RU2746550C2 - Fused serpine polypeptides and methods of their application - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to an isolated fused protein containing at least one human alpha-1-antitrypsin polypeptide functionally associated with a modified human IgG4 Fc polypeptide.

EFFECT: invention is effective for inhibiting or down-regulating impaired expression or activity of serine protease in a subject in need thereof.

10 cl, 4 dwg, 4 ex

Description

Related applications

[0001] This application claims priority from US Patent Application No. 14/524832, filed October 27, 2014, the entire contents of which are incorporated herein by reference.

Enabling a sequence list

[0002] The content of a text file named "INHI002002WO_SeqList.txt" created on October 26, 2015, size 228 KB is included in this application in full by reference.

The technical field to which the invention relates

[0003] The present invention relates to molecules, in particular to polypeptides, more particularly to fusion proteins, which comprise a serpin polypeptide or an amino acid sequence derived from serpin polypeptides and a second polypeptide. Additionally, the present invention relates to fusion proteins that contain a serpin polypeptide or an amino acid sequence derived from serpin polypeptides, a second polypeptide and a third polypeptide. In particular, the present invention relates to fusion proteins that contain at least one serpine polypeptide and a second polypeptide, or fusion proteins that contain at least one serpine polypeptide, a second polypeptide and a third polypeptide, wherein the second and third polypeptide of the fusion proteins of the present the invention may be at least one of the following: an Fc polypeptide or an amino acid sequence derived from an Fc polypeptide; a cytokine-acting polypeptide, or a sequence obtained from a cytokine-acting polypeptide; a polypeptide containing a WAP domain, or a sequence derived from a polypeptide containing a WAP domain; or an albumin polypeptide or an amino acid sequence derived from a serum albumin polypeptide. The present invention also relates to methods of using such molecules for a variety of therapeutic and diagnostic indications, as well as methods of making such molecules.

State of the art

[0004] Impaired serine protease activity or an imbalanced balance of protease and protease inhibitor can lead to protease-induced tissue destruction and an inflammatory response. Accordingly, there is a need for drugs and therapies that act on impaired serine protease activity and / or an impaired balance of protease and protease inhibitor. Also, the therapeutic effect can be increased by attenuating the impaired cytokine signal transduction pathway and impaired activity of serine proteases. Additionally, serpin proteins have been shown to have anti-infective activity, while acting on cytokines increases the risk of infection. The fusion proteins of the present invention have the ability to reduce the activity of inflammatory cytokines and limit the risk of infection.

Summary of the invention

[0005] The fusion proteins described in the present invention include at least a serpin polypeptide or amino acid sequence derived from a serpin polypeptide (Polypeptide 1) and a second polypeptide (Polypeptide 2). Additionally, fusion proteins described herein include a serpine 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 in this application, the terms "fusion protein" and "fusion polypeptide" refer to a serpin polypeptide or an amino acid sequence derived from a serpin polypeptide operably linked to at least one second polypeptide or to an amino acid sequence derived from at least one second polypeptide or the second polypeptide. The individual members of the fusion proteins can be linked in any of a variety of ways, including, for example, direct attachment, the use of an intermediate or spacer peptide, the use of a linker region, the use of a hinge region, or the use of both linker and hinge regions. In some embodiments, implementation of the present invention, the linker region may be part of the sequence of the hinge region, or, alternatively, the hinge region may be part of the sequence of the linker region. It is preferred that the linker region is a peptide sequence. For example, a linker peptide includes any number of amino acids from zero to 40 amino acids, for example, from zero to 35 amino acids, from zero to 30 amino acids, from zero to 25 amino acids, or from zero to 20 amino acids. Preferably, the hinge region is a peptide sequence. For example, a hinge peptide includes any number of amino acids from zero to 75 amino acids, for example, from zero to 70 amino acids, from zero to 65 amino acids, from zero to 62 amino acids. In those embodiments of the present invention in which the fusion protein includes both a linker region and a hinge region, it is preferred that both the linker region and the hinge region are peptide sequences. In these embodiments, the implementation of the present invention, the hinge peptide and the linker peptide together include any number of amino acids from zero to 90 amino acids, for example, from zero to 85 amino acids, or from zero to 82 amino acids.

[0006] In some embodiments, the serpine polypeptide and the second polypeptide may be linked by an intermediate binding protein. In some embodiments, implementation of the present invention, the serpin portion and the portion of the second polypeptide in the fusion protein may be connected non-covalently.

[0007] In some embodiments, implementation of the present invention, fusion proteins in accordance with the present invention may have one of the following formulas in the direction from the N-end to the C-end or in the direction from the C-end to the N-end:

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 zero to 20, where m is an integer from 1 to 62 and where a, b and c are integers of at least 1. These embodiments of the present invention include the above formulas and any variants and combinations thereof ... For example, the sequence of polypeptides in the formula also includes "Polypeptide 3 _(c) -Polypeptide 1 _(a) - Polypeptide 2 _(b) " _, "Polypeptide 2 _(b) -Polypeptide 3 _(c) - Polypeptide 1 _(a) " _, and any their variants and combinations.

[0008] In some embodiments of the present invention, the sequence of Polypeptide 1 comprises a serpin polypeptide. Serpins are a group of proteins with a similar structure, first identified as a group of proteins capable of inhibiting proteases. Serpin proteins suitable for use in the fusion proteins provided herein include, but are not limited to, for example, alpha-1 antitrypsin (AAT, alpha-1 antitrypsin), antitrypsin-related protein (SERPINA2), alpha-1 antichymotrypsin (SERPINA3), callistatin (SERPINA4), monocyte / neutrophil elastase inhibitor (SERPINB1), PI-6 (SERPINB6), antithrombin (SERPINC1), plasminogen activator inhibitor 1 (SERPINE1), alpha-2 antiplasmin) (SERPINF2 complement2 inhibitor) (SERPING1) and neuroserpine (SERPINI1).

[0009] In some embodiments, the sequence of Polypeptide 1 comprises an alpha-1 antitrypsin (AAT) polypeptide sequence or an amino acid sequence derived from AAT. In some embodiments, implementation of the present invention the sequence of Polypeptide 1 includes a portion of the AAT protein. In some embodiments, implementation of the present invention the sequence of Polypeptide 1 includes at least part of the loop of the reaction center of the AAT protein. In some embodiments, the loop portion of the AAT protein reaction center comprises at least the amino acid sequence: GTEAAGAMFLEAIPMSIPPEVKFNK SEQ ID NO: 1).

[00010] In a preferred embodiment of the present invention, the AAT polypeptide sequence or amino acid sequence derived from AAT is a human AAT polypeptide sequence or is derived from a human AAT polypeptide sequence.

[00011] In some embodiments, the fusion protein comprises a full-length human AAT polypeptide sequence with the following amino acid sequence:

1 EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI FFSPVSIATA

61 FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL RTLNQPDSQL QLTTGNGLFL

121 SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT EEAKKQINDY VEKGTQGKIV DLVKELDRDT

181 VFALVNYIFF KGKWERPFEV KDTEEEDFHV DQVTTVKVPM 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)

[00012] In some embodiments, the fusion protein comprises a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2.

[00013] In some embodiments, an AAT polypeptide sequence or amino acid sequence derived from an AAT polypeptide is or is derived from one or more human AAT polypeptide sequences with GenBank accession numbers AAB59495.1, CAJ15161.1, P01009.3, AAB59375 .1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_00112675.1, NP_00112675.1 , NP_001121172.1 and / or AAA51547.1.

[00014] In some embodiments, implementation of the present invention, the fusion proteins contain one or more mutations. For example, the fusion protein contains at least one mutation of a methionine (Met) residue in the serpin portion of the fusion protein. In such Met mutations, the Met residue can 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, for example, leucine (Leu, L). Without wishing to be bound by any theory, the present inventors believe that the Met mutation (s) prevent oxidation and subsequent inactivation of the inhibitory activity of the fusion proteins of the present invention. In some embodiments, implementation of the present invention the Met residue may be replaced with a charged residue, such as, for example, glutamate (Glu, E). In some embodiments, the Met mutation is at position 358 of the AAT polypeptide. For example, the Met mutation is Met358Leu (M358L). In some embodiments, the Met mutation is at position 351 of the AAT polypeptide. For example, the Met mutation is Met351Glu (M351E). In some embodiments, the Met mutations are at positions 351 and 358 of the AAT polypeptide, for example, Met mutations are Met351Glu (M351E) and Met358Leu (M358L). For example, the loop of the reaction center of such a variant AAT fusion polypeptide has the following sequence: GTEAAGA E FLEAIP L SIPPEVKFNK (SEQ ID NO: 32). In some embodiments, the Met mutations are at positions 351 and 358 of the AAT polypeptide, for example, Met mutations are Met351Leu (M351L) and Met358Leu (M358L). For example, the loop of the reaction center of such an AAT fusion polypeptide variant has the following sequence: GTEAAGA L FLEAIP L SIPPEVKFNK (SEQ ID NO: 33).

[00015] In some embodiments, the fusion protein comprises a full-length human AAT polypeptide sequence containing a reaction center loop variant modified at positions M351 and M358 with the following amino acid sequence:

[00016] В некоторых вариантах осуществления настоящего изобретения слитый белок включает полипептидную последовательность человеческого AAT, которая по меньшей мере на 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% или 99% идентичная аминокислотной последовательности SEQ ID NO: 80.

[00016] In some embodiments, the fusion protein comprises a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 80.

[00017] In some embodiments, the second polypeptide (Polypeptide 2) of the serpin fusion protein is an Fc polypeptide or is a polypeptide derived from an Fc polypeptide. In this application, these embodiments of the present invention are combined under the name "serpin-Fc fusion proteins"". Serpin-Fc fusion proteins described herein. Includes at least a serpine polypeptide or an amino acid sequence derived from a serpin and an Fc polypeptide or an amino acid sequence derived from an Fc polypeptide. In some embodiments, the serpin-Fc fusion protein comprises a single serpin polypeptide. In other embodiments, the serpin-Fc fusion protein comprises multiple serpin polypeptides, and these embodiments are combined under the name “serpin _{(a ')} -Fc fusion proteins”, where (a') is an integer, equal to at least 2. In some embodiments, the serpin polypeptides in the serpin _{(a ')} -Fc fusion protein all have the same amino acid sequence. In other embodiments, implementation of the present invention, each serpin polypeptide in the fusion protein "serpin _{(a ')} -Fc" includes serpin polypeptides with a different amino acid sequence. _{Serpin (a ')} -Fc fusion protein polypeptides can be located at any position of the fusion protein.

[00018] In some embodiments, the serpin-Fc fusion protein polypeptide comprises at least the amino acid sequence of a portion of the AAT protein reaction center loop. In some embodiments, implementation of the present invention part of the loop of the reaction center of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 1. In some embodiments, the serpin-Fc fusion protein polypeptide comprises at least the amino acid sequence of a variant loop portion of the AAT protein reaction center. In some embodiments, implementation of the present invention variant of the loop portion of the reaction center of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. In some embodiments, the serpin-Fc fusion protein polypeptide comprises at least the full length human AAT polypeptide sequence with the amino acid sequence SEQ ID NO: 2. In some embodiments, the serpin-Fc fusion protein polypeptide comprises at least the full length human AAT polypeptide sequence with the amino acid sequence SEQ ID NO: 80. In some embodiments, the serpin-Fc fusion protein polypeptide comprises a human AAT polypeptide sequence that is at least 50%, 60%, 65% , 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32, or 33, or 80.

[00019] In some embodiments, the serpin-Fc fusion protein polypeptide comprises an AAT polypeptide sequence or an amino acid sequence derived from an AAT polypeptide that is or is derived from one or more human AAT polypeptide sequences with GenBank accession numbers AAB59495. 1, CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, NP_001121176.16, NP_001121175.1, NP_001121174.1, NP_001121172.1 and / or AAA51547.1.

[00020] In some embodiments, the Fc fusion protein polypeptide is a human Fc polypeptide, eg, a human IgG Fc polypeptide or an amino acid sequence derived from a human IgG Fc polypeptide. For example, in some embodiments, the Fc polypeptide is a human IgG1 Fc polypeptide or an amino acid sequence derived from a human IgG1 Fc polypeptide. In some embodiments, the Fc polypeptide is a human IgG2 Fc polypeptide or an amino acid sequence derived from a human IgG2 Fc polypeptide. In some embodiments, the Fc polypeptide is a human IgG3 Fc polypeptide or an amino acid sequence derived from a human IgG3 Fc polypeptide. In some embodiments, the Fc polypeptide is a human IgG4 Fc polypeptide or an amino acid sequence derived from a human IgG4 Fc polypeptide. In some embodiments, the Fc polypeptide is a human IgM Fc polypeptide or an amino acid sequence derived from a human IgM Fc polypeptide.

[00021] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc fusion protein polypeptide comprises a human IgG1 Fc polypeptide sequence with 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)

[00022] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the fusion protein comprises a hinge region fused to the N-terminus of the Fc fusion protein polypeptide, wherein the Fc polypeptide comprises a human IgG1 Fc polypeptide sequence with the following amino acid 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)

[00023] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc fusion protein polypeptide comprises a human IgG1 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3 or 43.

[00024] In some embodiments of the present invention, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc polypeptide is mutated or modified to enhance FcRn binding. In such embodiments, the mutated or modified Fc polypeptide comprises the following mutations: Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E), or Met428Leu and Asn434Ser (M428L, N434S), or Met428Val (M428L, N434S), or Met428Val (M428L, N434S), or Met428Val (M428L, N434S), or Met428Val (M428L, N434S) by Kabat. In some embodiments, the mutated or modified Fc polypeptide comprises one or more mutations selected from the group consisting of Met252Tyr (M252Y), Ser254Thr (S256T), Thr256Glu (T256E), Met428Leu (M428L), Met428Val (M434Ser), N434S) and combinations thereof. In some embodiments, a portion of an Fc polypeptide is mutated or otherwise modified to prevent Fc mediated dimerization. In these embodiments, the implementation of the present invention, the fusion protein is monomeric in nature.

[00025] In some embodiments of the present invention, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc polypeptide is mutated or modified. In some embodiments, the mutated or modified Fc polypeptide comprises one or more mutations at a position selected from M252, T246, M428, and combinations thereof. In some embodiments, the mutated or modified Fc polypeptide comprises the following mutations: Met252Ile, Thr256Asp, Met428Leu (M252I, T256D, M428L) using the Kabat numbering system.

[00026] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes mutations at positions M252, T256 and M428 corresponding to residues 22, 26 and 198 of SEQ ID NO: 3 or residues 32, 36 and 208 of SEQ ID NO: 43 above and has the following amino acid sequence, in which mutated amino acid residues are outlined:

[00027] In some embodiments, in which the fusion protein of the present 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 includes mutations at positions M252, T256, and M428 corresponding to residues 22, 26 and 198 of the above SEQ ID NO: 3 or residues 32, 36 and 208 of the above SEQ ID NO: 43, and the fusion protein comprises at least the following amino acid sequence in which mutated amino acid residues are outlined:

[00028] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein comprises a modified human IgG1 Fc polypeptide sequence in which a G236 residue corresponding to residue 6 of SEQ ID NO: 3 or residue 16 of SEQ ID NO: 43 above is deleted 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)

[00029] In some embodiments, in which the fusion protein of the present 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 modified human IgG1 Fc polypeptide sequence in which a G236 residue corresponding to residue 6 of SEQ ID NO: 3 or residue 16 of SEQ ID NO: 43 above is deleted and the fusion protein comprises 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)

[00030] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein comprises mutations of residues L234 and L235 corresponding to residues 4 and 5 of SEQ ID NO: 3 or residues 14 and 15 SEQ ID NO: 43 above, and has the following amino acid sequence, in which mutated amino acid residues are outlined:

[00031] In some embodiments, in which the fusion protein of the present 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 mutations of residues L234 and L235 corresponding to residues 4 and 5 of SEQ ID NO: 3 or residues 14 and 15 of SEQ ID NO: 43 above, and the fusion protein comprises at least the following amino acid sequence, in which mutated amino acid residues are outlined:

[00032] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein comprises a deletion of residue G236 and mutations of residues L234 and L235, and the fusion protein comprises at least the following amino acid sequence, in which mutated amino acid residues are outlined:

[00033] In some embodiments, in which the fusion protein of the present 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 residue G236 and mutations of residues L234 and L235, and has the following amino acid sequence in which mutated amino acid residues are outlined:

[00034] In some embodiments, implementation of the present invention, in which the fusion protein of the present invention includes a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein includes a deletion of the G236 residue and mutations of residues L234, L235, M252, T256 and M428, and the fusion protein includes at least the following amino acid sequence, in which mutated amino acid residues are outlined:

[00035] In some embodiments, in which the fusion protein of the present 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 residue G236 and mutations of residues L234, L235, M252, T256 and M428, and has the following amino acid sequence, in which mutated amino acid residues are outlined:

[00036] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG1 Fc polypeptide, the modified IgG1 Fc polypeptide of the fusion protein comprises a modified human IgG1 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53.

[00037] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc fusion protein polypeptide comprises a human IgG2 Fc polypeptide sequence with the following amino acid sequence:

1 APPVAGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP EVQFNWYVDG VEVHNAKTKP

61 REEQFNSTFR VVSVLTVVHQ DWLNGKEYKC KVSNKGLPAP IEKTISKTKG QPREPQVYTL

121 PPSREEMTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPMLDSD GSFFLYSKLT

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

[00038] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc fusion protein polypeptide comprises a human IgG2 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4.

[00039] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG2 Fc polypeptide, the modified IgG2 Fc polypeptide of the fusion protein comprises a modified human IgG2 Fc polypeptide sequence with the following amino acid sequence, in which the mutated amino acid residues are boxed:

[00040] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG2 Fc polypeptide, the modified IgG2 Fc polypeptide of the fusion protein comprises a modified human IgG2 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 54.

[00041] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc fusion protein polypeptide comprises a human IgG3 Fc polypeptide sequence with 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)

[00042] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc fusion protein polypeptide comprises a human IgG3 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 5.

[00043] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein comprises a modified human IgG3 Fc polypeptide sequence with the following amino acid sequence, in which the mutated amino acid residues are boxed:

[00044] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein comprises a modified human IgG3 Fc polypeptide sequence, wherein a G236 residue corresponding to residue 6 of SEQ ID NO: 5 has been deleted and has 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)

[00045] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein comprises mutations of residues L234 and L235 corresponding to residues 4 and 5 of SEQ ID NO: 5 above and has the following amino acid sequence in which mutated amino acid residues are outlined:

[00046] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein comprises a deletion of residue G236 and mutations of residues L234 and L235 and has the following amino acid sequence:

[00047] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein comprises a deletion of residue G236 and mutations of residues L234, L235, M252, T256 and M428 and has the following amino acid sequence:

[00048] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG3 Fc polypeptide, the modified IgG3 Fc polypeptide of the fusion protein comprises a modified human IgG3 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 55, 56, 57, 58 or 59.

[00049] In some embodiments, the human IgG3 Fc region is modified at position Asn297 (Kabat numbering) to prevent glycosylation of an antibody, eg, Asn297Ala (N297A). In some embodiments, implementation of the present invention, the Fc region of human IgG3 is modified at position 435 to increase the half-life, for example, Arg435His (R435H). In some embodiments, implementation of the present invention, the Fc region of human IgG3 does not contain Lys447 (EC index according to Kabat et al 1991 Sequences of Proteins of Immunological Interest).

[00050] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc fusion protein polypeptide comprises a human IgG4 Fc polypeptide sequence with 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)

[00051] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc fusion protein polypeptide comprises a hinge region linked to the N-terminus of the Fc fusion protein polypeptide, wherein the Fc polypeptide comprises a human IgG4 Fc polypeptide sequence with the following amino acid 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)

[00052] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc fusion protein polypeptide comprises a human IgG4 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 6 or 60.

[00053] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes mutations at positions M252, T256 and M428 corresponding to residues 22, 26 and 197 of SEQ ID NO: 6 or residues 34, 38 and 210 of SEQ ID NO: 60 above and has the following amino acid sequence, in which mutated amino acid residues are outlined:

[00054] In some embodiments, in which the fusion protein of the present invention comprises a hinge region linked to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes mutations at positions M252, T256, and M428 corresponding to residues 22, 26 and 197 of SEQ ID NO: 6 or residues 34, 38 and 210 of SEQ ID NO: 60 above, and the fusion protein comprises at least the following amino acid sequence in which mutated amino acid residues are outlined:

[00055] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises a modified human IgG4 Fc polypeptide sequence in which a G236 residue corresponding to residue 6 of SEQ ID NO: 6 or residue 19 of SEQ ID NO: 60 above is deleted 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)

[00056] In some embodiments, in which the fusion protein of the present invention comprises a hinge region linked to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises a modified human IgG4 Fc polypeptide sequence in which a G236 residue corresponding to residue 6 of SEQ ID NO: 6 or residue 19 of SEQ ID NO: 60 above is deleted and the fusion protein comprises 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)

[00057] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises a mutation of residue L235 corresponding to residue 5 of SEQ ID NO: 6 above or residue 17 of SEQ ID above NO: 60, and has the following amino acid sequence, in which mutated amino acid residues are outlined:

[00058] In some embodiments, in which the fusion protein of the present invention comprises a hinge region linked to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises a mutation of residue L235 corresponding to residue 5 of SEQ ID NO: 6 or residue 17 of SEQ ID NO: 60 above, and the fusion protein comprises at least the following amino acid sequence, in which mutated amino acid residues are outlined:

[00059] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises mutations of residues L234 and L235 corresponding to residues 4 and 5 of SEQ ID NO: 6 above or residues 16 and 17 of the above SEQ ID NO: 60, and has the following amino acid sequence, in which mutated amino acid residues are outlined:

[00060] In some embodiments, in which the fusion protein of the present invention comprises a hinge region linked to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises mutations of residues L234 and L235 corresponding to residues 4 and 5 above SEQ ID NO: 6 or residues 16 and 17 of SEQ ID NO: 60 above, and the fusion protein comprises at least the following amino acid sequence in which mutated amino acid residues are outlined:

[00061] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises a mutation of residue S228 corresponding to residue 10 of SEQ ID NO: 60 above and has the following amino acid sequence, in which the mutated amino acid residue is framed:

[00062] In some embodiments, in which the fusion protein of the present invention comprises a hinge region linked to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises mutations of residues S228 and L235 corresponding to residues 10 and 17 above SEQ ID NO: 60, and the fusion protein includes at least the following amino acid sequence, with mutated amino acid residues outlined in boxes:

[00063] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes mutations at positions L235, M252, T256 and M428 corresponding to residues 5, 22, 26 and 197 of SEQ ID NO: 6 or residues 17, 34, 38 and 210 of the above SEQ ID NO: 60, and has the following amino acid sequence, in which mutated amino acid residues are outlined:

[00064] In some embodiments, in which the fusion protein of the present invention comprises a hinge region linked to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes mutations at positions L235, M252, T256 and M428 corresponding to residues 5, 22, 26 and 197 of the above SEQ ID NO: 6 or residues 17, 34, 38 and 210 of the above SEQ ID NO: 60, and the fusion protein comprises at least the following amino acid sequence, in which mutated amino acid residues are outlined:

[00065] In some embodiments, in which the fusion protein of the present invention comprises a hinge region linked to the N-terminus of a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein includes mutations at positions S228, L235, M252, T256 and M428, corresponding to residues 10, 17, 34, 38 and 210 of SEQ ID NO: 60 above, and the fusion protein comprises at least the following amino acid sequence, in which mutated amino acid residues are outlined:

[00066] In some embodiments, in which the fusion protein of the present invention comprises a modified IgG4 Fc polypeptide, the modified IgG4 Fc polypeptide of the fusion protein comprises a modified human IgG4 Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 or 73.

[00067] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc fusion protein polypeptide comprises a human IgM Fc polypeptide sequence with 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)

[00068] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc fusion protein polypeptide comprises a human IgM Fc polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 7.

[00069] In some embodiments, the serpin-Fc fusion protein comprises at least the amino acid sequence of a portion of the AAT protein reaction center loop operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of SEQ ID NO: 3 , 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the Fc polypeptide comprises an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48 , 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73 In some embodiments, the Fc polypeptide comprises an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99% ide an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73. In some embodiments, the loop portion of the AAT protein reaction center comprises at least the amino acid sequence of SEQ ID NO: 1. In some embodiments, the serpine polypeptide and the Fc polypeptide are operably linked through a linker region, for example, a glycine-serine linker or a glycine-serine-based linker. In some embodiments, implementation of the present invention serpentine polypeptide and Fc polypeptide are operably linked through a hinge region. In some embodiments, the serpine polypeptide and the Fc polypeptide are operably linked via a linker region and a hinge region. In other embodiments, implementation of the present invention serpine polypeptide and Fc polypeptide linked directly.

[00070] In some embodiments, the serpin-Fc fusion protein comprises at least the amino acid sequence of a variant portion of the AAT protein reaction center loop operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the Fc polypeptide comprises an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73. In some embodiments, the Fc polypeptide comprises an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% sludge and 99% identical to an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73. In some embodiments of the present invention, a variant loop portion of the AAT protein reaction center comprises at least the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. In some embodiments, the serpine polypeptide and the Fc polypeptide are operably linked through a linker region, for example, a glycine-serine linker or a glycine-serine-based linker. In some embodiments, implementation of the present invention serpentine polypeptide and Fc polypeptide are operably linked through a hinge region. In some embodiments, the serpine polypeptide and the Fc polypeptide are operably linked via a linker region and a hinge region. In other embodiments, implementation of the present invention serpine polypeptide and Fc polypeptide linked directly.

[00071] In some embodiments, the serpin-Fc fusion protein comprises at least the amino acid sequence of a variant loop portion of the AAT protein reaction center operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the Fc polypeptide comprises an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73. In some embodiments, the Fc polypeptide comprises an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% sludge and 99% identical to an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73. In some embodiments, the serpin-Fc fusion protein comprises a human AAT polypeptide sequence that is at least 50 %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical the amino acid sequence of SEQ ID NO: 2, operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 or 73. In some embodiments, the Fc polypeptide comprises an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 4 9, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 and 73. In some embodiments, the Fc polypeptide comprises an amino acid sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 , 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73. In some embodiments, the serpine and Fc polypeptide functionally linked through a linker region, for example, a glycine-serine linker or a glycine-serine linker. In some embodiments, implementation of the present invention serpentine polypeptide and Fc polypeptide are operably linked through a hinge region. In some embodiments, the serpine polypeptide and the Fc polypeptide are operably linked via a linker region and a hinge region. In other embodiments, implementation of the present invention serpine polypeptide and Fc polypeptide linked directly.

[00072] In some embodiments, the serpin-Fc fusion protein comprises at least a full length human AAT polypeptide sequence with the amino acid sequence SEQ ID NO: 80 operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the serpin-Fc fusion protein comprises a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% is identical to the amino acid sequence of SEQ ID NO: 80 operably linked to an Fc polypeptide sequence that includes or is derived from the amino acid sequence of any of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the serpine polypeptide and the Fc polypeptide are operably linked via a linker region, for example, a glycine-serine linker or a glycine-based linker -serina. In some embodiments, implementation of the present invention serpentine polypeptide and Fc polypeptide are operably linked through a hinge region. In some embodiments, the serpine polypeptide and the Fc polypeptide are operably linked via a linker region and a hinge region. In other embodiments, implementation of the present invention serpine polypeptide and Fc polypeptide linked directly.

[00073] In some embodiments of using the fusion proteins provided herein, the second polypeptide (Polypeptide 2) of the serpin fusion protein is a cytokine-acting polypeptide, or is a polypeptide derived from a cytokine-acting polypeptide. These embodiments of the present invention are combined in this application under the name "serpin-cytokine-acting polypeptide fusion proteins". The "serpin-cytokine-acting polypeptide" fusion proteins described herein include at least a serpin-polypeptide or an amino acid sequence derived from a serpin-polypeptide and a cytokine-acting polypeptide, or derivatives thereof. In some embodiments, the serpin-cytokine polypeptide fusion protein comprises a single serpin polypeptide. In other embodiments of the present invention, the serpin-cytokine-acting polypeptide fusion protein comprises several serpin-polypeptides, and these embodiments of the present invention are combined herein under the name "serpin _{(a ') -cytokine-acting polypeptide fusion proteins"} where (a ') is an integer of at least 2. In some embodiments, the serpin polypeptides in the serpin _(a') cytokine-acting polypeptide fusion protein all have the same amino acid sequence. In other embodiments, implementation of the present invention, each serpin polypeptide in the fusion protein "serpin _{(a ')} - acting on the cytokine polypeptide" includes serpin polypeptides with different amino acid sequences.

[00074] In some embodiments, the cytokine-acting polypeptide or serpin-cytokine-acting polypeptide fusion protein is a cytokine receptor or is derived from a cytokine receptor. In a preferred embodiment of the present invention, the cytokine-acting polypeptide or amino acid sequence derived from a cytokine receptor is or is derived from a human cytokine receptor sequence. In other embodiments, the cytokine-acting polypeptide is an antibody or antibody fragment, eg, an anti-cytokine antibody or an anti-cytokine antibody fragment. In a preferred embodiment of the present invention, the cytokine-acting polypeptide or amino acid sequence derived from an antibody or antibody fragment is derived from a chimeric, humanized or fully human antibody sequence. The term "antibody fragment" includes single chain antibody, Fab fragment, F (ab ') ₂ fragment, scFv, scAb, dAb, heavy chain single domain antibody and light chain single domain antibody.

[00075] In other embodiments, the cytokine-acting polypeptide binds to a cytokine receptor and prevents the cytokine from binding to the receptor. In other embodiments, the cytokine-acting polypeptide is an antibody or antibody fragment, eg, an anti-cytokine receptor antibody or an anti-cytokine receptor antibody fragment.

[00076] In some embodiments, the serpin-cytokine-acting polypeptide fusion protein polypeptide comprises at least the amino acid sequence of a portion of the AAT protein reaction center loop. In some embodiments, implementation of the present invention part of the loop of the reaction center of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 1. In some embodiments, the serpinated cytokine polypeptide fusion protein polypeptide comprises at least the amino acid sequence of a variant loop portion of the AAT protein reaction center. In some embodiments, implementation of the present invention variant of the loop portion of the reaction center of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. In some embodiments, the serpine cytokine-acting polypeptide fusion protein polypeptide comprises or is derived from at least the full length human AAT polypeptide sequence with the amino acid sequence SEQ ID NO: 2. In some embodiments, the serpine cytokine polypeptide fusion protein polypeptide serpin-cytokine-acting polypeptide "includes or is derived from at least the full-length polypeptide sequence of human AAT with the amino acid sequence SEQ ID NO: 80. In some embodiments, the serpin-cytokine-acting polypeptide" fusion protein polypeptide comprises a polypeptide sequence of a human AAT that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80.

[00077] In some embodiments of the present invention, a serpin-cytokine-acting polypeptide fusion protein polypeptide comprises an AAT polypeptide sequence or an amino acid sequence derived from an AAT polypeptide that is or is derived from one or more human AAT polypeptide sequences having accession numbers in GenBank 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.

[00078] A serpin-cytokine-acting polypeptide fusion protein may comprise a portion of a serpin-Fc fusion protein. For example, an antibody contains an Fc polypeptide. Thus, in some embodiments in which the cytokine-acting polypeptide is an anti-cytokine antibody, the serpin-cytokine-acting polypeptide fusion protein will comprise a portion of the serpin-Fc fusion protein. Also, most of the receptor fusion proteins used for therapeutic purposes are Fc fusion proteins. Thus, in some embodiments of the present invention, in which the serpin-cytokine-acting polypeptide fusion protein is a serpin-cytokine receptor fusion protein, the serpin-cytokine-receptor fusion protein may include an Fc polypeptide in addition to a serpin parts and parts of the cytokine receptor.

[00079] In some embodiments of the present invention, in which the "serpin-cytokine polypeptide" fusion protein comprises an Fc polypeptide sequence, the Fc polypeptide sequence includes or is derived from the amino acid sequence of any of SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 or 73. In some embodiments of the present invention, in which the serpin-cytokine polypeptide fusion protein comprises an Fc polypeptide sequence, an Fc polypeptide sequence of at least 50%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any of the amino acid sequences SEQ ID NO: 3, 4, 5, 6, 7, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, or 73. In some embodiments, the implementation on of the present invention, the serpine polypeptide and the cytokine-acting polypeptide are operably linked via a linker region, for example, via a glycine-serine linker or a glycine-serine-based linker. In some embodiments, the serpinated polypeptide and the cytokine-acting polypeptide are operably linked through a hinge region. In some embodiments, the serpine polypeptide and the cytokine-acting polypeptide are operably linked via a linker region and a hinge region. In other embodiments, the serpinated polypeptide and the cytokine-acting polypeptide are directly linked.

[00080] In some embodiments of the use of the fusion proteins provided herein, the second polypeptide (Polypeptide 2) of the serpin fusion protein is a polypeptide containing a WAP (whey acidic protein) domain or an amino acid sequence derived from a polypeptide containing a WAP. These embodiments of the present invention are combined in this application under the general name "WAP serpin-domain fusion proteins"". Serpin-WAP domain fusion proteins include at least a serpin polypeptide or at least an amino acid sequence derived from a serpin, a polypeptide comprising a WAP domain, or an amino acid sequence derived from a polypeptide comprising a WAP domain. In some embodiments, the WAP serpin domain fusion protein comprises a single serpin polypeptide. In other embodiments, implementation of the present invention the fusion protein "serpin-acting on a WAP polypeptide" includes multiple serpin polypeptides. These embodiments of the present invention are collectively referred to herein as "serpin _{(a ')} WAP domain fusion proteins", where (a') is an integer of at least 2. In some embodiments, serpin polypeptides fusion protein "serpin _{(a ')} -domain WAP" have the same amino acid sequence. In other embodiments, the serpin _{(a ')} -cytokine-acting polypeptide fusion protein polypeptides comprise serpin polypeptides with different amino acid sequences.

[00081] These "serpin _{(a ')} WAP domain" fusion proteins include a WAP domain containing polypeptide or a polypeptide sequence that is or is derived from a WAP domain containing polypeptide. The WAP domain is an evolutionarily conserved 50 amino acid sequence containing eight cysteines arranged in a characteristic 4 disulfide bond structure (also called a core 4 disulfide bond motif). The WAP domain sequence motif is a functional motif characterized by serine protease inhibitory activity in a variety of proteins.

[00082] WAP domain containing polypeptides suitable for use in the fusion proteins provided herein include, but are not limited to, for example, secretory leukocyte protease inhibitor (SLPI), elafin (Elafin) and eppin (Eppin ).

[00083] In some embodiments, the WAP domain polypeptide sequence of the fusion protein comprises a secretory leukocyte protease inhibitor (SLPI) polypeptide sequence or an amino acid sequence derived from an SLPI. These embodiments of the present invention are combined in this application under the general name "fusion proteins derived from" serpin-SLPI ". In some embodiments, the SLPI polypeptide sequence comprises a portion of an SLPI protein, such as, for example, a WAP2 domain or portion thereof. In a preferred embodiment of the present invention, an SLPI polypeptide sequence or amino acid sequence derived from an SLPI is or is derived from a human SLPI polypeptide sequence.

[00084] In some embodiments of the serpin-SLPI fusion proteins of the present invention, the SLPI sequence or sequence derived from an SLPI fusion protein comprises a full length human SLPI polypeptide sequence with 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)

[00085] In some embodiments of the serpin-SLPI fusion proteins of the present invention, the SLPI sequence, or sequence derived from an SLPI, fusion protein comprises a full length human SLPI polypeptide sequence that is at least 50%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8.

[00086] In some embodiments of the serpin-SLPI fusion proteins of the present invention, an SLPI sequence, or a sequence derived from an SLPI, a fusion protein comprises a portion of a full length human SLPI polypeptide sequence with the following amino acid sequence:

1 SGKSFKAGVC PPKKSAQCLR YKKPECQSDW QCPGKKRCCP DTCGIKCLDP VDTPNPTRRK

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

[00087] In some embodiments of using the serpin-SLPI fusion proteins of the present invention, the SLPI sequence, or sequence derived from SLPI, the fusion protein comprises a human SLPI polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% are identical to the amino acid sequence of SEQ ID NO: 9.

[00088] In some embodiments of the serpin-SLPI fusion protein of the present invention, the SLPI sequence, or sequence derived from an SLPI, of the fusion protein comprises the WAP2 domain of a full length human SLPI polypeptide sequence with the following amino acid sequence of the WAP2 domain:

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

[00089] In some embodiments of the serpin-SLPI fusion protein of the present invention, the SLPI sequence, or sequence derived from SLPI, the fusion protein comprises a human SLPI polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% are identical to the amino acid sequence of SEQ ID NO: 10.

[00090] In certain uses of the serpin-SLPI fusion proteins of the present invention, an SLPI polypeptide sequence or amino acid sequence derived from an SLPI polypeptide is or is derived from one or more human SLPI polypeptide sequences having GenBank accession numbers CAA28187.1, NP_003055 .1, EAW75869.1, P03973.2, AAH20708.1, CAB64235.1, CAA28188.1, AAD19661.1 and / or BAG35125.1.

[00091] In some embodiments of the serpin-SLPI fusion proteins of the present invention, an SLPI polypeptide sequence or sequence derived from an SLPI fusion protein comprises a human SLPI polypeptide sequence modified at a methionine (Met) residue. In such Met mutations, the Met residue can be substituted with any amino acid. For example, a Met residue may be substituted with an amino acid with a hydrophobic side chain, such as, for example, leucine (Leu, L) or valine (Val, V). Without wishing to be bound by any theory, the present inventors believe that the Met mutation (s) prevent oxidation and subsequent inactivation of the inhibitory activity of the fusion proteins of the present invention. In some embodiments, the Met mutation is at position 98 of the SLPI polypeptide. For example, the modified serpin-SLPI SLPI polypeptide sequence contains the M98L or M98V mutation in SEQ ID NO: 8.

[00092] In other embodiments of the present invention, the polypeptide sequence containing the WAP domain of the fusion protein comprises an elafin polypeptide sequence or an amino acid sequence derived from elafin. These embodiments of the present invention are combined in this application under the general name "serpin-elafin fusion proteins". In some embodiments, the elafin polypeptide sequence comprises a portion of an elafin protein, such as, for example, a WAP domain or portion thereof. In a preferred embodiment of the present invention, the elafin polypeptide sequence or amino acid sequence derived from elafin is or is derived from a human elafin polypeptide sequence.

[00093] In some embodiments of the serpin-elafin fusion proteins, the fusion protein comprises a full-length human elafin polypeptide sequence with the following amino acid sequence:

1 MRASSFLIVV VFLIAGTLVL EAAVTGVPVK GQDTVKGRVP FNGQDPVKGQ VSVKGQDKVK

61 AQEPVKGPVS TKPGSCPIIL IRCAMLNPPN RCLKDTDCPG IKKCCEGSCG MACFVPQ

(SEQ ID NO: 11)

[00094] In some uses of the serpin-elafin fusion proteins, the fusion protein comprises a full-length human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11.

[00095] In some embodiments of the serpin-elafin fusion proteins, the fusion protein comprises a portion of a full-length human elafin polypeptide sequence with the following amino acid sequence:

1 AVTGVPVKGQ DTVKGRVPFN GQDPVKGQVS VKGQDKVKAQ EPVKGPVSTK PGSCPIILIR

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

[00096] In some uses of the serpin-elafin fusion proteins, the fusion protein comprises a human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 12.

[00097] In some embodiments of serpin-elafin fusion proteins, the fusion protein comprises a WAP domain of a full-length human elafin polypeptide sequence with the following amino acid sequence:

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

[00098] In some uses of the serpin-elafin fusion proteins, the fusion protein comprises a human elafin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13.

[00099] In certain uses of the serpin-elafin fusion proteins, an elafin polypeptide sequence or an amino acid sequence derived from an elafin polypeptide is derived from one or more human elafin polypeptide sequences having GenBank accession numbers P19957.3, NP_002629.1, BAA02441 .1, EAW75814.1, EAW75813.1, Q8IUB2.1 and / or NP_542181.1.

[000100] In other embodiments, the polypeptide sequence containing the WAP domain of the fusion protein comprises an eppin polypeptide sequence or an amino acid sequence derived from eppin. These embodiments of the present invention are combined in this application under the general name "serpin _{(a ')} - eppin fusion proteins". In some embodiments, the eppin polypeptide sequence of the serpin-eppin fusion protein comprises a portion of an eppin protein, such as, for example, a WAP domain or portion thereof. In a preferred embodiment of the present invention, the eppin polypeptide sequence or amino acid sequence derived from eppin is or is derived from a human eppin polypeptide sequence.

[000101] In some embodiments of the use of serpin-eppin fusion proteins, an eppin polypeptide sequence or amino acid sequence derived from an eppin polypeptide is or is derived from one or more human eppin polypeptide sequences having GenBank accession numbers O95925.1, NP_065131.1 , AAH44829.2, AAH53369.1, AAG00548.1, AAG00547.1 and / or AAG00546.1.

[000102] In some embodiments, the serpin WAP serpin fusion polypeptide comprises at least the amino acid sequence of a portion of the AAT protein reaction center loop. In some embodiments, implementation of the present invention part of the loop of the reaction center of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 1. In some embodiments, the serpin WAP domain fusion protein polypeptide comprises at least the amino acid sequence of a variant loop portion of the AAT protein reaction center. In some embodiments, implementation of the present invention variant of the loop portion of the reaction center of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. In some embodiments, the serpin WAP serpin fusion protein polypeptide comprises at least the full length human AAT polypeptide sequence with the amino acid sequence SEQ ID NO: 2. In some embodiments, the serpin acting on cytokine polypeptide "includes or is derived from at least the full length human AAT polypeptide sequence with the amino acid sequence SEQ ID NO: 80. In some embodiments, the serpine WAP serpin domain fusion protein polypeptide comprises a human AAT polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99 % identical to the amino acid sequence of SEQ ID NO: 2 or 32 or 33 or 80.

[000103] In some embodiments, a serpin WAP serpin fusion protein polypeptide comprises an AAT polypeptide sequence or an amino acid sequence derived from an AAT polypeptide derived from one or more human AAT polypeptide sequences with GenBank accession numbers AAB59495.1 , CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, NP_001121277.1, NP_0011212 .16, NP_001121175.1, NP_001121174.1, NP_001121172.1 and / or AAA51547.1.

[000104] In some embodiments, the serpin-WAP domain fusion protein may also include an Fc polypeptide or an amino acid sequence derived from an Fc polypeptide. These embodiments of the present invention are combined in this application under the general name "serpin-Fc-domain WAP fusion proteins" ". In these embodiments of the present invention, this term does not limit a particular order. For example, the sequence of the fusion protein may be WAP serpin Fc domain, WAP serpin Fc domain, or any combination thereof. The serpin-Fc-WAP domain fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence derived from serpin, a polypeptide comprising a WAP domain, or an amino acid sequence derived from a polypeptide comprising a WAP domain, and a polypeptide Fc or an amino acid sequence derived from an Fc polypeptide.

[000105] In some embodiments of the present invention, in which the serpin-WAP domain fusion protein comprises an Fc polypeptide sequence, the Fc polypeptide sequence may have the amino acid sequence of SEQ ID NOs: 3-7 and 43-73. In other embodiments, implementation of the present invention, in which the fusion protein "serpin-WAP domain" comprises an Fc polypeptide sequence, an Fc polypeptide sequence of at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NOs 3-7 and 43-73. In some embodiments, the serpin-WAP domain fusion protein may also include an albumin polypeptide or an amino acid sequence derived from an albumin polypeptide. These embodiments of the present invention are combined in this application under the general name "serpin-albumin-WAP domain fusion proteins". In these embodiments of the present invention, this term does not limit a particular order. For example, the sequence of the fusion protein can be WAP serpin albumin domain, WAP serpin albumin domain, or any combination thereof. The serpin-albumin-WAP domain fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence derived from serpin, a polypeptide containing a WAP domain, or an amino acid sequence derived from a polypeptide comprising a WAP domain, and an albumin a polypeptide or amino acid sequence derived from an albumin polypeptide.

[000106] In some embodiments of the present invention, in which the serpin-WAP domain fusion protein comprises an albumin polypeptide sequence, the albumin polypeptide sequence comprises the amino acid sequence SEQ ID NO: 14-15 described herein. In other embodiments, implementation of the present invention, in which the fusion protein "serpin-domain WAP" comprises an albumin polypeptide sequence, an albumin polypeptide sequence of at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% are identical to any of the amino acid sequences of SEQ ID NO: 14 or 15.

[000107] In some embodiments, the second polypeptide (Polypeptide 2) of the serpin fusion protein is an albumin polypeptide or is derived from an albumin polypeptide. These embodiments of the present invention are combined in this application under the general name "serpin _{(a ')} -albumin fusion proteins". Serpin-albumin fusion proteins described herein include at least a serpin polypeptide or amino acid sequence derived from serpin and an albumin polypeptide or amino acid sequence derived from an albumin polypeptide. Additionally, the present invention relates to serpin-binding albumin polypeptide fusion proteins in which albumin is operably linked to serpin via an intermediate binding molecule. In the present application, serpin is non-covalently or covalently linked to human serum albumin (HSA).

[000108] In embodiments in which the fusion protein of the present invention comprises an albumin polypeptide sequence, the albumin fusion protein polypeptide sequence is a human serum albumin polypeptide or an amino acid sequence derived from human serum albumin. In some embodiments, the fusion protein comprises a human serum albumin polypeptide sequence with the following amino acid sequence:

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 14)

[000109] In embodiments in which the fusion protein of the present invention comprises an albumin polypeptide sequence, the albumin fusion protein polypeptide sequence comprises a human serum albumin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14.

[000110] In embodiments in which the fusion protein of the present invention comprises an albumin polypeptide sequence, the albumin fusion protein polypeptide sequence comprises domain 3 of the human serum albumin polypeptide sequence with the following amino acid sequence:

EEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA (SEQ ID NO: 15)

[000111] In embodiments in which the fusion protein of the present invention comprises an albumin polypeptide sequence, the albumin fusion protein polypeptide sequence comprises a human serum albumin polypeptide sequence that is at least 50%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 15.

[000112] In some embodiments of the present invention, in which the fusion protein of the present invention comprises an albumin polypeptide sequence, the fusion protein is linked to human serum albumin via an albumin-binding polypeptide intermediate. The albumin binding polypeptide can be an antibody or antibody fragment, or can be derived from an antibody or antibody fragment. In a preferred embodiment of the present invention, the albumin binding polypeptide or amino acid sequence derived from an antibody or from an antibody fragment is derived from a chimeric, humanized or fully human antibody sequence. The term "antibody fragment" includes single chain antibody, Fab fragment, F (ab ') ₂ fragment, scFv, scAb, dAb, and single domain antibody heavy chain and single domain antibody light chain. Additionally, the albumin binding polypeptide can be an albumin binding peptide. Another embodiment of the present invention is a serpin-fused albumin-binding polypeptide, wherein the albumin-binding polypeptide is domain 3 of streptococcal protein G or a sequence derived from domain 3 of streptococcal protein G.

[000113] In some embodiments, the serpin _{(a ')} -albumin fusion protein polypeptide comprises at least the amino acid sequence of a portion of the AAT protein reaction center loop. In some embodiments, implementation of the present invention part of the loop of the reaction center of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 1. In some embodiments, the serpin-albumin fusion protein polypeptide comprises at least the amino acid sequence of a variant loop portion of the AAT protein reaction center. In some embodiments, implementation of the present invention variant of the loop portion of the reaction center of the AAT protein includes at least the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. In some embodiments, the serpin-albumin fusion protein polypeptide comprises at least the full-length human AAT polypeptide sequence with the amino acid sequence SEQ ID NO: 2. In some embodiments, the serpin-cytokine polypeptide fusion protein polypeptide "Includes or is derived from at least the full length human AAT polypeptide sequence with the amino acid sequence of SEQ ID NO: 80. In some embodiments, the serpin-albumin fusion protein polypeptide comprises at least 50% human AAT polypeptide sequence. , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to amino acid SEQ ID NO: 2 or 32 or 33 or 80.

[000114] In some embodiments, the serpin-albumin fusion protein polypeptide comprises an AAT polypeptide sequence or an amino acid sequence derived from an AAT polypeptide that is or is derived from one or more human AAT polypeptide sequences with GenBank accession numbers AAB59495.1 , CAJ15161.1, P01009.3, AAB59375.1, AAA51546.1, CAA25838.1, NP_001002235.1, CAA34982.1, NP_001002236.1, NP_000286.3, NP_001121179.1, NP_001121178.1, NP_001121177.1, NP_001121277.1, NP_0011212 .16, NP_001121175.1, NP_001121174.1, NP_001121172.1 and / or AAA51547.1.

[000115] In some embodiments of the present invention, the fusion proteins are modified to increase or otherwise inhibit proteolytic cleavage, for example, by mutating one or more proteolytic cleavage sites. In some embodiments, implementation of the present invention, the fusion proteins are modified to alter or otherwise modulate the effector function of the Fc fusion protein, while maintaining the binding and inhibitory function compared to the unchanged fusion protein. The effector functions of Fc include, but are not limited to, for example, Fc receptor binding, prevention of the release of a proinflammatory mediator upon Fc receptor binding, phagocytosis, modified antibody-dependent cell-mediated cytotoxicity, modified complement-dependent cytotoxicity, modified glycosylation index Kabat Asn al 1991 Sequences of Proteins of Immunological Interest) of an Fc polypeptide. In some embodiments, implementation of the present invention, the fusion proteins are mutated or otherwise modified to regulate the binding of the Fc receptor. In some embodiments, the Fc polypeptide is modified to enhance FcRn binding. Examples of Fc polypeptide mutations that enhance binding to FcRn are Met252Tyr, Ser254Thr, Thr256Glu (M252Y, S256T, T256E) (Kabat numbering, Dall'Acqua et al 2006, J. Biol Chem Vol 281 (3324) 23514-235 and Asn434Ser (M428L, N434S) (Zalevsky et al 2010 Nature Biotech, Vol. 28 (2) 157-159) (EU index according to Kabat et al 1991 Sequences of Proteins of Immunological Interest) or Met428Val and Asn434Ser (M428V, N434S) at using the Kabat numbering system. In some embodiments, the mutated or modified Fc polypeptide comprises one or more mutations selected from the group consisting of Met252Tyr (M252Y), Ser254Thr (S256T), Thr256Glu (T256E), Met428Leu (M428L), Met428Val (M434Ser), N434S) and combinations thereof. In some embodiments, a portion of an Fc polypeptide is mutated or otherwise modified to prevent Fc mediated dimerization (Ying et al 2012 J. Biol Chem 287 (23): 19399-19408). In these embodiments, the implementation of the present invention, the fusion protein is monomeric in nature.

[000116] The fusion proteins and their variants provided herein have inhibitory activity, for example, inhibiting serine proteases such as human neutrophil elastase, chymotrypsin fold serine protease secreted by neutrophils during an inflammatory response. The fusion proteins provided herein completely or partially reduce or otherwise modulate the expression or activity of serine proteases when they bind or otherwise interact with a serine protease, eg, a human serine protease. The decrease or modulation of the biological function of the serine protease occurs in whole or in part through the interaction of fusion proteins and a protein, polypeptide and / or peptide of human serine protease. Fusion proteins are considered to completely inhibit serine protease expression or activity if the level of serine protease expression or activity in the presence of the fusion protein is reduced by at least 95%, e.g. 96%, 97%, 98%, 99% or 100% compared to the level of expression or activity of serine protease in the absence of interaction, eg, binding, with the fusion protein described in this application. Fusion proteins are considered to partially inhibit serine protease expression or activity if the level of serine protease expression or activity in the presence of the fusion protein is reduced by less than 95%, e.g. 10%, 20%, 25%, 30%, 40%. 50%, 60%, 75%, 80%, 85% or 90% compared to the level of expression or activity of serine protease in the absence of interaction, eg, binding, with the fusion protein described in this application.

[000117] The fusion proteins described herein are useful for a variety of therapeutic, diagnostic and prophylactic indications. For example, fusion proteins are useful in treating a variety of diseases and disorders in a subject. In some embodiments, serpin fusion proteins, including the fusion proteins described herein, are useful for treating, reducing symptoms, alleviating and / or delaying the onset of a disease or disorder in a subject suffering from a disease or disorder or at increased risk of developing a disease or disorder , selected from the lack of alpha-1-antitrypsin (AAT, alpha-1-antitrypsin), emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory failure syndrome, allergic asthma, cystic fibrosis, lung cancer, ischemia-reperfusion injury, including ischemia-reperfusion injury after heart transplant, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and / or II diabetes, bacterial infections, fungal infections, viral infections, pneumonia, sepsis, graft versus host reactions the effects of wounds, systemic lupus erythematosus and multiple sclerosis.

[000118] Pharmaceutical compositions in accordance with the present invention include the fusion protein of the present invention, including modified fusion proteins and other variants, as well as a suitable carrier. These pharmaceutical compositions can be included in kits such as, for example, diagnostic kits.

Brief Description of Drawings

[000119] Figure 1A is a schematic representation of some embodiments of the use of serpin-Fc fusion proteins in accordance with the present invention. Serpin can be in any position in the fusion protein. Also provided are serpin-Fc fusion proteins comprising multiple serpin polypeptides. Figure 1B is a photograph of the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) showing serum-derived AAT (lane 1), AAT-Fc1 (lane 2, human IgG1 Fc) and AAT-EL-Fc1 (lane 3, Met351Glu mutations , Met358Leu in AAT, human IgG1 Fc). Figure 1C is a graph showing the inhibition of neutrophil elastase activity by AAT-Fc fusion proteins. Figure 1D is a photograph of the SDS PAGE result showing a tetravalent AAT-Fc-AAT containing two AAT polypeptides per Fc polypeptide. Figure 1 E is a graph showing the inhibition of neutrophil elastase activity by a tetravalent fusion protein AAT-Fc-AAT. Figure 1F is a graph showing the effect of elution from protein A resin at low pH, where the ability of the fusion protein "AAT-Fc" eluted at low pH to inhibit neutrophil elastase is greatly reduced. Figure 1G is a graph showing that AAT-EL-Fc double mutant (Met351Glu, Met358Leu mutations) is resistant to H ₂ O ₂ inactivation (conc.) Compared to wild-type AAT and AAT-EM-Fc single mutant (Met351Glu ). Figure 1H is a graph showing the serum clearance rate of serum derived AAT (sdAAT, serum derived AAT) versus AAT-Fc in rats dosed with mg / kg protein (3 rats / test protein). The half-life of AAT-Fc is significantly longer than that of sdAAT.

[000120] Figure 2A is a schematic representation of some embodiments of the use of serpin-cytokine polypeptide fusion proteins of the present invention. Serpin can be linked to either the heavy chain or light chain, or both the heavy and light chains of an antibody. Serpin-cytokine receptor fusion proteins are also shown. Figure 2B is a photograph of the SDS PAGE result showing the D2E7 antibody (lane 1) and the D2E7 heavy chain fusion with AAT (lane 2). Figure 2C is a graph showing the inhibition of neutrophil elastase activity by D2E7 antibody fused to AAT. Serum-isolated AAT is shown as a positive control, while D2E7 alone is shown as a negative control for inhibition of neutrophil elastase.

[000121] Figure 3A is a schematic representation of some embodiments of the use of serpin-Fc-WAP fusion proteins. Figure 3B is a photograph of the SDS PAGE result showing AAT-Fc-ELAFIN (lane 1) and AAT-Fc-SLPI (lane 2). Figure 3C is a graph showing the inhibition of neutrophil elastase activity by AAT-Fc-elafin fusion protein and AAT-Fc-SLPI fusion protein. AAT-Fc fusion protein and serum isolated AAT are included for comparison.

[000122] Figure 4A is a schematic representation of some embodiments of the use of "AAT-HSA" fusion proteins. Figure 4B is a photograph of the result of SDS PAGE showing the "AAT-HSA" fusion protein. Figure 4C is a graph showing the inhibition of neutrophil elastase activity by "AAT-HSA" versus serum-isolated AAT.

Detailed description of the invention

[000123] Human neutrophil elastase is a serine protease with a chymotrypsin domain, secreted during inflammation. Impaired neutrophil elastase activity leads to progressive degradation of elastin tissues and slow destruction of the alveolar structures of the lungs, which causes emphysema and pulmonary fibrosis (Lungarella et al 2008 Int. J. Biochem Cell Biol 40: 1287). Often, the impaired activity of neutrophil elastase is due to an imbalance between the protease and its natural inhibitor alpha-1-antitrypsin (AAT, alpha1-antitrypsin). This imbalance is due to increased infiltration of neutrophils in the lungs, which is observed in the lungs of smokers and patients with cystic fibrosis or acute respiratory failure syndrome. In turn, due to lack of AAT, usually caused by a point mutation, due to which ATT aggregates and accumulates in the liver, uncontrolled activity of neutrophil elastase is observed in the lungs. Individuals with AAT deficiency are at increased risk of developing emphysema, COPD, liver disease, and several other conditions.

[000124] AAT deficiency occurs in approximately 100,000 Americans (estimated by the Alpha-1 Foundation), and many die between the ages of 30-50. Currently, there are only a few FDA (Food and Drug Administration) approved drugs for the treatment of ATT deficiency (Prolastin®, Aralast ™, Zemaira®, Glassia ™). Each drug is a naturally occurring AAT derived from pooled human blood plasma, which is obviously insufficient to meet clinical needs. Also, these products have a short serum half-life (T _1/2 is approximately 5 days) and require weekly high dose infusions (60 mg / kg bw). The market for these drugs is currently estimated at US $ 400 million. The market for AAT-like drugs is probably much larger, judging by the fact that 95% of individuals with AAT deficiency remain undiagnosed and given the fact that these drugs may be effective in the treatment of pathologies characterized by increased activity of neutrophil elastase in individuals without AAT deficiency (eg, cystic fibrosis, smoking-induced acute respiratory distress syndrome, emphysema and / or COPD).

[000125] AAT is believed to have a broad spectrum of 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, there has been evidence of the potential use of AAT in the treatment of many human pathologies not related to commonly assumed inflammatory conditions of the airways. Human AAT has been shown to protect mice from the clinical and histopathological signs of experimental autoimmune encephalomyelitis, suggesting that it can be used to treat autoimmune diseases such as multiple sclerosis or systemic lupus erythematosus (Subramanian et al 2011 Metab Brain Dis 26: 107 -113). Serum AAT has been shown to be active in rodent graft versus host models (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 a steroid refractory acute graft versus host reaction (NCT01523821). Additionally, AAT has been effective in animal models of type I and II diabetes, in reducing inflammation, protecting pancreatic islet cells from apoptosis, and promoting pancreatic islet cell allograft resistance (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 Islets 2: 185-189). Many early phase clinical trials are underway in people with type I diabetes using serum-derived AAT products (NCT01183468, NCT01319331, NCT01304537).

[000126] Currently existing serum-derived AAT products are thoroughly purified and tested to ensure the removal of pathogenic viruses, however, the risk of contamination with infectious agents cannot be completely excluded. Moreover, the amount of serum is limited, which limits the ability to obtain AAT from serum. Efforts to solve the problems associated with serum-derived products and their production have focused on the expression of recombinant AAT. However, in 20 years of work, the generation of therapeutically available recombinant AAT never hit the market (Karnaukhova et al 2006 Amino Acids 30: 317). Like plasma-derived products, recombinant AAT variants have a short half-life, low production yields, and poor distribution in the lungs.

[000127] The fusion proteins of the present invention have improved functionality over the unmodified AAT molecule. Binding of the AAT polypeptide to a second polypeptide that interacts with the neonatal Fc receptor (FcRn) serves to increase the serum half-life, which provides the dosing advantage needed by patients. These FcRn-interacting fusion protein polypeptides include immunoglobulin (Ig) Fc polypeptides derived from human IgG1, IgG2, IgG3, IgG4 or IgM and derivatives of human albumin. In some embodiments, the fusion protein includes mutations in a portion of the AAT that render the molecule more resistant to oxidative inactivation. For example, Met351Glu, Met358Leu (AAT-EL-Fc) show resistance to inactivation by H ₂ O ₂ oxidation (Figure 1G). While AAT is a naturally occurring anti-inflammatory protein, some embodiments of the present invention have an increased ability to reduce inflammation by binding of the AAT polypeptide to the cytokine-acting polypeptide. The binding of the two anti-inflammatory functions of AAT and the second polypeptide will provide a more pronounced activity of the therapeutic protein than either polypeptide alone. Additionally, binding the anti-infective activity of AAT will reduce the risk of infection with most cytokine-acting biologics. Some embodiments of the present invention provide more active anti-inflammatory and anti-infective proteins by linking the AAT polypeptide and the WAP domain containing polypeptide. The fusion proteins of the present invention are expected to have greater therapeutic utility and be superior to currently available serum-derived AAT products.

[000128] To increase the half-life of recombinant AAT, recombinant DNA technology was used to generate an AAT gene fused to the Fc domain of human IgG1, IgG2, IgG3, IgG4, IgM or HSA such that the Fc domain (( AAT-Fc (IgG1), AAT-Fc (IgG2), AAT-Fc (IgG3), AAT-Fc (IgG4), AAT-Fc (IgM)) or AAT would be followed by HSA. While it was known that binding of HSA Fc domains to certain proteins, protein domains or peptides can increase their half-life (see, e.g., Jazayeri et al. BioDrugs 22, 11-26, Huang et al. (2009) Curr Opin Biotechnol 20, 692-699 , Kontermann et al. (2009) BioDrugs 23, 93-109, Schmidt et al. (2009) Curr Opin Drug Discov Devel 12, 284-295), it was not known that binding of the Fc or HSA domain to AAT provides proper folding and maintenance of neutrophil elastase inhibiting activity or prolonging the half-life of recombinant AAT. tenia are potent inhibitors of neutrophil elastase, have an increased half-life, and are oxidatively stable in some embodiments. In other embodiments, implementation of the present invention, the fusion proteins described in this application have certain properties by including other functional polypeptides, including polypeptides acting on cytokines and polypeptides containing the WAP domain.

[000129] In neutrophils, the main source of neutrophil elastase, an oxidative burst often occurs simultaneously with the secretion of neutrophil elastase. Thus, an important property of the fusion proteins of the present invention is activity in an oxidizing environment. Oxidation of AAT within the loop of the reaction center by Met351 and / or Met358 reduces the ability of AAT to inhibit neutrophil elastase. As shown in Figure 1G, oxidative inactivation can be reduced by the Met351Glu and Met358Leu (M351E / M358L) mutations shown in SEQ ID NO: 32. It has also been shown that oxidation of the Fc region at Met252 and Met428 decreases FcRn binding and subsequently shortens the serum half-life of the Fc-containing protein. The Met252 and Met428 mutation reduces oxidation of the Fc region. The present invention discloses and optimizes an "AAT-Fc" fusion protein that is resistant to oxidative inactivation in the AAT portion of the fusion protein due to M351 and M358 mutations, and to oxidative disruption of interaction with FcRn due to M252 and M428 mutations, and has an increased half-life , thanks to a set of mutations M252I, T256D and M428L.

[000130] In some embodiments of the present invention, in which the fusion protein of the present invention comprises 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: Met252Ile, Thr256Asp, and Met428Leu (M252I, T256D, M428L) using the Kabat numbering system.

[000131] In some embodiments, in which the fusion protein of the present 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.

[000132] In some embodiments, in which the fusion protein of the present 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.

[000133] In some embodiments, in which the fusion protein of the present invention comprises an Fc polypeptide, the Fc polypeptide is mutated or modified to reduce binding to Fc-gamma receptors (FcgR, Fc-gamma receptor). In some embodiments, the reduction of FcgR binding can be achieved by modifying Fc glycosylation at Asn297. For example, with the mutation Asn297Ala (N297A) or Asn297Gln (N297Q). In some embodiments, implementation of the present invention, the reduction of FcgR binding can be achieved by modifying the lower Fc hinge region. In some embodiments, the Fc polypeptide is derived from human IgG1. In some of these embodiments, the lower hinge region is modified to mimic the IgG2 hinge region by mutation Leu234Val and Leu235Ala (L235V / L235A) and deletion Gly236 (ΔG236) using the Kabat numbering system:

In some of these embodiments, the fusion protein comprises at least the amino acid sequence of SEQ ID NO: 51.

[000134] In some embodiments, the Fc polypeptide is derived from human IgG4. In some of these embodiments, the lower hinge region is modified by the Leu235Glu (L235E) mutation. Additionally, in embodiments of the present invention in which the Fc polypeptide is derived from IgG4, the hinge region is modified by the Ser228Pro (S228P) stabilizing mutation using the Kabat numbering system:

In some of these embodiments, the fusion protein comprises at least the amino acid sequence of SEQ ID NO: 70.

[000135] The fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence derived from serpin and a second polypeptide. In some embodiments, implementation of the present invention, for example, the present invention provides a serpinous polypeptide associated with derivatives of human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc or HSA. Serpin fusion proteins described herein are expected to be useful in treatment for a variety of indications, including but not limited to, for example, alpha-1-antitrypsin (AAT, alpha-1-antitrypsin) deficiency, emphysema, chronic obstructive disease lungs (COPD), acute respiratory failure syndrome, allergic asthma, cystic fibrosis, lung cancer, ischemia-reperfusion injury, including ischemia-reperfusion injury after heart transplant, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis Crohn's disease, psoriasis, type I and / or II diabetes, bacterial infections, fungal infections, viral infections, pneumonia, sepsis, graft versus host disease, wound healing, systemic lupus erythematosus, and multiple sclerosis.

[000136] In some embodiments, the fusion proteins described herein comprise at least an alpha-1-antitrypsin (AAT, alpha-1-antitrypsin) polypeptide or an amino acid sequence derived from AAT, and a second polypeptide. For example, the present invention provides alpha-1-antitrypsin (AAT) linked to derivatives of human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, IgM-Fc, or HSA.

[000137] In some embodiments, the fusion proteins described herein comprise at least a serpine polypeptide or an amino acid sequence derived from a serpin polypeptide and a cytokine-acting polypeptide or an amino acid sequence derived from a cytokine-acting polypeptide. For example, the present invention provides a serpine polypeptide or a sequence derived from a serpin polypeptide fused to a human cytokine receptor or a derivative thereof. Another embodiment of the present invention is a serpine polypeptide or a sequence derived from a serpin polypeptide fused to a cytokine-acting antibody, e.g., an anti-cytokine antibody, or a sequence derived from a cytokine-acting antibody, e.g., an anti-cytokine antibody or a sequence derived from a fragment of a cytokine-acting antibody, e.g. from an anti-cytokine antibody fragment. For example, the present invention provides a serpine polypeptide or a sequence derived from a serpin polypeptide fused to a cytokine-acting polypeptide, wherein the cytokine-acting polypeptide binds any of the following human cytokines: TNFα (tumor necrosis factor), IgE, IL-12 (interleukin, interleukin), IL-23, IL-6, IL-1α, IL-1β, IL-17, IL-13, IL-4, IL-10, IL-2, IL-18, IL-27 or IL-32.

[000138] For example, in some embodiments, the cytokine-acting polypeptide acts on TNFα and includes any of the following TNFα-acting polypeptides or sequences derived from the following TNFα-acting polypeptides: Remicade®, Humira®, Simponi®, Cimiza®, Enbrel® or ATN-103 and ATN-192.

[000139] For example, in some embodiments, the cytokine-acting polypeptide acts on IgE and includes any of the following IgE-acting polypeptides or sequences derived from the following IgE-acting polypeptides: Xolair or FcεRI.

[000140] For example, in some embodiments, the cytokine-acting polypeptide acts on the p40 subunit common to IL-12 and IL-23 and includes a Stelara® polypeptide or sequences derived from a Stelara® polypeptide.

[000141] For example, a cytokine-acting Stelara® polypeptide acts on IL-13 and includes a CDP7766 polypeptide or sequences derived from a CDP7766 polypeptide.

[000142] In some embodiments, the fusion proteins described herein comprise at least an alpha-1-antitrypsin (AAT) polypeptide or an amino acid sequence derived from AAT and a cytokine-acting polypeptide, or an amino acid sequence derived from a cytokine-acting polypeptide. For example, the present invention provides an alpha-1 antitrypsin inhibitor (AAT) fused to a cytokine-acting polypeptide, wherein the cytokine-acting polypeptide binds any of the following human cytokines: TNFα, IgE, IL-6, IL-1α, IL-1β , IL-12, IL-17, IL-13, IL-23, IL-4, IL-10, IL-2, IL-18, IL-27 or IL-32.

[000143] In some embodiments, the cytokine-acting polypeptide binds to a cytokine receptor and prevents cytokine binding. For example, the present invention includes serpin associated with an antibody acting on a cytokine receptor. For example, the present invention provides an α-1 antitrypsin inhibitor (AAT) fused to a cytokine acting polypeptide, wherein the cytokine acting polypeptide binds any of the following human cytokines: TNFα, IgE, IL-6, IL-1α, IL-1β , IL-12, IL-17, IL-13, IL-23, p40 subunit of IL-12 and IL-23, IL-4, IL-10, IL-2, IL-18, IL-27 or IL-32 ...

[000144] For example, in some embodiments of the present invention, the cytokine-acting polypeptide acts on the IL-6 receptor and includes an Actemra® polypeptide (as described in patent publication EP0628639) or an ALX-0061 polypeptide (as described in WO2010 / 115998), or sequences derived from Actemra® polypeptide or from ALX-0061 polypeptide.

[000145] For example, the cytokine-acting Actemra® polypeptide acts on the IL-6 receptor and includes the tocilizumab polypeptide or sequences derived from the tocilizumab polypeptide.

[000146] The action on inflammatory cytokines and immunostimulatory agents by protein drugs has been shown to be clinically effective in a variety of inflammatory conditions. The most common proteins used as cytokine-acting agents are soluble cytokine receptors and monoclonal antibodies and their fragments. A significant disadvantage when acting on cytokines is the risk of infection in patients, as evidenced by the data on the biological drugs Remicade®, Humira®, Simponi®, Cimiza® and Enbrel® acting on TNFα and the Stelara antibody acting on IL-12/23 p40 ®. This is likely a common problem with inflammatory cytokines that result in immunosuppression in patients. AAT and other serpinated proteins are interesting in that they have both anti-infective and anti-inflammatory activity. Thus, the "serpin-cytokine polypeptide" fusion proteins of the present invention can suppress abnormal cytokine activity, reducing the risk of infection.

[000147] In some embodiments, the fusion proteins described herein comprise a serpin polypeptide or an amino acid sequence derived from serpin, a WAP domain containing polypeptide or amino acid sequence derived from a WAP domain containing polypeptide, and an Fc polypeptide or amino acid sequence, derived from an Fc polypeptide. For example, the present invention provides a serpinated polypeptide comprising a WAP domain polypeptide and human IgG1-Fc, IgG2-Fc, IgG3-Fc, IgG4-Fc, or IgM-Fc derivatives operably linked to each other in any functional combination. In some embodiments, implementation of the present invention containing the WAP domain protein is human SLPI or derived from human SLPI. In other embodiments, the WAP domain containing protein is human elafin or is derived from human elafin. In some embodiments, implementation of the present invention, the fusion proteins described in this application include at least an alpha-1-antitrypsin (AAT, alpha-1-antitrypsin) polypeptide or amino acid sequence derived from AAT, and an SLPI polypeptide or amino acid sequence derived from SLPI. In some embodiments, the fusion proteins described herein comprise at least an AAT polypeptide or amino acid sequence derived from AAT and an elafin polypeptide or amino acid sequence derived from elafin.

[000148] SLPI and elafin are WAP domain containing proteins having serine protease inhibitory activity. Both of these proteins have anti-inflammatory functions. In addition, these proteins have a wide range of anti-infective properties against a number of strains of bacteria, viruses and fungi.

[000149] In some embodiments, the fusion proteins described herein include at least a serpin polypeptide or an amino acid sequence derived from serpin and a human serum albumin polypeptide or an amino acid sequence derived from a human serum albumin polypeptide. Other embodiments of the present invention include serpin-albumin-binding polypeptide fusion proteins, in which the albumin-binding polypeptide is responsible for binding serpin and human serum albumin. Thus, the present invention includes both covalent and non-covalent linkages of a serpine polypeptide and an HSA polypeptide, or sequences derived from a serpine polypeptide or HSA polypeptide. For example, the present invention provides a serpinated polypeptide associated with human HSA or HSA derivatives or HSA binding peptide or polypeptides.

[000150] In some embodiments, the fusion proteins described herein comprise at least an alpha-1-antitrypsin (AAT, alpha-1-antitrypsin) polypeptide or amino acid sequence derived from AAT and an HSA polypeptide or amino acid sequence derived from an HSA polypeptide. For example, the present invention provides alpha-1-antitrypsin (AAT) linked to HSA or a fragment derived from HSA or an albumin binding polypeptide.

[000151] In some embodiments, the fusion proteins described herein include a serpine polypeptide or an amino acid sequence derived from serpin, an HSA polypeptide, or an amino acid sequence derived from an HSA polypeptide and comprising a WAP domain polypeptide or an amino acid sequence derived from containing a WAP domain polypeptide. In some embodiments, implementation of the present invention, the fusion proteins described in this application include at least an alpha-1-antitrypsin (AAT, alpha-1-antitrypsin) polypeptide or an amino acid sequence derived from an AAT, an HSA polypeptide, or an amino acid sequence derived from a polypeptide HSA, and an SLPI polypeptide or amino acid sequence derived from an SLPI polypeptide. In other embodiments, implementation of the present invention, the fusion proteins described in this application include at least an alpha-1-antitrypsin (AAT, alpha-1-antitrypsin) polypeptide or an amino acid sequence derived from an AAT, an HSA polypeptide, or an amino acid sequence derived from a polypeptide HSA, and an elafin polypeptide or amino acid sequence derived from elafin.

[000152] The fusion proteins of the present invention can be readily produced in mammalian cell expression systems. For example, Chinese hamster ovary cells (CHO, Chinese Hamster Ovary), human embryonic kidney 293 cells (HEK, Human Embryonic Kidney), COS cells, PER.C6® cells, NS0, SP2 / 0, YB2 / 0 cells can be successfully used for the expression of serpin fusion proteins described in this application. Importantly, mammalian cell expression systems produce proteins that are generally more suitable for therapeutic use. Unlike expression systems based on bacteria, insects or yeast, expression systems based on mammalian cells produce proteins whose glycosylation pattern is similar to the glycosylation pattern of naturally occurring proteins. Proper glycosylation of a protein can be important for serum stability, biodistribution pharmacokinetics, folding, and protein functionality. Thus, the ability to obtain therapeutic proteins in mammalian expression systems has certain advantages over other systems. Also, the volume of expression systems based on mammalian cells (e.g., CHO, NS0, PER.C6® cells) can be easily scaled up commercially to provide therapeutic proteins to meet clinical needs. The fusion proteins described herein have improved functionality over the native form of AAT and can be produced in mammalian cell expression systems for clinical and commercial supplies. Some embodiments of the present invention include a purification system that allows the release of serpin fusion proteins that retain the ability to inhibit neutrophil elastase. It is important to note that the purification process of the present invention can easily be incorporated into current commercial mammalian cell production processes.

[000153] Unless otherwise defined, scientific and technical terms used in connection with the present invention will have the meanings generally accepted by those skilled in the art. Also, unless otherwise specified by context, singular terms include the plural, and plural terms also include the singular. Typically, the nomenclature and techniques of cell and tissue culture, molecular biology, protein, oligo- or polynucleotide chemistry, and hybridization as described herein are well known and widely used in the art. Standard techniques are used for the synthesis of recombinant DNA and oligonucleotides, for tissue culture, and for transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, or as is customary in the art or as described herein. The following techniques and procedures are generally carried out in accordance with standard methods well known in the art and described in various general and specific references, which are cited and discussed throughout the present disclosure. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). Used in the context of the nomenclature, laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry described in this application are well known and widely used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceuticals, dosage form, drug delivery, and patient care. The term "patient" includes human and veterinary subjects.

[000154] It will be appreciated that the therapeutic agents of the present invention will be administered with suitable carriers, buffers, excipients, and other agents that are formulated to optimize transport, delivery, tolerance, and the like. A variety of suitable forms can be found in the reference book known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), especially Chapter 87 by Blaug, Seymour, ibid. These forms include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as Lipofectin ™), DNA conjugates, anhydrous adsorbent pastes, oil-in-water emulsions and water in oil ”, carbovax emulsions (polyethylene glycols of different molecular weights), semi-solid gels and semi-solid mixtures containing carbovax. Any of these mixtures may be suitable for treatment and therapy in accordance with the present invention, given that the active ingredient in the form is not inactivated by the dosage form, and that the dosage form is physiologically compatible with the route of administration and is tolerated. See 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 references cited therein for additional information regarding formulations, excipients and carriers well known to pharmaceutical chemists.

[000155] Therapeutic forms of the present invention, including the fusion protein of the present invention, are used to treat or reduce a symptom associated with a disease or disorder associated with a disruption of serine protease activity in a subject. The present invention also provides methods for treating or ameliorating a symptom associated with a disease or disorder associated with a disruption of serine protease activity in a subject. A treatment regimen is carried out by identifying a subject, eg, a human, suffering from (or at high risk of developing) a disease or disorder associated with a disruption of serine protease activity using standard methods, including any of a number of clinical and / or laboratory procedures. The term "patient" includes human and veterinary subjects. The term "subject" includes humans and other mammals.

[000156] The efficacy of the treatment is determined using any known method for diagnosing or treating a specific disease or disorder associated with a disruption in serine protease activity. A decrease in one or more symptoms of a disease or disorder associated with a disruption of serine protease activity indicates that the fusion protein is clinically effective.

[000157] Methods for screening fusion proteins having the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA), enzymatic assays, flow cytometry, and other immunological techniques known in the art.

[000158] The fusion proteins described herein can be used in methods known in the art for localizing and / or quantifying a target such as a serine protease, eg, for use in measuring the levels of these targets in appropriate physiological samples, for use in diagnostic methods, for use in protein imaging, and the like). The terms "physiological sample" and "biological sample", used interchangeably in this application, include tissues, cells and body fluids taken from a subject, as well as tissues, cells and fluids found in the body of a subject. Thus, the terms "physiological sample" and "biological sample" encompass blood and a fraction or component of blood, including blood serum, blood plasma, or lymph.

[000159] In a specific embodiment of the present invention, fusion proteins specific to a particular target or derivatives, fragments, analogs or homologues thereof containing a target binding domain are used as pharmacologically active compounds (hereinafter referred to as therapeutic agents).

[000160] The fusion protein of the present invention can be used to isolate a particular target using standard techniques such as immunoaffinity techniques, chromatography, or immunoprecipitation. Detection can also be carried out by linking (i.e., physically linking) the fusion protein to the substance to be detected. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include oxycoumarin, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a suitable luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ³⁵ S, or ³ H.

[000161] The therapeutically effective amount of the fusion protein of the present invention generally refers to the amount required to achieve the therapeutic goal. As indicated above, this can be the binding of the fusion protein to the target, which in certain cases disrupts the function of the target. The amount to be administered will also depend on the binding affinity of the fusion protein to a particular target, and will also depend on the rate at which the administered fusion protein is cleared from the subject to which it was administered. Typical ranges of therapeutically effective dose of the fusion protein of the present invention or fragment thereof may be, for example, from about 0.1 mg / kg body weight to about 250 mg / kg body weight, but are not limited to. The standard frequency of dosing can vary, for example, from twice a day to once a month.

[000162] When using fragments of fusion proteins, it is preferable to use the inhibitory fragment of the smallest size that specifically binds to the target. For example, you can create peptide molecules that retain the ability to bind a target. Such peptides can be chemically synthesized and / or produced using recombinant DNA technology. (See, eg, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The dosage form can also contain several active compounds in accordance with a specific indication for therapeutic use, preferably with complementary activity, which do not have an undesirable effect on each other. Alternatively or additionally, the composition may contain a function enhancing agent such as, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent, a growth inhibitory agent, an anti-inflammatory agent, or an anti-infective agent. Such molecules are present in combination in an amount effective for a given purpose.

[000163] Also, the active ingredients can be encapsulated in microcapsules obtained, for example, by coacervation or interface polymerization techniques, for example, in hydroxymethylcellulose or gelatin microcapsules and polymethylmethacrylate microcapsules, respectively, in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in a macroemulsion.

[000164] Dosage forms for use for in vivo administration must be sterile. This is easily achieved by filtration through sterile filter membranes.

[000165] Delayed release agents can be obtained. Suitable examples of delayed release agents include semi-permeable matrices of solid hydrophobic polymers containing a fusion protein, said matrices in a specific form, eg, in the form of films or microcapsules. Examples of delayed release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable copolymers of lactic and glycolic acid such as LUPRON DEPOT ™ (injection microspheres consisting of a copolymer of lactic and glycolic acid and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and lactic / glycolic acid copolymers release molecules over 100 days, certain hydrogels release proteins for shorter periods of time.

Pharmaceutical compositions

[000166] Fusion proteins of the present invention (also referred to as active compounds in this application) and their derivatives, fragments, analogs and homologues can be included in pharmaceutical compositions suitable for administration. Such compositions typically comprise a fusion protein and a pharmaceutically acceptable carrier. As used in this application, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and adsorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, the standard reference literature in the art, incorporated herein by reference. Preferred examples of such carriers or solvents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and anhydrous inert carriers such as fatty oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of such compositions is contemplated, unless any standard medium or agent is incompatible with the active compound. Additional active compounds can also be included in the compositions.

[000167] The pharmaceutical composition of the present invention is formulated in a form that is compatible with the desired route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile solvent such as water for injection, saline, fatty oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral agents can be enclosed in ampoules, disposable syringes, or multi-dose containers made of glass or plastic.

[000168] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (for water-soluble compositions) or dispersions and sterile powders for the extemporaneous preparation of sterile solutions or dispersions for injection. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL ™ (BASF, Parsippany, N.J.), or phosphate buffered saline. In all cases, the composition must be sterile and liquid to allow for syringe administration. It must be stable under the conditions of receipt and storage and must contain preservatives to prevent the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersing medium containing, for example, water, ethanol, polyhydric alcohol (eg, glycerol, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the desired particle size in the case of dispersion, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents in the composition, for example, sugars, polyhydric alcohols such as mannitol, sorbitol, sodium chloride. Prolonged adsorption of injectable compositions can be achieved by the inclusion in the composition of an adsorption delaying agent, for example, aluminum monostearate and gelatin.

[000169] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount of an appropriate solvent with one ingredient listed above, or a combination of the ingredients listed above, as required, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile inert carrier containing a basic dispersing medium and other necessary ingredients from among those overdone above. In the case of sterile powders for the preparation of sterile solutions for injection, the methods of preparation are vacuum drying and lyophilization to form a powder of the active ingredient with any additional desired ingredients from a solution thereof previously sterilized by filtration.

[000170] Oral compositions typically include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or formed into tablets. For oral therapeutic administration, excipients can be added to the active compound and used in the form of tablets, troches or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, in which a compound in a liquid carrier is orally administered, rinsed, spit out, or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: a binding agent such as microcrystalline cellulose, traganic gum or gelatin; an excipient such as starch or lactose; a disintegrant such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a flow regulator such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate, or orange flavoring.

[000171] For inhalation administration, the compounds are delivered in the form of an aerosol sprayed from a pressurized container or dispenser containing a suitable propellant, eg, a gas such as carbon dioxide, or from a nebulizer.

[000172] Systemic administration can also be transmucosal or transdermal. For transmucosal or transdermal administration, a wetting agent is used in the form. Such wetting agents are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be carried out using nasal sprays or suppositories. For transdermal administration, the active compounds are formulated as ointments, balms, gels or creams as is well known in the art.

[000173] the Compounds can also be formulated in the form of suppositories (eg, with standard suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[000174] In one embodiment of the present invention, the active compounds are prepared with carriers that protect the compound from rapid elimination from the body, such as controlled release forms, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used such as vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for constructing such forms are familiar to those skilled in the art. The materials can also be purchased from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions can also be used as pharmaceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

[000175] It is particularly convenient to formulate oral or parenteral compositions in dosage form for ease of administration and uniformity of dosage. As used in this application, dosage form means physically separated units corresponding to single doses for a subject undergoing treatment; each unit contains a predetermined amount of active compound calculated to provide the desired therapeutic effect associated with the desired pharmaceutical carrier. The technical characteristics of the dosage form of the present invention are determined by and depend on the unique characteristics of the active compound and the particular therapeutic effect to be achieved and the limitations known in the art of formulating such an active compound for the treatment of individuals.

[000176] The pharmaceutical compositions can be included in a container, package, or dispenser along with instructions for administration.

[000177] The present invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: "AAT-Fc" fusion proteins and their variants

[000178] By way of example, and not limitation, "AAT-Fc" fusion proteins in accordance with the present invention include the following sequences. While these examples include a hinge sequence and / or linker sequence, the fusion proteins of the present invention may be prepared using any hinge sequence and / or linker sequence suitable in length and / or flexibility. Alternatively, fusion proteins can be produced without the use of a hinge and / or linker sequence. For example, you can directly connect the polypeptide components.

[000179] An exemplary "AAT-Fc" fusion protein is AAT-hFc1 (human IgG1 Fc) described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2) and the IgG-Fc polypeptide that is part of the fusion protein is in oblique font (SEQ ID NO: 3).

AAT-hFc1 (Human IgG1 Fc)

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK EPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16)

[000180] An exemplary "AAT-Fc" fusion protein is AAT-hFc2 (human IgG2 Fc) described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2) and the IgG-Fc polypeptide that is part of the fusion protein is in oblique font (SEQ ID NO: 4).

AAT-hFc2 (Human IgG2 Fc)

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17)

[000181] An exemplary "AAT-Fc" fusion protein is AAT-MM-EL-hFc1 (Human IgG1 Fc, Met351Glu / Met358Leu) described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 34), the IgG-Fc polypeptide that is part of the fusion protein is in oblique (SEQ ID NO: 3), the Met351Glu mutation is boxed, and the Met358Leu mutation highlighted in gray.

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

[000182] An exemplary "AAT-Fc" fusion protein is AAT-MM-EL-hFc2 (Human IgG2 Fc, Met351Glu / Met358Leu) described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 34), the IgG-Fc polypeptide that is part of the fusion protein is in oblique (SEQ ID NO: 4), the Met351Glu mutation is boxed, and the Met358Leu mutation highlighted in gray.

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

[000183] An exemplary "AAT-Fc" fusion protein is AAT-MM-LL-hFc1 (human IgG1 Fc, Met351Leu / Met358Leu) described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 35), the IgG-Fc polypeptide that is part of the fusion protein is in oblique type (SEQ ID NO: 3), the Met351Leu mutation is highlighted in black, and the Met358Leu mutation highlighted in gray.

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

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGALFLEAIPLSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36)

[000184] An exemplary "AAT-Fc" fusion protein is AAT-MM: LL-hFc2 (Human IgG2 Fc, Met351Leu / Met358Leu) described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 35), the IgG-Fc polypeptide that is part of the fusion protein is in oblique type (SEQ ID NO: 4), the Met351Leu mutation is highlighted in black, and the Met358Leu mutation highlighted in gray.

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

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGALFLEAIPLSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK ERKCCVECPPCP APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20)

[000185] An exemplary "AAT-Fc" fusion protein is AAT-hFc1-AAT (human IgG1) described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2), the IgG-Fc polypeptide that is part of the fusion protein is in oblique font (SEQ ID NO: 3).

AAT-hFc1-AAT (human IgG1)

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK EPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ASTGS EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVK FNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO: 21)

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

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

[000186] These exemplary "AAT-Fc" fusion proteins were prepared using the following techniques.

[000187] The gene encoding human AAT was amplified by PCR with human liver cDNA (Zyagen). Specific point mutations in the gene encoding the AAT or Fc region were obtained by PCR with overlapping primers. The gene encoding AAT was cloned in frame with the gene encoding the hinge region, followed by the CH2 domain and the CH3 domain of human IgG1, IgG2, IgG3, IgG4 or IgM, into a mammalian expression vector containing the mammalian secretory signal sequence upstream of the site insertion of the AAT gene. These expression vectors were transfected into mammalian cells (namely HEK293 or CHO cells) and grown for several days at 8% CO ₂ at 37 ° C. Recombinant AAT-Fc fusion proteins were isolated from the supernatant of expressing cells using protein A chromatography. It is important to note that the elution of the AAT-Fc fusion protein from the protein A resin was performed with a buffer with neutral pH (Gentle Ag / Ab Elution Buffer, Thermo Scientific). The “AAT-Fc” fusion protein cannot be eluted from the Protein A resin using standard low pH elution because the ability of AAT to inhibit neutrophil elastase is impaired by low pH treatment, likely due to low pH mediated oligomerization of AAT. Figure 1F shows the effects that low pH elution has on the ability of AAT to inhibit neutrophil elastase. The “AAT-Fc” fusion protein can be purified with either Protein A and Low pH Elution Buffer or CaptureSelect® Alpha-1 Antitrypsin Affinity Matrix (BAC BV).

[000188] Purified fusion proteins "AAT-Fc" were tested for activity, determining their ability to inhibit neutrophil elastase. Figures 1B and 1D show the results of electrophoresis of serum-purified AAT (sdAAT) and “AAT-Fc” fusion proteins in SDS PAGE under reducing conditions (Fig.1B-lane 1: sdAAT, lane 2: AAT-Fc (SEQ ID NO : 16) lane 3: AAT-EL-Fc (SEQ ID NO: 18) Fig. 1D AAT-Fc-AAT (SEQ ID NO: 20) Proteins were visualized by Coomassie blue staining.

[000189] To study the activity of elastase of human neutrophils used the study of fluorescence in microplates. Inhibitory activity was measured by the concomitant decrease in the residual activity of neutrophil elastase, conducting the following study. The assay buffer consists of 100 mM Tris pH 7.4, 500 mM NaCl and 0.0005% Triton X-100. Human neutrophil elastase is used at a final concentration of 5 nM (a concentration of 1-20 nM can also be used). AAVP-AMC fluorescent peptide substrate was used at a final concentration of 100 μM in this assay. A Gemini EM plate reader from Molecular Devices was used to read kinetic studies at excitation and emission wavelengths of 370 nm and 440 nm, respectively, and a cut-off value of 420 nm. The results are read for 10 minutes at room temperature, scanning every 5-10 seconds. The Vmax value per second corresponds to the residual activity of neutrophil elastase; it is plotted on the graph for each inhibitor concentration. The intersection with the abscissa indicates the concentration of inhibitor required to completely inactivate the baseline concentration of neutrophil elastase in the study. Serum derived AAT (sdAAT) was used as a positive control in these studies. Fusion proteins "AAT-Fc" have the ability to inhibit neutrophil elastase, as shown in Figure 1C. When two AAT polypeptides bind to a single Fc polypeptide (AAT-Fc-AAT), the ability to inhibit neutrophil elastase is enhanced over both sdAAT and the "AAT-Fc" fusion protein containing a single AAT polypeptide (Figure 1E). The studies presented here demonstrate for the first time that AAT can bind to the Fc region while retaining the ability to inhibit neutrophil elastase. Of particular interest is that the "AAT-Fc-AAT" fusion protein is a stronger inhibitor of neutrophil elastase.

[000190] Figure 1F shows the resistance of the fusion protein "AAT-EL-Fc (M351E, M358L)" to oxidative inactivation. AAT, AAT-Fc (wt), AAT-EL-Fc (M351E, M358L) and AAT-EM-Fc (M351E) fusion proteins were treated with 33 mM H ₂ O ₂ and compared to untreated fusion proteins in neutrophil elastase inhibition studies. The inhibition of neutrophil elastase AAT-EL-Fc was not impaired by oxidation, in contrast to other studied proteins.

[000191] Also, the "AAT-Fc" fusion protein had a longer half-life from rat serum compared to serum-derived AAT (Figure 1H). In these studies, three rats in each study were injected intravenously with 10 mg / kg sdAAT or AAT-Fc. Serum samples were taken at various time points over a 48 hour period. Serum ATT concentration was determined by ELISA. These results demonstrate that the fusion proteins of the present invention have improved pharmacokinetic properties and a therapeutic form superior to serum-derived AAT for many human inflammatory conditions and infectious diseases.

Example 2: Fusion Proteins "AAT-Acting TNF α Molecule"

[000192] The studies presented in this application describe several non-limiting examples of recombinant AAT derivatives containing human AAT linked to an anti-TNFα antibody or a TNFα receptor derivative. These examples are presented below to further illustrate the various properties of the present invention. The examples also illustrate a suitable methodology for carrying out the present invention. These examples do not limit the claimed invention and are not intended to limit the claimed invention.

[000193] The fusion proteins below include sequences of cytokine-acting polypeptides from (i) anti-TNFα D2E7 antibody (also known as adalimumab or Humira®) or (ii) the extracellular domain of TNFα type 2 receptor (TNFR2-ECD) or derived from (i ) antibodies to TNFα D2E7 (also known as adalimumab or Humira®) or (ii) the extracellular domain of the TNFα type 2 receptor (TNFR2-ECD). The AAT polypeptide that is part of the fusion protein is underlined, antibody constant regions (CH1-hinge-CH2-CH3 or CL) are in oblique, and 2E7-VH, D2E7-VK and TNFR2-ECD are in bold in the text. While these examples include a hinge sequence and / or linker sequence, the fusion proteins of the present invention may be prepared using any hinge sequence and / or linker sequence suitable in length and / or flexibility. Alternatively, fusion proteins can be produced without the use of a hinge and / or linker sequence.

[000194] An exemplary "AAT-TNFα" fusion protein is "D2E7-light chain-AAT linker (G ₃ S) ₂ " described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VK is in bold in the text (SEQ ID NO: 37), and antibody constant regions are in bold (SEQ ID NO: 38 ).

D2E7-light chain-AAT linker (G ₃ S) ₂

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC GGGSGGGS EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO : 22)

[000195] An exemplary "AAT-TNFα" fusion protein is the "D2E7-light chain-AAT linker ASTGS" described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VK is in bold in the text (SEQ ID NO: 37), and antibody constant regions are in bold (SEQ ID NO: 38 ).

D2E7-light chain-AAT linker ASTGS

DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC ASTGS EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO : 23)

[000196] An exemplary "AAT-TNFα" fusion protein is the "D2E7-heavy chain-AAT linker (G ₃ S) ₂ " described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VH is in bold (SEQ ID NO: 39), and antibody constant regions are in oblique (SEQ ID NO: 40 ).

D2E7-heavy chain-AAT linker (G ₃ S) ₂

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGSGGGS EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO : 24)

[000197] An exemplary "AAT-TNFα" fusion protein is the "D2E7-heavy chain-AAT linker ASTGS" described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2), D2E7-VH is in bold (SEQ ID NO: 39), and antibody constant regions are in oblique (SEQ ID NO: 40 ).

D2E7- heavy chain-AAT linker ASTGS

EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ASTGS EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO : 25)

[000149] An exemplary "AAT-TNFα" fusion protein is "TNFR2-ECD-Fc1-AAT linker (G ₃ S) ₂ " described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2), TNFR2-ECD is in bold in the text (SEQ ID NO: 41), and antibody constant regions are in oblique font (SEQ ID NO: 42 ).

TNFR2-ECD-Fc1-AAT linker (G ₃ S) ₂

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK GGGSGGGSEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO: 26)

[000150] An exemplary "AAT-TNFα" fusion protein is "TNFR2-ECD-Fc1-AAT linker ASTGS" described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2), TNFR2-ECD is in bold in the text (SEQ ID NO: 41), and antibody constant regions are in bold (SEQ ID NO: 42 ).

TNFR2-ECD-Fc1-AAT linker ASTGS

LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPQVKFNWYVDGVQVHNAKTKPREQQYNSTYRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ASTGS EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK (SEQ ID NO : 27)

[000198] These exemplified fusion proteins "AAT-acting on TNFα molecule" were obtained using the following techniques.

[000199] The genes encoding the variable heavy (VH) and variable kappa (VK) regions of the anti-TNFα antibody D2E7 were obtained by gene synthesis. The D2E7-VH gene was cloned in frame with a gene encoding a human IgG1 heavy chain constant region consisting of a CH1 domain, a hinge domain, a CH2 domain and a CH3 domain into a mammalian expression vector containing the mammalian secretory signal sequence upstream of the insertion site the VH domain (D2E7-HC). The D2E7-VK gene was cloned in frame with a human antibody kappa light chain (CL) constant domain into a mammalian expression vector containing the mammalian secretory signal sequence upstream of the VK domain insertion site (D2E7-LC). The gene encoding AAT and the 5'-adjacent linker sequence were cloned in frame 3'-end of the coding sequence of either the CH3 domain of the D2E7 heavy chain gene (D2E7-HC-AAT) or the CL domain of the D2E7 light chain gene (D2E7- LC-AAT) into the mammalian expression vectors described above. The extracellular domain of the TNFα 2 receptor (TNFR2-ECD) was obtained by gene synthesis and cloned in frame with the gene encoding the hinge region, behind which is the CH2 domain and the CH3 domain of human IgG1 (hFc1) into a mammalian expression vector containing the mammalian secretory signal sequence located upstream of the TNFR2-ECD insertion site. The gene encoding AAT and the 5'-adjacent linker sequence was cloned in frame from the 3'-end of the gene encoding TNFR2-ECD-hFc1 into a mammalian expression vector (TNFR2-ECD-hFc1-AAT).

[000200] Mammalian cells (namely HEK293 or CHO cells) were co-transfected with the D2E7-HC-AAT expression vector, either with the D2E7-LC expression vector or with the D2E7-LC-AAT expression vector to generate the D2E7 antibody with AAT linked to C- the end of the heavy chain or the C-terminus of both the heavy and light chains, respectively. D2E7-LC-AAT mammalian cells were co-transfected with the D2E7-HC expression vector to obtain D2E7 antibody with AAT linked to the C-terminus of the light chain. Mammalian cells were transfected with the TNFR2-hFc1-AAT expression vector. The transfected cells were grown for several days at 8% CO ₂ at 37 ° C.

[000201] Recombinant AAT TNFα fusion proteins were 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 AAT TNFα fusion proteins proteins from resin with protein A.

[000202] Figure 2B shows the results of SDS PAGE with D2E7 antibody alone (lane 1) and with a variant in which AAT is fused to the D2E7 heavy chain (lane 2). Proteins were visualized by Coomassie blue staining.

[000203] Purified fusion proteins "AAT-acting on TNFα molecule" were investigated for activity, determining their ability to inhibit neutrophil elastase. Serum derived AAT (sdAAT, serum derived AAT) was used as a positive control in these studies. Neutrophil elastase inhibition assays were performed as described above. Figure 2C shows the data for sdAAT: the fusion protein "AAT-TNFα-acting molecule" exhibits similar inhibition of neutrophil elastase, indicating that the inhibitory capacity of AAT is not impaired by antibody binding.

Example 3: "AAT-Fc-SLPI" and "AAT-Fc-elafin"

[000204] The studies presented in this application describe several non-limiting examples of recombinant AAT derivatives containing human AAT linked to a protein containing a WAP domain. These examples are presented below to further illustrate the various properties of the present invention. The examples also illustrate a useful methodology for carrying out the present invention. The AAT polypeptide that is part of the fusion protein is underlined, the Fc that is part of the fusion protein is in oblique, and the polypeptide containing the WAP domain is in bold. While these examples include a hinge sequence and / or linker sequence, the fusion proteins of the present invention may be prepared using any hinge sequence and / or linker sequence suitable in length and / or flexibility. Alternatively, fusion proteins can be produced without the use of a hinge and / or linker sequence. For example, you can directly connect the polypeptide components.

[000205] An exemplary "AAT-Fc-SLPI" fusion protein is AAT-hFc1-SLPI (human IgG1 Fc) described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2), the Fc that is part of the fusion protein is in oblique (SEQ ID NO: 3), and the polypeptide containing the WAP domain is in bold ( SEQ ID NO: 9).

AAT-hFc1-SLPI (Human IgG1 Fc)

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGS SGKSFKAGVCPPKKSAQCLRYKKPECQSDWQCPGKKRCCPDTCGIKCLDPVDTPNPTRRKPGKCPVTYGQCLMLNPPNFCEMDGQCKRDLKCCMGMCGKSCVSPVKA (SEQ ID NO: 28)

[000206] An exemplary "AAT-Fc-SLPI" fusion protein is AAT-hFc1-SLPI (human IgG1 Fc) described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2), the Fc that is part of the fusion protein is in oblique (SEQ ID NO: 3), and the polypeptide containing the WAP domain is in bold ( SEQ ID NO: 12).

AAT-hFc1-elafin (human IgG1 Fc)

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKASTGS AVTGVPVKGQDTVKGRVPFNGQDPVKGQVSVKGQDKVKAQEPVKGPVSTKPGSCPIILIRCAMLNPPNRCLKDTDCPGIKKCCEGSCGMACFVPQ (SEQ ID NO: 29)

[000207] Genes encoding SLPI and elafin were amplified by PCR from human spleen cDNA (Zyagen). These genes were cloned into the mammalian expression vector from Example 1, in which the SLPI or elafin gene was inserted in frame with the AAT-Fc gene. These expression vectors were transfected into mammalian cells (namely HEK293 or CHO cells) and grown for several days at 8% CO ₂ at 37 ° C. Recombinant fusion proteins “AAT-Fc-domain of WAP” were isolated from the supernatant of expressing cells by chromatography on protein A. A buffer with close to neutral pH was used (Gentle Ag / Ab Elution Buffer, Thermo Scientific) to elute the fusion protein “AAT-Fc -domain WAP ”from resin with protein A.

[000208] Figure 3B shows the results of PAGE in the presence of sodium dodecyl sulfate fusion proteins "AAT-Fc-WAP domain" (lane 1: AAT-Fc-elafin, lane 2: AAT-Fc-SLPI). Proteins were visualized by Coomassie blue staining. The purified WAP AAT-Fc domain fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase. Neutrophil elastase inhibition assays were performed as described above. Serum derived AAT (sdAAT, serum derived AAT) and "AAT-Fc" fusion protein were used as positive controls in these studies. Relative to sdAAT, the fusion protein "AAT-Fc-WAP-acting molecule" exhibits more pronounced activity in inhibiting neutrophil elastase (Figure 3C).

Example 4: "AAT-albumin"

[000209] The studies presented in this application describe several non-limiting examples of recombinant AAT derivatives containing human AAT linked to an albumin polypeptide. These examples are presented below to further illustrate the various properties of the present invention. The examples also illustrate a useful methodology for carrying out the present invention. These examples do not limit the present invention and are not intended to limit the claimed invention. AAT that is part of the fusion protein is underlined, albumin that is part of the fusion protein is in oblique. For example, you can directly connect the polypeptide components.

[000210] An exemplary "AAT-albumin" fusion protein is AAT-HSA, as described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2), the albumin polypeptide that is part of the fusion protein is in oblique type (SEQ ID NO: 14).

AAT-HSA

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK ASTGS DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 30)

[000211] An exemplary "AAT-albumin" fusion protein is "HSA AAT-domain 3" described herein. As shown below, the AAT polypeptide that is part of the fusion protein is underlined (SEQ ID NO: 2), the albumin polypeptide that is part of the fusion protein is in oblique type (SEQ ID NO: 15).

AAT domain 3 HSA

EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK ASTGS EEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVA (SEQ ID NO: 31)

[000212] The gene encoding human serum albumin (HSA) was amplified by PCR with human liver cDNA (Zyagen). A mammalian expression vector was prepared in which the gene encoding HSA or HSA domain 3 was cloned in frame from the 3 'end of the AAT-encoding gene containing the mammalian secretory signal sequence upstream of the AAT.

[000213] These expression vectors were transfected into mammalian cells (namely HEK293 or CHO cells) and grown for several days at 8% CO ₂ at 37 ° C. Recombinant AAT-HSA fusion proteins were isolated from the supernatant of expressing cells using CaptureSelect® alpha-1-antitrypsin affinity matrix (BAC BV), the binding buffer consisted of 20 mM Tris, 150 mM NaCl, pH 7.4 and the elution buffer consisted of 20 mM Tris, 2M MgCl ₂ pH 7.4.

[000214] Figure 4B shows the results of electrophoresis in PAGE in the presence of sodium dodecyl sulfate with a fusion protein "AAT-HSA". Proteins were visualized by Coomassie blue staining. The purified "AAT-HSA" fusion proteins were tested for activity by determining their ability to inhibit neutrophil elastase. Neutrophil elastase inhibition assays were performed as described above. Serum derived AAT (sdAAT) was used as a positive control in these studies. With respect to sdAAT, the “AAT-HSA” fusion protein showed similar activity in inhibiting neutrophil elastase, indicating that binding to albumin does not prevent AAT from inhibiting neutrophil elastase (Figure 4C).

Other embodiments of the present invention

[000215] While the present invention has been described in conjunction with its detailed description, the above description is intended to illustrate, but not to limit the scope of the invention as defined by the scope of the appended claims. Other aspects, advantages and modifications are included within the scope of the following claims.

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SEQUENCE LIST

<110> Inhibrx LP

Eckelman, Brendan P.

Timmer, John C.

Deveraux, Quinn

<120> FUSED SERPINE POLYPEPTIDES AND METHODS OF THEIR APPLICATION

<130> INHI-002 / 002WO

<150> US 14 / 524,832

<151> 2014-10-27

<160> 80

<170> PatentIn version 3.5

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<223> reactive site loop sequence

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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

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<212> PROTEIN

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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

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Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

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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

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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

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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 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

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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

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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

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<212> PROTEIN

<213> Homo sapiens

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Met Lys Ser Ser Gly Leu Phe Pro Phe Leu Val Leu Leu Ala Leu Gly

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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> PROTEIN

<213> Homo sapiens

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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

fifty

<210> 11

<211> 117

<212> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<213> Artificial sequence

<220>

<223> D2E7-light chain-AAT linker (G3S) 2

<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> PROTEIN

<213> Artificial sequence

<220>

<223> D2E7-light chain-AAT linker ASTGS

<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 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> PROTEIN

<213> Artificial sequence

<220>

<223> D2E7-heavy chain-AAT linker (G3S) 2

<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 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> PROTEIN

<213> Artificial sequence

<220>

<223> D2E7-heavy chain-AAT linker ASTGS

<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> PROTEIN

<213> Artificial sequence

<220>

<223> TNFR2-ECD-Fc1-AAT linker (G3S) 2

<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 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> PROTEIN

<213> Artificial sequence

<220>

<223> TNFR2-ECD-Fc1-AAT linker ASTGS

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<213> Artificial sequence

<220>

<223> AAT domain 3 HSA

<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> PROTEIN

<213> Artificial sequence

<220>

<223> reactive site loop sequence 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> PROTEIN

<213> Artificial sequence

<220>

<223> reactive site loop sequence 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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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 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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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 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> PROTEIN

<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> PROTEIN

<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 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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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 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> PROTEIN

<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> PROTEIN

<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 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> PROTEIN

<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 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> PROTEIN

<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 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> PROTEIN

<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> PROTEIN

<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 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> PROTEIN

<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 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> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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

twenty

<210> 77

<211> 21

<212> PROTEIN

<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

twenty

<210> 78

<211> 624

<212> PROTEIN

<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> PROTEIN

<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> PROTEIN

<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 (10)

1. An isolated fusion protein comprising at least one human alpha-1-antitrypsin (AAT) polypeptide operably linked to a modified human IgG4 Fc polypeptide, wherein said modified human IgG4 Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 60, 69 or 70 wherein said modified human IgG4 Fc polypeptide comprises a mutation at position M252Y, which corresponds to residue 34 of SEQ ID NO: 60, and a mutation M428L, which corresponds to residue 210 of SEQ ID NO: 60, for use in a method of inhibiting or down-regulating a disturbed expression or activity serine protease in a subject in need thereof. 2. The isolated fusion protein of claim 1, wherein the AAT polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 80. 3. The isolated fusion protein of claim 1, wherein the AAT polypeptide comprises an AAT reaction center loop comprising the amino acid sequence of SEQ ID NO: 1. 4 The isolated fusion protein of claim 1, wherein the AAT polypeptide comprises a mutant AAT reaction center loop comprising the amino acid sequence of SEQ ID NO: 32 or 33. 5. The isolated fusion protein of claim 1, wherein said modified IgG4 Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 73. 6. The isolated fusion protein of claim 1, wherein the serpinated polypeptide and said modified IgG4 Fc polypeptide are operably linked by a linker region, optionally wherein the linker region comprises a peptide sequence. 7. The isolated fusion protein of claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 79. 8. The isolated fusion protein of claim 1, wherein the subject is a human. 9. The isolated fusion protein of claim 1, wherein said subject has an inflammatory disease or disorder selected from the following: alpha-1 antitrypsin deficiency, emphysema, chronic obstructive pulmonary disease (COPD), acute respiratory failure syndrome (OCPF), allergic asthma, cystic fibrosis, lung cancer, ischemia-reperfusion injury, including ischemia-reperfusion injury after heart transplant, myocardial infarction, rheumatoid arthritis, septic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, psoriasis, type I and / or type II diabetes, pneumonia, sepsis, graft versus host disease, wound healing, systemic lupus erythematosus, and multiple sclerosis. 10. The isolated protein of claim 1, wherein said subject has an infection or risk of infection, wherein said infection is selected from bacterial infections, fungal infections, or viral infections.

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