> PRT <213> Artificial Sequence <220> <223> antibody constant region <400> 40 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 1 5 10 15 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 20 25 30 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 35 40 45 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 50 55 60 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 65 70 75 80 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 85 90 95 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 100 105 110 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 115 120 125 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 130 135 140 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 145 150 155 160 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 165 170 175 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 180 185 190 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 195 200 205 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 210 215 220 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 225 230 235 240 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 245 250 255 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 260 265 270 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 275 280 285 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 290 295 300 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 305 310 315 320 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 335 <210> 41 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> TNFR2-extracellular domain <400> 41 Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser 1 5 10 15 Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys 20 25 30 Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr 35 40 45 Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu 50 55 60 Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser 65 70 75 80 Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys 85 90 95 Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys 100 105 110 Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala 115 120 125 Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro 130 135 140 Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His 145 150 155 160 Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala 165 170 175 Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val 180 185 190 His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr 195 200 205 Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly 210 215 220 Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp 225 230 235 <210> 42 <211> 232 <212> PRT <213> Artificial Sequence <220> <223> antibody constant region <400> 42 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp Val Ser His Glu Asp Pro Gln Val Lys Phe Asn Trp Tyr Val 50 55 60 Asp Gly Val Gln Val His Asn Ala Lys Thr Lys Pro Arg Glu Gln Gln 65 70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 85 90 95 Asn Trp Leu Asp Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 130 135 140 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 43 <211> 227 <212> PRT <213> Homo sapiens <400> 43 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 44 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG1 Fc <400> 44 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Ile Ile Ser Arg Asp Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 45 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG1 Fc <400> 45 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Ile 20 25 30 Ile Ser Arg Asp Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 46 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG1 Fc <400> 46 Ala Pro Glu Leu Leu Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 180 185 190 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 47 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG1 Fc <400> 47 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 20 25 30 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 35 40 45 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 50 55 60 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 65 70 75 80 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 85 90 95 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 100 105 110 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 115 120 125 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 130 135 140 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 145 150 155 160 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 165 170 175 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 180 185 190 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 195 200 205 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 210 215 220 Gly Lys 225 <210> 48 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG1 Fc <400> 48 Ala Pro Glu Val Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 49 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG1 Fc <400> 49 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Val Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 50 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG1 Fc <400> 50 Ala Pro Glu Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 180 185 190 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 51 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG1 Fc <400> 51 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Val Ala Gly 1 5 10 15 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 20 25 30 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 35 40 45 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 50 55 60 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 65 70 75 80 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 85 90 95 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 100 105 110 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 115 120 125 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 130 135 140 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 145 150 155 160 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 165 170 175 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 180 185 190 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 195 200 205 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 210 215 220 Gly Lys 225 <210> 52 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG1 Fc <400> 52 Ala Pro Glu Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Ile Ile Ser Arg Asp Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 180 185 190 Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 53 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG1 Fc <400> 53 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Val Ala Gly 1 5 10 15 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Ile Ile 20 25 30 Ser Arg Asp Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 35 40 45 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 50 55 60 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 65 70 75 80 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 85 90 95 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 100 105 110 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 115 120 125 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 130 135 140 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 145 150 155 160 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 165 170 175 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 180 185 190 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His 195 200 205 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 210 215 220 Gly Lys 225 <210> 54 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG2 Fc <400> 54 Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Ile Ile Ser Arg Asp Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 180 185 190 Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 55 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG3 Fc <400> 55 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Ile Ile Ser Arg Asp Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Asn Thr Thr Pro Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 180 185 190 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn Arg Phe Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 56 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG3 Fc <400> 56 Ala Pro Glu Leu Leu Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe 180 185 190 Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 57 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG3 Fc <400> 57 Ala Pro Glu Val Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 58 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG3 Fc <400> 58 Ala Pro Glu Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe 180 185 190 Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 59 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG3 Fc <400> 59 Ala Pro Glu Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Ile Ile Ser Arg Asp Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 85 90 95 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe 180 185 190 Ser Cys Ser Val Leu His Glu Ala Leu His Asn Arg Phe Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210>60 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400>60 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 61 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400> 61 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Ile Ile Ser Arg Asp Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 62 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400>62 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Ile Ile Ser Arg Asp Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 63 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400> 63 Ala Pro Glu Phe Leu Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly 85 90 95 Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 100 105 110 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 115 120 125 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 130 135 140 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 145 150 155 160 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 165 170 175 Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe 180 185 190 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 195 200 205 Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 64 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400>64 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 65 70 75 80 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90 95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 180 185 190 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220 Ser Leu Gly Lys 225 <210> 65 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400>65 Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 66 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400> 66 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 67 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400> 67 Ala Pro Glu Val Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 68 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400> 68 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Val 1 5 10 15 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 69 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400> 69 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210>70 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400>70 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 71 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400> 71 Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Ile Ile Ser Arg Asp Pro Glu Val Thr Cys Val 20 25 30 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 165 170 175 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 <210> 72 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400> 72 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Ile Ile Ser Arg Asp Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 73 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> modified human IgG4 Fc <400> 73 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Ile Ile Ser Arg Asp Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 74 <211> 19 <212> PRT <213> Homo sapiens <400> 74 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser <210> 75 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> IgG1-VA delta G hinge <400> 75 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Val Ala Gly 1 5 10 15 Pro Ser <210> 76 <211> 21 <212> PRT <213> Homo sapiens <400> 76 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser 20 <210> 77 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> IgG4-hinge PE <400> 77 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser 20 <210> 78 <211> 624 <212> PRT <213> Artificial Sequence <220> <223> AAT-EL-Fc-IgG1-DV, delta G, IDL <400> 78 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Glu Phe 340 345 350 Leu Glu Ala Ile Pro Leu Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Gly Gly Gly Gly Asp Lys 385 390 395 400 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Val Ala Gly Pro Ser 405 410 415 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Ile Ile Ser Arg 420 425 430 Asp Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 435 440 445 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 450 455 460 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 465 470 475 480 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 485 490 495 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 500 505 510 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 515 520 525 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 530 535 540 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 545 550 555 560 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 565 570 575 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 580 585 590 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 595 600 605 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 610 615 620 <210> 79 <211> 623 <212> PRT <213> Artificial Sequence <220> <223> AAT-EL-Fc-IgG4-PE,IDL <400> 79 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Glu Phe 340 345 350 Leu Glu Ala Ile Pro Leu Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Glu Ser Lys Tyr Gly Pro 385 390 395 400 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val 405 410 415 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Ile Ile Ser Arg Asp 420 425 430 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 435 440 445 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 450 455 460 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 465 470 475 480 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 485 490 495 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 500 505 510 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 515 520 525 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 530 535 540 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 545 550 555 560 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 565 570 575 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 580 585 590 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu 595 600 605 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 610 615 620 <210>80 <211> 394 <212> PRT <213> Homo sapiens <400>80 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Glu Phe 340 345 350 Leu Glu Ala Ile Pro Leu Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys 385 390

Claims (17)

An isolated fusion protein comprising one or more human serpin polypeptides operably linked to a modified human IgG4 Fc polypeptide,
wherein the modified human IgG4 Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 69 or 70, wherein the modified human IgG4 Fc polypeptide comprises mutations at positions M252 and M428,
An isolated fusion protein, wherein the human serpin polypeptide is a human alpha-1 antitrypsin (AAT) polypeptide or is derived from a human AAT polypeptide.
According to paragraph 1,
An isolated fusion protein, wherein the modified human IgG4 Fc polypeptide contains a mutation at the T256 position.
According to paragraph 1,
The isolated fusion protein further comprises an additional polypeptide selected from the group consisting of the following polypeptides:
A cytokine targeting polypeptide or a sequence derived from a cytokine targeting polypeptide;
A polypeptide comprising a WAP domain or a sequence derived from a polypeptide comprising a WAP domain; or
An amino acid sequence derived from albumin polypeptide or serum albumin polypeptide.
According to paragraph 1,
The isolated fusion protein, wherein the AAT polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 80.
According to paragraph 1,
An isolated fusion protein, wherein the AAT polypeptide comprises a reactive site loop of AAT comprising the amino acid sequence of SEQ ID NO: 1.
According to paragraph 1,
The isolated fusion protein, wherein the AAT polypeptide comprises a reactive site loop of the mutated AAT comprising the amino acid sequence of SEQ ID NO: 32 or 33.
According to paragraph 1,
The isolated fusion protein, wherein the IgG4 Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 70.
In clause 7,
The IgG4 Fc polypeptide is an isolated fusion protein comprising an M252Y or M252I mutation at the M252 position and an M428L or M428V mutation at the M428 position.
According to clause 8,
The isolated fusion protein, wherein the IgG4 Fc polypeptide comprises the amino acid sequence of SEQ ID: 73.
According to paragraph 1,
An isolated fusion protein, wherein the serpin polypeptide and the modified human IgG4 Fc polypeptide are operably linked through a hinge region, a linker region, or a hinge region and a linker region.
According to clause 10,
An isolated fusion protein, wherein the hinge region, linker region, or hinge region and linker region comprise a peptide sequence.
According to paragraph 1,
An isolated fusion protein, wherein the fusion protein comprises the amino acid sequence of SEQ ID: 79.
A composition for treating or alleviating the symptoms of a disease or disorder in a subject in need thereof, comprising the isolated fusion protein according to any one of claims 1 to 12,
A composition wherein the disease or disorder is selected from:
Emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), allergic asthma, cystic fibrosis, lung cancer of the lung, ischemia-reperfusion injury, ischemia/reperfusion injury following cardiac transplantation, myocardial infarction, rheumatoid arthritis, purulent arthritis (septic arthritis), psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and/or type II diabetes (type I and/or type II) diabetes, pneumonia, sepsis, graft versus host disease (GVHD), wound healing, Systemic lupus erythematosus, and Multiple sclerosis .
According to clause 13,
A composition, wherein the subject has inflammation or symptoms of an inflammatory disease or disorder.
According to clause 13,
A composition wherein the subject is at risk of infection.
According to clause 13,
A composition wherein the subject is a human.
According to clause 15,
The composition of claim 1, wherein the infection is an infection selected from bacterial infection, fungal infection, or viral infection.