C1 esterase inhibitor fusion protein and its use

Abstract

The present invention provides, inter alia, methods and compositions for treating complement-mediated diseases, particularly chronic diseases requiring preventive and / or maintenance therapy. In one aspect, a C1-INH fusion protein having a longer half-life than native plasma-derived C1-INH is provided. In some embodiments, the methods according to the present invention comprise administering an effective amount of a recombinant C1-INH fusion protein to an individual suffering from or susceptible to a complement-mediated disease, such that at least one symptom or feature of the complement-mediated disease is prevented and / or reduced in intensity, severity or frequency.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of the Chinese invention patent application with application number 201580067942.4, application date October 31, 2015, and invention name “C1 esterase inhibitor fusion protein and its use”. The original application is the Chinese national phase application of the PCT international application with international application number PCT / US2015 / 058521, which claims the priority of the U.S. provisional patent application serial number 62 / 073,657 filed on October 31, 2014. The disclosure of the U.S. provisional patent application is hereby incorporated by reference in its entirety. background

[0003] C1-inhibitor (C1-INH), also known as C1 esterase inhibitor, is the largest member of the serpin protein superfamily. It is a heavily glycosylated serine protease inhibitor with the primary function of inhibiting spontaneous activation of the complement system. C1-INH regulates the complement cascade system, plays a key role in regulating the contact (kallikrein-kinin) amplification cascade, and is involved in regulating the coagulation and fibrinolysis systems. Karnaukhova, E., C1-Esterase Inhibitor: Biological Activities and Therapeutic Applications. J Hematol Thromb Dis, 1: 113 (2013).

[0004] C1-INH dysfunction and / or deficiency in subjects has been associated with a variety of autoimmune diseases due to the failure of C1-INH to inhibit the activation of the complement system. An example of such a disease is hereditary angioedema (HAE), a rare but potentially life-threatening condition characterized by unpredictable and recurrent inflammation. Symptoms of HAE attacks include swelling of the face, mouth, and / or airways that occurs spontaneously or is triggered by minor trauma. Such swelling may also occur in any part of the body. In some cases, HAE is associated with low plasma levels of C1 inhibitor, while in other cases, the protein circulates in normal or elevated amounts, but it is dysfunctional. In addition to inflammatory attacks, this can also lead to more severe or life-threatening indications, such as autoimmune diseases or lupus erythematosus.

[0005] As a human plasma-derived C1 esterase inhibitor, it has been approved for prophylactic use and for the treatment of acute HAE attacks. (also a plasma-derived human C1-INH, CSL Behring) is indicated for the treatment of acute HAE attacks. The supply of human plasma-derived C1 esterase inhibitors is dependent on the availability of blood and plasma donations. (conestat alfa, Pharming NV) is a recombinant C1-INH expressed in engineered rabbits and is indicated for intravenous (IV) administration to treat acute HAE attacks. It is produced in rabbits, so its glycosylation profile is different from that of human plasma-derived C1-INH. As a result, Ruconest has an extremely short half-life of approximately 2.4-2.7 hours. FDA labeling and prescribing information.

[0006] Therefore, there remains a need in the art for improved C1 esterase inhibitors for the treatment of various C1 esterase-mediated indications. SUMMARY OF THE INVENTION

[0007] In particular, the present invention provides improved long-acting recombinant C1 esterase inhibitors that are useful for the effective treatment of various complement-mediated disorders and that can be manufactured in a cost-effective manner.

[0008] Specifically, the present invention provides C1 esterase inhibitor fusion proteins exhibiting longer half-lives. In some embodiments, the C1 inhibitor fusion proteins of the present invention exhibit similar or longer half-lives compared to plasma-derived C1-INH. For example, the inventors have demonstrated that certain exemplary C1 inhibitor fusion proteins according to the present invention have an extended serum half-life of at least 4 days. The long serum half-life of the recombinant C1 inhibitor is expected to result in superior in vivo efficacy and allow for better dosing regimens and routes of administration. In certain embodiments, the C1 inhibitor fusion proteins of the present invention can be administered subcutaneously with less frequency compared to approved C1 inhibitors while still achieving the desired efficacy (e.g., prevention and treatment). In addition, the C1 inhibitor fusion proteins of the present invention can be recombinantly produced in host cells so that the disclosed C1 inhibitor fusion proteins are independent of the blood supply, do not pose a risk of transmitting infectious agents, and are less expensive to manufacture. Because they are recombinantly produced in host cells, they provide greater consistency in production and final products compared to those products purified from human blood, human blood components (e.g., plasma), or animal milk. In addition, the C1 inhibitor fusion proteins provided herein are independent of animal husbandry considerations, including animal age and / or maturity, milk production, animal diseases, etc., all of which can affect both the quantity and quality (e.g., glycosylation characteristics, heterogeneity of expressed proteins, etc.) of C1-INH expressed by rabbits. Therefore, the present invention provides a cost-effective and reliable manufacture of recombinant C1 esterase inhibitors, as well as safer and more effective treatments for HAE and other complement-mediated disorders. Other features, objects, and advantages of the present invention will be apparent from the detailed description that follows. However, it should be understood that although embodiments of the present invention are indicated, the detailed description is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description.

[0009] In one aspect, the present invention provides a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain. In some embodiments, the human C1-inhibitor polypeptide comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the full-length human C1-inhibitor having SEQ ID NO: 1.

[0010] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 70% identity with the full-length human C1-inhibitor protein SEQ ID NO: 1. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 80% identity with the full-length human C1-inhibitor protein SEQ ID NO: 1. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 90% identity with the full-length human C1-inhibitor protein SEQ ID NO: 1. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 95% identity with the full-length human C1-inhibitor protein SEQ ID NO: 1. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to the full-length human C1-inhibitor protein SEQ ID NO: 1. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 1.

[0011] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to the full-length human C1-inhibitor protein SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to the full-length human C1-inhibitor protein SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to the full-length human C1-inhibitor protein SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to the full-length human C1-inhibitor protein SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is identical to the full-length human C1-inhibitor protein SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 2.

[0012] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain is connected to the N-terminus of the human C1-inhibitor polypeptide. In some embodiments, the Fc domain is a human IgG1 Fc domain. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain is connected to the N-terminus of the human C1-inhibitor polypeptide. In some embodiments, the Fc domain is derived from a human IgG1 Fc domain. In some embodiments, an Fc domain suitable for the present invention comprises an amino acid sequence having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity with SEQ ID NO: 3. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 70% identity with the human IgG1 Fc domain SEQ ID NO: 3. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 80% identity with the human IgG1 Fc domain SEQ ID NO: 3. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 90% identity with the human IgG1 Fc domain SEQ ID NO: 3. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 95% identity with the human IgG1 Fc domain SEQ ID NO: 3. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to the human IgG1 Fc domain SEQ ID NO: 3. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 3.

[0013] In some embodiments, an Fc domain suitable for the present invention comprises an L234A mutation. In some embodiments, an Fc domain suitable for the present invention comprises an L235A mutation. In some embodiments, an Fc domain suitable for the present invention comprises an L234A mutation and an L235A mutation. In some embodiments, an Fc domain suitable for the present invention comprises an L234A mutation or an L235A mutation.

[0014] In some embodiments, an Fc domain suitable for the present invention comprises the amino acid sequence SEQ ID NO: 4. In some embodiments, an Fc domain suitable for the present invention comprises one or more mutations that extend the half-life of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain. In some embodiments, the mutations in the Fc domain comprise one or more mutations selected from one or more positions corresponding to Thr250, Met 252, Ser254, Thr256, Thr 307, Glu380, Met428, His 433, and / or Asn434 of human IgG1.

[0015] In some embodiments, an Fc domain suitable for the present invention comprises an H433K mutation. In some embodiments, an Fc domain suitable for the present invention comprises an N434F mutation. In some embodiments, an Fc domain suitable for the present invention comprises an H433K mutation and an N434F mutation. In some embodiments, an Fc domain suitable for the present invention comprises an H433K mutation or an N434F mutation.

[0016] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 5. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 5. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 5. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 5. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to SEQ ID NO: 5. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 5.

[0017] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 6. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 6. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 6. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 6. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 6. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to SEQ ID NO: 6. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 6.

[0018] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 7. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 7. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 7. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 7. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to SEQ ID NO: 7. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 7.

[0019] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0020] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 70% identity to any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

[0021] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain is linked to the N-terminus of the human C1-inhibitor polypeptide. In some embodiments, the Fc domain is a human IgG4 Fc domain. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain is linked to the N-terminus of the human C1-inhibitor polypeptide. In some embodiments, the Fc domain is derived from a human IgG4 Fc domain.

[0022] In some embodiments, an Fc domain suitable for the present invention comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 9. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to a human IgG4 Fc domain, SEQ ID NO: 9. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to a human IgG4 Fc domain, SEQ ID NO: 9. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to a human IgG4 Fc domain, SEQ ID NO: 9. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 95% identity to the human IgG4 Fc domain SEQ ID NO: 9. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to the human IgG4 Fc domain SEQ ID NO: 9. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 9.

[0023] In some embodiments, an Fc domain suitable for the present invention comprises an S241P mutation. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO: 10. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 10. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 10. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 10. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 10. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is identical to SEQ ID NO: 10. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 10.

[0024] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 11. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 11. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 11. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 11. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to SEQ ID NO: 11. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 11.

[0025] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 12. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 12. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 12. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 12. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 12. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to SEQ ID NO: 12. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 12.

[0026] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 13. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 13. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 13. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 13. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to SEQ ID NO: 13. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 13.

[0027] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 14. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 14. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 14. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 14. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 14. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to SEQ ID NO: 14. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 14.

[0028] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 15. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 15. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 15. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 15. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 15. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to SEQ ID NO: 15. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 15.

[0029] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 16. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 16. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 16. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 16. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises an amino acid sequence identical to SEQ ID NO: 16. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 16.

[0030] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein comprises a linker between the human C1-inhibitor polypeptide and the Fc domain. In some embodiments, the linker is a peptide comprising 3-100 amino acids.

[0031] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein binds to FcRN. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein inhibits C1 esterase activity.

[0032] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain comprises a mutation that reduces or eliminates ADCC activity. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain comprises one or more mutations that reduce or eliminate ADCC activity. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain comprises a mutation that reduces or eliminates CDC activity. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain comprises one or more mutations that reduce or eliminate CDC activity.

[0033] In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain comprises a mutation that reduces or eliminates FcγR binding. In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain comprises one or more mutations that reduce or eliminate FcγR binding. In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain comprises a mutation that reduces or eliminates FcγR effector function. In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain comprises one or more mutations that reduce or eliminate FcγR effector function.

[0034] In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain comprises a mutation that reduces or eliminates C1q binding. In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the Fc domain comprises one or more mutations that reduce or eliminate C1q binding. In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the mutation that reduces or eliminates C1q binding is in the Fc domain.

[0035] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein inhibits and / or inactivates C1r and / or C1s protease activity. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein inhibits lysis of red blood cells in vitro.

[0036] In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein has a longer half-life than plasma-derived human C1-inhibitor. In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein has a half-life of at least 4 days. In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein has a half-life of at least 5 days. In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein has a half-life of at least 6 days. In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein has a half-life of at least 7 days.

[0037] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein has an in vivo half-life of or greater than about 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, or 10 days. In some embodiments, the recombinant C1-INH fusion protein has a lifespan of between 0.5 and 10 days, between 1 day and 10 days, between 1 day and 9 days, between 1 day and 8 days, between 1 day and 7 days, between 1 day and 6 days, between 1 day and 5 days, between 1 day and 4 days, between 1 day and 3 days, between 2 days and 10 days, between 2 days and 9 days, between 2 days and 8 days, between 2 days and 7 days, between 2 days and 6 days, between 2 days and 5 days, between 2 days and 4 days. days, between 2 days and 3 days, between 2.5 days and 10 days, between 2.5 days and 9 days, between 2.5 days and 8 days, between 2.5 days and 7 days, between 2.5 days and 6 days, between 2.5 days and 5 days, between 2.5 days and 4 days, between 3 days and 10 days, between 3 days and 9 days, between 3 days and 8 days, between 3 days and 7 days, between 3 days and 6 days, between 3 days and 5 days, between 3 days and 4 days, between 3.5 days and 10 days, between Between 3.5 days and 9 days, Between 3.5 days and 8 days, Between 3.5 days and 7 days, Between 3.5 days and 6 days, Between 3.5 days and 5 days, Between 3.5 days and 4 days, Between 4 days and 10 days, Between 4 days and 9 days, Between 4 days and 8 days, Between 4 days and 7 days, Between 4 days and 6 days, Between 4 days and 5 days, Between 4.5 days and 10 days, Between 4.5 days and 9 days, Between 4.5 days and 8 days, Between 4.5 days and 7 days, Between In vivo half-life of between 4.5 days and 6 days, between 4.5 days and 5 days, between 5 days and 10 days, between 5 days and 9 days, between 5 days and 8 days, between 5 days and 7 days, between 5 days and 6 days, between 5.5 days and 10 days, between 5.5 days and 9 days, between 5.5 days and 8 days, between 5.5 days and 7 days, between 5.5 days and 6 days, between 6 days and 10 days, between 7 days and 10 days, between 8 days and 10 days, and between 9 days and 10 days.

[0038] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein is monovalent. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, the fusion protein is dimeric.

[0039] In one aspect, the present invention provides a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide. In some embodiments, the human C1-inhibitor polypeptide comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the full-length human C1-inhibitor having SEQ ID NO: 1. In some embodiments, the fusion protein comprising the human C1-inhibitor polypeptide and the albumin polypeptide comprises an amino acid sequence that is at least 70% identical to the full-length human C1-inhibitor protein SEQ ID NO: 1. In some embodiments, the fusion protein comprising the human C1-inhibitor polypeptide and the albumin polypeptide comprises an amino acid sequence that is at least 80% identical to the full-length human C1-inhibitor protein SEQ ID NO: 1. In some embodiments, the fusion protein comprising the human C1-inhibitor polypeptide and the albumin polypeptide comprises an amino acid sequence that is at least 90% identical to the full-length human C1-inhibitor protein SEQ ID NO: 1. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 95% identical to the full-length human C1-inhibitor protein SEQ ID NO: 1. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is identical to the full-length human C1-inhibitor protein SEQ ID NO: 1. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 1.

[0040] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 70% identical to the full-length human C1-inhibitor protein SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 80% identical to the full-length human C1-inhibitor protein SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 90% identical to the full-length human C1-inhibitor protein SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 95% identical to the full-length human C1-inhibitor protein SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is identical to the full-length human C1-inhibitor protein SEQ ID NO: 2. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 2.

[0041] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the albumin is linked to the N-terminus of the human C1-inhibitor polypeptide. . In some embodiments, the albumin polypeptide comprises one or more domains of human serum albumin. In some embodiments, the albumin polypeptide comprises the D3 domain of human serum albumin. In some embodiments, the albumin polypeptide is derived from human serum albumin. . In some embodiments, the albumin polypeptide is human serum albumin.

[0042] In some embodiments, an albumin polypeptide suitable for the present invention comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 20. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 70% identical to the human albumin polypeptide SEQ ID NO: 20. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 80% identical to the human albumin polypeptide SEQ ID NO: 20. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 90% identical to the human albumin polypeptide SEQ ID NO: 20. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 95% identical to the human albumin polypeptide SEQ ID NO: 20. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is identical to the human albumin polypeptide SEQ ID NO: 20. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 20.

[0043] In some embodiments, an albumin polypeptide suitable for the present invention comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 17. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 70% identical to the human albumin polypeptide SEQ ID NO: 17. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 80% identical to the human albumin polypeptide SEQ ID NO: 17. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 90% identical to the human albumin polypeptide SEQ ID NO: 17. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 95% identical to the human albumin polypeptide SEQ ID NO: 17. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is identical to the human albumin polypeptide SEQ ID NO: 17. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 17.

[0044] In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 18. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 18. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 18. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 18. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 18. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence identical to SEQ ID NO: 18. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 18.

[0045] In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 19. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 19. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 19. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 19. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 19. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence identical to SEQ ID NO: 19. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 19.

[0046] In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 21. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 21. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 21. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 21. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 21. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence identical to SEQ ID NO: 21. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 21.

[0047] In some embodiments of a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 22. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 22. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 22. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 22. In some embodiments, a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 22. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence identical to SEQ ID NO: 22. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 22.

[0048] In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence that is at least 70% identical to any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises an amino acid sequence identical to any one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide comprises one or more truncations, deletions, mutations, or insertions compared to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22.

[0049] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein binds to FcRN. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein inhibits C1 esterase activity. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein inhibits C1r and / or C1s protease activity and / or inactivates C1r and / or C1s protease activity. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein inhibits the lysis of red blood cells in vitro.

[0050] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein comprises a linker between the human C1-inhibitor polypeptide and the albumin polypeptide. In some embodiments, the linker is a peptide comprising 3-100 amino acids. In some embodiments, the linker comprises the sequence GGG. In some embodiments, the linker comprises the sequence SEQ ID NO: 27.

[0051] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the albumin polypeptide does not comprise one or more mutations selected from the group consisting of 464His, 510His, 535His and / or combinations thereof.

[0052] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein has a longer half-life compared to plasma-derived human C1-inhibitor. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein has a half-life of at least 4 days. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein has a half-life of at least 5 days. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein has a half-life of at least 6 days. In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein has a half-life of at least 7 days.

[0053] In some embodiments of the fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, the fusion protein has an in vivo half-life of or greater than about 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, or 10 days. In some embodiments, the recombinant C1-INH fusion protein has a lifespan of between 0.5 and 10 days, between 1 day and 10 days, between 1 day and 9 days, between 1 day and 8 days, between 1 day and 7 days, between 1 day and 6 days, between 1 day and 5 days, between 1 day and 4 days, between 1 day and 3 days, between 2 days and 10 days, between 2 days and 9 days, between 2 days and 8 days, between 2 days and 7 days, between 2 days and 6 days, between 2 days and 5 days, between 2 days and 4 days. days, between 2 days and 3 days, between 2.5 days and 10 days, between 2.5 days and 9 days, between 2.5 days and 8 days, between 2.5 days and 7 days, between 2.5 days and 6 days, between 2.5 days and 5 days, between 2.5 days and 4 days, between 3 days and 10 days, between 3 days and 9 days, between 3 days and 8 days, between 3 days and 7 days, between 3 days and 6 days, between 3 days and 5 days, between 3 days and 4 days, between 3.5 days and 10 days, between Between 3.5 days and 9 days, Between 3.5 days and 8 days, Between 3.5 days and 7 days, Between 3.5 days and 6 days, Between 3.5 days and 5 days, Between 3.5 days and 4 days, Between 4 days and 10 days, Between 4 days and 9 days, Between 4 days and 8 days, Between 4 days and 7 days, Between 4 days and 6 days, Between 4 days and 5 days, Between 4.5 days and 10 days, Between 4.5 days and 9 days, Between 4.5 days and 8 days, Between 4.5 days and 7 days, Between In vivo half-life of between 4.5 days and 6 days, between 4.5 days and 5 days, between 5 days and 10 days, between 5 days and 9 days, between 5 days and 8 days, between 5 days and 7 days, between 5 days and 6 days, between 5.5 days and 10 days, between 5.5 days and 9 days, between 5.5 days and 8 days, between 5.5 days and 7 days, between 5.5 days and 6 days, between 6 days and 10 days, between 7 days and 10 days, between 8 days and 10 days, and between 9 days and 10 days.

[0054] In one aspect, the present invention provides a nucleic acid encoding a fusion protein, which is any one of the fusion proteins comprising a human C1-inhibitor polypeptide and an Fc domain disclosed herein. In another aspect, the present invention provides a nucleic acid encoding a fusion protein, which is any one of the fusion proteins comprising a human C1-inhibitor polypeptide and an albumin polypeptide disclosed herein.

[0055] In one aspect, the present invention provides a cell comprising a nucleic acid encoding a fusion protein, wherein the fusion protein is any one of the fusion proteins comprising a human C1-inhibitor polypeptide and an Fc domain disclosed herein. In another aspect, the present invention provides a cell comprising a nucleic acid encoding a fusion protein, wherein the fusion protein is any one of the fusion proteins comprising a human C1-inhibitor polypeptide and an albumin polypeptide disclosed herein.

[0056] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the cell is engineered to modify the glycosylation of a protein expressed by the cell. In some embodiments, the cell is engineered to modify the glycosylation of a protein expressed by the cell compared to the same cell that has not been engineered. In some embodiments, the cell is engineered to enhance, improve, increase and / or humanize the glycosylation of a protein expressed by the cell. In some embodiments, the cell is engineered to enhance, improve, increase and / or humanize the glycosylation of a protein expressed by the cell compared to the same cell that has not been engineered. In some embodiments, the cell is engineered to modify the sialylation of a protein expressed by the cell. In some embodiments, the cell is engineered to modify the sialylation of a protein expressed by the cell compared to the same cell that has not been engineered. In some embodiments, the cell is engineered to enhance, improve, increase and / or humanize the sialylation of a protein expressed by the cell. In some embodiments, the cell is engineered to enhance, improve, increase, and / or humanize sialylation of a protein expressed by the cell compared to the same cell that has not been engineered.

[0057] In one aspect, the present invention provides a method for producing a fusion protein, comprising the step of culturing or growing a cell comprising a nucleic acid encoding a fusion protein, wherein the fusion protein is any one of the fusion proteins comprising a human C1-inhibitor polypeptide and an Fc domain disclosed herein. In one aspect, the present invention provides a method for producing a fusion protein, comprising the step of culturing or growing a cell comprising a nucleic acid encoding a fusion protein, wherein the fusion protein is any one of the fusion proteins comprising a human C1-inhibitor polypeptide and an albumin polypeptide disclosed herein.

[0058] In another aspect, the present invention provides a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, and expressed or produced by cells engineered to modify, enhance, improve, increase and / or humanize glycosylation. In another aspect, the present invention provides a fusion protein comprising a human C1-inhibitor polypeptide and an Fc domain, and expressed or produced by cells engineered to modify, enhance, improve, increase and / or humanize sialylation. In another aspect, the present invention provides a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, and expressed or produced by cells engineered to modify, enhance, improve, increase and / or humanize glycosylation. In another aspect, the present invention provides a fusion protein comprising a human C1-inhibitor polypeptide and an albumin polypeptide, and expressed or produced by cells engineered to modify, enhance, improve, increase and / or humanize sialylation.

[0059] In another aspect, the present invention provides a pharmaceutical composition comprising a fusion protein and a pharmaceutically acceptable carrier, wherein the fusion protein is any one of the fusion proteins comprising a human C1-inhibitor polypeptide and an Fc domain disclosed herein. In another aspect, the present invention provides a pharmaceutical composition comprising a fusion protein and a pharmaceutically acceptable carrier, wherein the fusion protein is any one of the fusion proteins comprising a human C1-inhibitor polypeptide and an albumin polypeptide disclosed herein.

[0060] In one aspect, the present invention provides a method for treating a complement-mediated disorder, comprising administering to a subject in need of treatment a pharmaceutical composition comprising a fusion protein and a pharmaceutically acceptable carrier, wherein the fusion protein is any one of the fusion proteins comprising a human C1-inhibitor polypeptide and an Fc domain disclosed herein. In one aspect, the present invention provides a method for treating a complement-mediated disorder, comprising administering to a subject in need of treatment a pharmaceutical composition comprising a fusion protein and a pharmaceutically acceptable carrier, wherein the fusion protein is any one of the fusion proteins comprising a human C1-inhibitor polypeptide and an albumin polypeptide disclosed herein.

[0061] In some embodiments, the subject in need of treatment has a complement-mediated disorder. In some embodiments, the complement-mediated disorder is selected from hereditary angioedema, antibody-mediated rejection, neuromyelitis optica spectrum disorder, traumatic brain injury, spinal cord injury, ischemic brain injury, burn injury, toxic epidermal necrolysis, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, stroke, chronic inflammatory demyelinating polyneuropathy (CIDP), myasthenia gravis, multifocal motor neuropathy. Sequence Listing <110> Takeda Pharmaceutical Company Limited <120> C1 esterase inhibitor fusion protein and its use <130> 2006685-1208 <140> PCT / US2015 / 058521 <141> 2015-10-31 <150> 62 / 073,657 <151> 2014-10-31 <160> 38 <170> PatentIn version 3.5 <210> 1 <211> 478 <212> PRT <213> Homo sapiens <400> 1 Asn Pro Asn Ala Thr Ser Ser Ser Ser Gln Asp Pro Glu Ser Leu Gln 1 5 10 15 Asp Arg Gly Glu Gly Lys Val Ala Thr Thr Val Ile Ser Lys Met Leu 20 25 30 Phe Val Glu Pro Ile Leu Glu Val Ser Ser Leu Pro Thr Thr Asn Ser 35 40 45 Thr Thr Asn Ser Ala Thr Lys Ile Thr Ala Asn Thr Thr Asp Glu Pro 50 55 60 Thr Thr Gln Pro Thr Thr Glu Pro Thr Thr Gln Pro Thr Ile Gln Pro 65 70 75 80 Thr Gln Pro Thr Thr Gln Leu Pro Thr Asp Ser Pro Thr Gln Pro Thr 85 90 95 Thr Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser Asp Leu Glu 100 105 110 Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val Asp Phe Ser 115 120 125 Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val Glu Thr Asn 130 135 140 Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr Gln Val Leu 145 150 155 160 Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser Ile Leu Ser 165 170 175 Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys Gly Phe Thr 180 185 190 Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser Pro Asp Leu 195 200 205 Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu Tyr Ser Ser 210 215 220 Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu Glu Leu Ile 225 230 235 240 Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser Arg Leu Leu 245 250 255 Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn Ala Ile Tyr 260 265 270 Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys Thr Arg Met 275 280 285 Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro Met Met Asn 290 295 300 Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr Leu Lys Ala 305 310 315 320 Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu Val Ile Leu 325 330 335 Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu Gln Ala Leu 340 345 350 Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu Met Ser Lys 355 360 365 Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val Thr Thr Ser 370 375 380 Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe Asp Phe Ser 385 390 395 400 Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp Leu Gln Val 405 410 415 Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu Thr Gly Val 420 425 430 Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr Leu Leu Val 435 440 445 Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp Gln Gln His 450 455 460 Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg Ala 465 470 475 <210> 2 <211> 381 <212> PRT <213> Homo sapiens <400> 2 Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser Asp Leu Glu Ser 1 5 10 15 His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val Asp Phe Ser Leu 20 25 30 Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val Glu Thr Asn Met 35 40 45 Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr Gln Val Leu Leu 50 55 60 Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser Ile Leu Ser Tyr 65 70 75 80 Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys Gly Phe Thr Thr 85 90 95 Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser Pro Asp Leu Ala 100 105 110 Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu Tyr Ser Ser Ser 115 120 125 Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu Glu Leu Ile Asn 130 135 140 Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser Arg Leu Leu Asp 145 150 155 160 Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn Ala Ile Tyr Leu 165 170 175 Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys Thr Arg Met Glu 180 185 190 Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro Met Met Asn Ser 195 200 205 Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr Leu Lys Ala Lys 210 215 220 Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu Val Ile Leu Val 225 230 235 240 Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu Gln Ala Leu Ser 245 250 255 Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu Met Ser Lys Phe 260 265 270 Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val Thr Thr Ser Gln 275 280 285 Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe Asp Phe Ser Tyr 290 295 300 Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp Leu Gln Val Ser 305 310 315 320 Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu Thr Gly Val Glu 325 330 335 Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr Leu Leu Val Phe 340 345 350 Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp Gln Gln His Lys 355 360 365 Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg Ala 370 375 380 <210> 3 <211> 227 <212> PRT <213> Homo sapiens <400> 3 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> 4 <211> 227 <212> PRT <213> Homo sapiens <400> 4 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 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> 5 <211> 227 <212> PRT <213> Homo sapiens <400> 5 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 Lys Phe His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 6 <211> 227 <212> PRT <213> Homo sapiens <400> 6 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 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 Lys Phe His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 7 <211> 247 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 7 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 65 70 75 80 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 145 150 155 160 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220 Cys Ser Val Met His Glu Ala Leu Lys Phe His Tyr Thr Gln Lys Ser 225 230 235 240 Leu Ser Leu Ser Pro Gly Lys 245 <210> 8 <211> 247 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 8 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 65 70 75 80 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 145 150 155 160 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220 Cys Ser Val Met His Glu Ala Leu Lys Phe His Tyr Thr Gln Lys Ser 225 230 235 240 Leu Ser Leu Ser Pro Gly Lys 245 <210> 9 <211> 229 <212> PRT <213> Homo sapiens <400> 9 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> 10 <211> 229 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 10 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> 11 <211> 705 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 11 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 Asn Pro Asn Ala Thr Ser Ser Ser Ser Gln Asp Pro Glu 225 230 235 240 Ser Leu Gln Asp Arg Gly Glu Gly Lys Val Ala Thr Thr Val Ile Ser 245 250 255 Lys Met Leu Phe Val Glu Pro Ile Leu Glu Val Ser Ser Leu Pro Thr 260 265 270 Thr Asn Ser Thr Thr Asn Ser Ala Thr Lys Ile Thr Ala Asn Thr Thr 275 280 285 Asp Glu Pro Thr Thr Gln Pro Thr Thr Glu Pro Thr Thr Gln Pro Thr 290 295 300 Ile Gln Pro Thr Gln Pro Thr Thr Gln Leu Pro Thr Asp Ser Pro Thr 305 310 315 320 Gln Pro Thr Thr Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser 325 330 335 Asp Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val 340 345 350 Asp Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val 355 360 365 Glu Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr 370 375 380 Gln Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser 385 390 395 400 Ile Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys 405 410 415 Gly Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser 420 425 430 Pro Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu 435 440 445 Tyr Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu 450 455 460 Glu Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser 465 470 475 480 Arg Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn 485 490 495 Ala Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys 500 505 510 Thr Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro 515 520 525 Met Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr 530 535 540 Leu Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu 545 550 555 560 Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu 565 570 575 Gln Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu 580 585 590 Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val 595 600 605 Thr Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe 610 615 620 Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp 625 630 635 640 Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu 645 650 655 Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr 660 665 670 Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp 675 680 685 Gln Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg 690 695 700 Ala 705 <210> 12 <211> 608 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 12 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 Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser Asp 225 230 235 240 Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val Asp 245 250 255 Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val Glu 260 265 270 Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr Gln 275 280 285 Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser Ile 290 295 300 Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys Gly 305 310 315 320 Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser Pro 325 330 335 Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu Tyr 340 345 350 Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu Glu 355 360 365 Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser Arg 370 375 380 Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn Ala 385 390 395 400 Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys Thr 405 410 415 Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro Met 420 425 430 Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr Leu 435 440 445 Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu Val 450 455 460 Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu Gln 465 470 475 480 Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu Met 485 490 495 Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val Thr 500 505 510 Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe Asp 515 520 525 Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp Leu 530 535 540 Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu Thr 545 550 555 560 Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr Leu 565 570 575 Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp Gln 580 585 590 Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg Ala 595 600 605 <210> 13 <211> 705 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 13 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 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 Asn Pro Asn Ala Thr Ser Ser Ser Ser Gln Asp Pro Glu 225 230 235 240 Ser Leu Gln Asp Arg Gly Glu Gly Lys Val Ala Thr Thr Val Ile Ser 245 250 255 Lys Met Leu Phe Val Glu Pro Ile Leu Glu Val Ser Ser Leu Pro Thr 260 265 270 Thr Asn Ser Thr Thr Asn Ser Ala Thr Lys Ile Thr Ala Asn Thr Thr 275 280 285 Asp Glu Pro Thr Thr Gln Pro Thr Thr Glu Pro Thr Thr Gln Pro Thr 290 295 300 Ile Gln Pro Thr Gln Pro Thr Thr Gln Leu Pro Thr Asp Ser Pro Thr 305 310 315 320 Gln Pro Thr Thr Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser 325 330 335 Asp Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val 340 345 350 Asp Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val 355 360 365 Glu Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr 370 375 380 Gln Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser 385 390 395 400 Ile Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys 405 410 415 Gly Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser 420 425 430 Pro Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu 435 440 445 Tyr Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu 450 455 460 Glu Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser 465 470 475 480 Arg Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn 485 490 495 Ala Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys 500 505 510 Thr Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro 515 520 525 Met Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr 530 535 540 Leu Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu 545 550 555 560 Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu 565 570 575 Gln Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu 580 585 590 Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val 595 600 605 Thr Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe 610 615 620 Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp 625 630 635 640 Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu 645 650 655 Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr 660 665 670 Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp 675 680 685 Gln Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg 690 695 700 Ala 705 <210> 14 <211> 608 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 14 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 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 Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser Asp 225 230 235 240 Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val Asp 245 250 255 Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val Glu 260 265 270 Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr Gln 275 280 285 Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser Ile 290 295 300 Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys Gly 305 310 315 320 Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser Pro 325 330 335 Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu Tyr 340 345 350 Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu Glu 355 360 365 Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser Arg 370 375 380 Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn Ala 385 390 395 400 Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys Thr 405 410 415 Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro Met 420 425 430 Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr Leu 435 440 445 Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu Val 450 455 460 Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu Gln 465 470 475 480 Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu Met 485 490 495 Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val Thr 500 505 510 Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe Asp 515 520 525 Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp Leu 530 535 540 Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu Thr 545 550 555 560 Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr Leu 565 570 575 Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp Gln 580 585 590 Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg Ala 595 600 605 <210> 15 <211> 707 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 15 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 Asn Pro Asn Ala Thr Ser Ser Ser Ser Gln Asp 225 230 235 240 Pro Glu Ser Leu Gln Asp Arg Gly Glu Gly Lys Val Ala Thr Thr Val 245 250 255 Ile Ser Lys Met Leu Phe Val Glu Pro Ile Leu Glu Val Ser Ser Leu 260 265 270 Pro Thr Thr Asn Ser Thr Thr Asn Ser Ala Thr Lys Ile Thr Ala Asn 275 280 285 Thr Thr Asp Glu Pro Thr Thr Gln Pro Thr Thr Glu Pro Thr Thr Gln 290 295 300 Pro Thr Ile Gln Pro Thr Gln Pro Thr Thr Gln Leu Pro Thr Asp Ser 305 310 315 320 Pro Thr Gln Pro Thr Thr Gly Ser Phe Cys Pro Gly Pro Val Thr Leu 325 330 335 Cys Ser Asp Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala 340 345 350 Leu Val Asp Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys 355 360 365 Lys Val Glu Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu 370 375 380 Leu Thr Gln Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu 385 390 395 400 Glu Ser Ile Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala 405 410 415 Leu Lys Gly Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe 420 425 430 His Ser Pro Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg 435 440 445 Thr Leu Tyr Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala 450 455 460 Asn Leu Glu Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys 465 470 475 480 Ile Ser Arg Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu 485 490 495 Leu Asn Ala Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro 500 505 510 Lys Lys Thr Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys 515 520 525 Val Pro Met Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp 530 535 540 Gln Thr Leu Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu 545 550 555 560 Ser Leu Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp 565 570 575 Met Glu Gln Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys 580 585 590 Leu Glu Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile 595 600 605 Lys Val Thr Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu 610 615 620 Phe Phe Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp 625 630 635 640 Pro Asp Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu 645 650 655 Thr Glu Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala 660 665 670 Arg Thr Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu 675 680 685 Trp Asp Gln Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp 690 695 700 Pro Arg Ala 705 <210> 16 <211> 610 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 16 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 Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys 225 230 235 240 Ser Asp Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu 245 250 255 Val Asp Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys 260 265 270 Val Glu Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu 275 280 285 Thr Gln Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu 290 295 300 Ser Ile Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu 305 310 315 320 Lys Gly Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His 325 330 335 Ser Pro Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr 340 345 350 Leu Tyr Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn 355 360 365 Leu Glu Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile 370 375 380 Ser Arg Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu 385 390 395 400 Asn Ala Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys 405 410 415 Lys Thr Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val 420 425 430 Pro Met Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln 435 440 445 Thr Leu Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser 450 455 460 Leu Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met 465 470 475 480 Glu Gln Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu 485 490 495 Glu Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys 500 505 510 Val Thr Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe 515 520 525 Phe Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro 530 535 540 Asp Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr 545 550 555 560 Glu Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg 565 570 575 Thr Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp 580 585 590 Asp Gln Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro 595 600 605 Arg Ala 610 <210> 17 <211> 609 <212> PRT <213> Homo sapiens <400> 17 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 On Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Lys Glu Cys Cys Glu 290,295,300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Thr Thr Thr Thr Thr Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 405 410 415 Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 595 600 605 Leu <210> 18 <211> 1087 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 18 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 On Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Leu Lys Glu Cys Cys Glu 290,295,300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 To Let Arg Lead to Lys Thr Tyr Lys Thr Thr Thr Thr Thr Thr to Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 405 410 415 Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 595 600 605 Leu Asn Pro Asn Ala Thr Ser Ser Ser Ser Gln Asp Pro Glu Ser Leu 610 615 620 Gln Asp Arg Gly Glu Gly Lys Val Ala Thr Thr Val Ile Ser Lys Met 625 630 635 640 Leu Phe Val Glu Pro Ile Leu Glu Val Ser Ser Leu Pro Thr Thr Asn 645 650 655 Ser Thr Thr Asn Ser Ala Thr Lys Ile Thr Ala Asn Thr Thr Asp Glu 660 665 670 Pro Thr Thr Gln Pro Thr Thr Glu Pro Thr Thr Gln Pro Thr Ile Gln 675 680 685 Pro Thr Gln Pro Thr Thr Gln Leu Pro Thr Asp Ser Pro Thr Gln Pro 690 695 700 Thr Thr Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser Asp Leu 705 710 715 720 Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val Asp Phe 725 730 735 Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val Glu Thr 740 745 750 Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr Gln Val 755 760 765 Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser Ile Leu 770 775 780 Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys Gly Phe 785 790 795 800 Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser Pro Asp 805 810 815 Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu Tyr Ser 820 825 830 Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu Glu Leu 835 840 845 Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser Arg Leu 850 855 860 Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn Ala Ile 865 870 875 880 Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys Thr Arg 885 890 895 Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro Met Met 900 905 910 Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr Leu Lys 915 920 925 Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu Val Ile 930 935 940 Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu Gln Ala 945 950 955 960 Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu Met Ser 965 970 975 Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val Thr Thr 980 985 990 Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe Asp Phe 995 1000 1005 Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp Leu 1010 1015 1020 Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu 1025 1030 1035 Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg 1040 1045 1050 Thr Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu 1055 1060 1065 Trp Asp Gln Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr 1070 1075 1080 Asp Pro Arg Ala 1085 <210> 19 <211> 990 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: Synthetic polypeptide <400> 19 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 On Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Leu Lys Glu Cys Cys Glu 290,295,300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Thr Thr Thr Thr Thr Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 405 410 415 Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 595 600 605 Leu Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser Asp Leu Glu 610 615 620 Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val Asp Phe Ser 625 630 635 640 Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val Glu Thr Asn 645 650 655 Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr Gln Val Leu 660 665 670 Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser Ile Leu Ser 675 680 685 Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys Gly Phe Thr 690 695 700 Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser Pro Asp Leu 705 710 715 720 Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu Tyr Ser Ser 725 730 735 Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu Glu Leu Ile 740 745 750 Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser Arg Leu Leu 755 760 765 Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn Ala Ile Tyr 770 775 780 Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys Thr Arg Met 785 790 795 800 Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro Met Met Asn 805 810 815 Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr Leu Lys Ala 820 825 830 Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu Val Ile Leu 835 840 845 Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu Gln Ala Leu 850 855 860 Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu Met Ser Lys 865 870 875 880 Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val Thr Thr Ser 885 890 895 Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe Asp Phe Ser 900 905 910 Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp Leu Gln Val 915 920 925 Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu Thr Gly Val 930 935 940 Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr Leu Leu Val 945 950 955 960 Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp Gln Gln His 965 970 975 Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg Ala 980 985 990 <210> 20 <211> 225 <212> PRT <213> Homo sapiens <400> 20 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 20 25 30 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 35 40 45 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 50 55 60 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 65 70 75 80 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 85 90 95 Your Asn Gln Your Cys Will Your Glu Lys Thr Pro Will Be Asp Arg 100 105 110 Val Asn Arg Arg Pro Cys Phe 115 120 125 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 130 135 140 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 145 150 155 160 Arg Gln Ile Lys Gln Thr Ala Leu Val Glu Leu Val Lys Lys Lys 165 170 175 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 180 185 190 Ala Phe Val Glu Lys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 195 200 205 Glu Glu Gly Lys Lys Leu Val Val Ser Arg Ala Leu Gly 210 215 220 Leu 225 <210> 21 <211> 703 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> twenty one Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 20 25 30 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 35 40 45 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 50 55 60 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 65 70 75 80 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 85 90 95 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 100 105 110 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 115 120 125 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 130 135 140 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 145 150 155 160 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 165 170 175 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 180 185 190 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 195 200 205 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 210 215 220 Leu Asn Pro Asn Ala Thr Ser Ser Ser Ser Gln Asp Pro Glu Ser Leu 225 230 235 240 Gln Asp Arg Gly Glu Gly Lys Val Ala Thr Thr Val Ile Ser Lys Met 245 250 255 Leu Phe Val Glu Pro Ile Leu Glu Val Ser Ser Leu Pro Thr Thr Asn 260 265 270 Ser Thr Thr Asn Ser Ala Thr Lys Ile Thr Ala Asn Thr Thr Asp Glu 275 280 285 Pro Thr Thr Gln Pro Thr Thr Glu Pro Thr Thr Gln Pro Thr Ile Gln 290 295 300 Pro Thr Gln Pro Thr Thr Gln Leu Pro Thr Asp Ser Pro Thr Gln Pro 305 310 315 320 Thr Thr Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser Asp Leu 325 330 335 Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val Asp Phe 340 345 350 Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val Glu Thr 355 360 365 Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr Gln Val 370 375 380 Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser Ile Leu 385 390 395 400 Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys Gly Phe 405 410 415 Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser Pro Asp 420 425 430 Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu Tyr Ser 435 440 445 Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu Glu Leu 450 455 460 Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser Arg Leu 465 470 475 480 Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn Ala Ile 485 490 495 Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys Thr Arg 500 505 510 Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro Met Met 515 520 525 Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr Leu Lys 530 535 540 Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu Val Ile 545 550 555 560 Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu Gln Ala 565 570 575 Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu Met Ser 580 585 590 Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val Thr Thr 595 600 605 Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe Asp Phe 610 615 620 Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp Leu Gln 625 630 635 640 Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu Thr Gly 645 650 655 Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr Leu Leu 660 665 670 Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp Gln Gln 675 680 685 His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg Ala 690 695 700 <210> 22 <211> 606 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 22 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 20 25 30 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 35 40 45 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 50 55 60 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 65 70 75 80 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 85 90 95 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 100 105 110 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 115 120 125 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 130 135 140 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 145 150 155 160 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 165 170 175 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 180 185 190 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 195 200 205 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 210 215 220 Leu Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser Asp Leu Glu 225 230 235 240 Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val Asp Phe Ser 245 250 255 Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val Glu Thr Asn 260 265 270 Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr Gln Val Leu 275 280 285 Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser Ile Leu Ser 290 295 300 Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys Gly Phe Thr 305 310 315 320 Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser Pro Asp Leu 325 330 335 Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu Tyr Ser Ser 340 345 350 Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu Glu Leu Ile 355 360 365 Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser Arg Leu Leu 370 375 380 Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn Ala Ile Tyr 385 390 395 400 Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys Thr Arg Met 405 410 415 Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro Met Met Asn 420 425 430 Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr Leu Lys Ala 435 440 445 Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu Val Ile Leu 450 455 460 Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu Gln Ala Leu 465 470 475 480 Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu Met Ser Lys 485 490 495 Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val Thr Thr Ser 500 505 510 Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe Asp Phe Ser 515 520 525 Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp Leu Gln Val 530 535 540 Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu Thr Gly Val 545 550 555 560 Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr Leu Leu Val 565 570 575 Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp Gln Gln His 580 585 590 Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg Ala 595 600 605 <210> 23 <211> 1090 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic sequence description: Synthetic polypeptide <400> 23 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 On Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Lys Glu Cys Cys Glu 290,295,300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Lys Thr Thr Leu Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 595 600 605 Leu Gly Gly Gly Asn Pro Asn Ala Thr Ser Ser Ser Ser Gln Asp Pro 610 615 620 Glu Ser Leu Gln Asp Arg Gly Glu Gly Lys Val Ala Thr Thr Val Ile 625 630 635 640 Ser Lys Met Leu Phe Val Glu Pro Ile Leu Glu Val Ser Ser Leu Pro 645 650 655 Thr Thr Asn Ser Thr Thr Asn Ser Ala Thr Lys Ile Thr Ala Asn Thr 660 665 670 Thr Asp Glu Pro Thr Thr Gln Pro Thr Thr Glu Pro Thr Thr Gln Pro 675 680 685 Thr Ile Gln Pro Thr Gln Pro Thr Thr Gln Leu Pro Thr Asp Ser Pro 690 695 700 Thr Gln Pro Thr Thr Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys 705 710 715 720 Ser Asp Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu 725 730 735 Val Asp Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys 740 745 750 Val Glu Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu 755 760 765 Thr Gln Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu 770 775 780 Ser Ile Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu 785 790 795 800 Lys Gly Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His 805 810 815 Ser Pro Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr 820 825 830 Leu Tyr Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn 835 840 845 Leu Glu Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile 850 855 860 Ser Arg Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu 865 870 875 880 Asn Ala Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys 885 890 895 Lys Thr Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val 900 905 910 Pro Met Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln 915 920 925 Thr Leu Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser 930 935 940 Leu Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met 945 950 955 960 Glu Gln Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu 965 970 975 Glu Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys 980 985 990 Val Thr Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe 995 1000 1005 Phe Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp 1010 1015 1020 Pro Asp Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu 1025 1030 1035 Leu Thr Glu Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser 1040 1045 1050 Val Ala Arg Thr Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu 1055 1060 1065 Phe Val Leu Trp Asp Gln Gln His Lys Phe Pro Val Phe Met Gly 1070 1075 1080 Arg Val Tyr Asp Pro Arg Ala 1085 1090 <210> twenty four <211> 993 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> twenty four Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 On Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Leu Lys Glu Cys Cys Glu 290,295,300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Thr Thr Thr Thr Thr Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 405 410 415 Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Your Asn Gln Your Cys Will Your Glu Lys Thr Pro Will Be Asp Arg 485,490,495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 595 600 605 Leu Gly Gly Gly Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser 610 615 620 Asp Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val 625 630 635 640 Asp Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val 645 650 655 Glu Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr 660 665 670 Gln Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser 675 680 685 Ile Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys 690 695 700 Gly Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser 705 710 715 720 Pro Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu 725 730 735 Tyr Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu 740 745 750 Glu Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser 755 760 765 Arg Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn 770 775 780 Ala Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys 785 790 795 800 Thr Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro 805 810 815 Met Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr 820 825 830 Leu Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu 835 840 845 Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu 850 855 860 Gln Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu 865 870 875 880 Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val 885 890 895 Thr Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe 900 905 910 Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp 915 920 925 Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu 930 935 940 Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr 945 950 955 960 Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp 965 970 975 Gln Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg 980 985 990 Ala <210> 25 <211> 706 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 25 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 20 25 30 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 35 40 45 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 50 55 60 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 65 70 75 80 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 85 90 95 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 100 105 110 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 115 120 125 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 130 135 140 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 145 150 155 160 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 165 170 175 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 180 185 190 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 195 200 205 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 210 215 220 Leu Gly Gly Gly Asn Pro Asn Ala Thr Ser Ser Ser Ser Gln Asp Pro 225 230 235 240 Glu Ser Leu Gln Asp Arg Gly Glu Gly Lys Val Ala Thr Thr Val Ile 245 250 255 Ser Lys Met Leu Phe Val Glu Pro Ile Leu Glu Val Ser Ser Leu Pro 260 265 270 Thr Thr Asn Ser Thr Thr Asn Ser Ala Thr Lys Ile Thr Ala Asn Thr 275 280 285 Thr Asp Glu Pro Thr Thr Gln Pro Thr Thr Glu Pro Thr Thr Gln Pro 290 295 300 Thr Ile Gln Pro Thr Gln Pro Thr Thr Gln Leu Pro Thr Asp Ser Pro 305 310 315 320 Thr Gln Pro Thr Thr Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys 325 330 335 Ser Asp Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu 340 345 350 Val Asp Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys 355 360 365 Val Glu Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu 370 375 380 Thr Gln Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu 385 390 395 400 Ser Ile Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu 405 410 415 Lys Gly Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His 420 425 430 Ser Pro Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr 435 440 445 Leu Tyr Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn 450 455 460 Leu Glu Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile 465 470 475 480 Ser Arg Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu 485 490 495 Asn Ala Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys 500 505 510 Lys Thr Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val 515 520 525 Pro Met Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln 530 535 540 Thr Leu Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser 545 550 555 560 Leu Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met 565 570 575 Glu Gln Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu 580 585 590 Glu Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys 595 600 605 Val Thr Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe 610 615 620 Phe Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro 625 630 635 640 Asp Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr 645 650 655 Glu Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg 660 665 670 Thr Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp 675 680 685 Asp Gln Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro 690 695 700 Arg Ala 705 <210> 26 <211> 609 <212> PRT <213> Synthetic sequence <220> <223> Synthetic sequence description: Synthetic polypeptide <400> 26 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 20 25 30 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 35 40 45 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 50 55 60 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 65 70 75 80 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 85 90 95 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 100 105 110 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 115 120 125 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 130 135 140 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 145 150 155 160 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 165 170 175 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 180 185 190 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 195 200 205 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 210 215 220 Leu Gly Gly Gly Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys Ser 225 230 235 240 Asp Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu Val 245 250 255 Asp Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys Val 260 265 270 Glu Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu Thr 275 280 285 Gln Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu Ser 290 295 300 Ile Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu Lys 305 310 315 320 Gly Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His Ser 325 330 335 Pro Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr Leu 340 345 350 Tyr Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn Leu 355 360 365 Glu Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile Ser 370 375 380 Arg Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu Asn 385 390 395 400 Ala Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys Lys 405 410 415 Thr Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val Pro 420 425 430 Met Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln Thr 435 440 445 Leu Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser Leu 450 455 460 Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met Glu 465 470 475 480 Gln Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu Glu 485 490 495 Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys Val 500 505 510 Thr Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe Phe 515 520 525 Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro Asp 530 535 540 Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr Glu 545 550 555 560 Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr 565 570 575 Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp 580 585 590 Gln Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg 595 600 605 Ala <210> 27 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 27 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 28 <211> 1097 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 28 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 On Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Lys Glu Cys Cys Glu 290,295,300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 To Let Arg Lead to Lys Thr Tyr Lys Thr Thr Thr Thr Thr Thr to Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 405 410 415 Leu Phe Glu Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 595 600 605 Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Pro Asn Ala Thr 610 615 620 Ser Ser Ser Ser Gln Asp Pro Glu Ser Leu Gln Asp Arg Gly Glu Gly 625 630 635 640 Lys Val Ala Thr Thr Val Ile Ser Lys Met Leu Phe Val Glu Pro Ile 645 650 655 Leu Glu Val Ser Ser Leu Pro Thr Thr Asn Ser Thr Thr Asn Ser Ala 660 665 670 Thr Lys Ile Thr Ala Asn Thr Thr Asp Glu Pro Thr Thr Gln Pro Thr 675 680 685 Thr Glu Pro Thr Thr Gln Pro Thr Ile Gln Pro Thr Gln Pro Thr Thr 690 695 700 Gln Leu Pro Thr Asp Ser Pro Thr Gln Pro Thr Thr Gly Ser Phe Cys 705 710 715 720 Pro Gly Pro Val Thr Leu Cys Ser Asp Leu Glu Ser His Ser Thr Glu 725 730 735 Ala Val Leu Gly Asp Ala Leu Val Asp Phe Ser Leu Lys Leu Tyr His 740 745 750 Ala Phe Ser Ala Met Lys Lys Val Glu Thr Asn Met Ala Phe Ser Pro 755 760 765 Phe Ser Ile Ala Ser Leu Leu Thr Gln Val Leu Leu Gly Ala Gly Glu 770 775 780 Asn Thr Lys Thr Asn Leu Glu Ser Ile Leu Ser Tyr Pro Lys Asp Phe 785 790 795 800 Thr Cys Val His Gln Ala Leu Lys Gly Phe Thr Thr Lys Gly Val Thr 805 810 815 Ser Val Ser Gln Ile Phe His Ser Pro Asp Leu Ala Ile Arg Asp Thr 820 825 830 Phe Val Asn Ala Ser Arg Thr Leu Tyr Ser Ser Ser Pro Arg Val Leu 835 840 845 Ser Asn Asn Ser Asp Ala Asn Leu Glu Leu Ile Asn Thr Trp Val Ala 850 855 860 Lys Asn Thr Asn Asn Lys Ile Ser Arg Leu Leu Asp Ser Leu Pro Ser 865 870 875 880 Asp Thr Arg Leu Val Leu Leu Asn Ala Ile Tyr Leu Ser Ala Lys Trp 885 890 895 Lys Thr Thr Phe Asp Pro Lys Lys Thr Arg Met Glu Pro Phe His Phe 900 905 910 Lys Asn Ser Val Ile Lys Val Pro Met Asn Ser Lys Lys Tyr Pro 915 920 925 Val Ala His Phe Ile Asp Gln Thr Leu Lys Ala Lys Val Gly Gln Leu 930 935 940 Gln Leu Ser His Asn Leu Ser Leu Val Ile Leu Val Pro Gln Asn Leu 945 950 955 960 Lys His Arg Leu Glu Asp Met Glu Gln Ala Leu Ser Pro Ser Val Phe 965 970 975 Lys Ala Ile Met Glu Lys Leu Glu Met Ser Lys Phe Gln Pro Thr Leu 980 985 990 Leu Thr Leu Pro Arg Ile Lys Val Thr Thr Ser Gln Asp Met Leu Ser 995 1000 1005 Ile Met Glu Lys Leu Glu Phe Phe Asp Phe Ser Tyr Asp Leu Asn 1010 1015 1020 Leu Cys Gly Leu Thr Glu Asp Pro Asp Leu Gln Val Ser Ala Met 1025 1030 1035 Gln His Gln Thr Val Leu Glu Leu Thr Glu Thr Gly Val Glu Ala 1040 1045 1050 Ala Ala Ala Ser Ala Ile Ser Val Ala Arg Thr Leu Leu Val Phe 1055 1060 1065 Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp Asp Gln Gln His 1070 1075 1080 Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro Arg Ala 1085 1090 1095 <210> 29 <211> 1000 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic polypeptide <400> 29 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 On Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Lys Glu Cys Cys Glu 290,295,300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 To Let Arg Lead to Lys Thr Tyr Lys Thr Thr Thr Thr Thr Thr to Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 405 410 415 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 565 570 575 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 580 585 590 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 595 600 605 Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Phe Cys Pro 610 615 620 Gly Pro Val Thr Leu Cys Ser Asp Leu Glu Ser His Ser Thr Glu Ala 625 630 635 640 Val Leu Gly Asp Ala Leu Val Asp Phe Ser Leu Lys Leu Tyr His Ala 645 650 655 Phe Ser Ala Met Lys Lys Val Glu Thr Asn Met Ala Phe Ser Pro Phe 660 665 670 Ser Ile Ala Ser Leu Leu Thr Gln Val Leu Leu Gly Ala Gly Glu Asn 675 680 685 Thr Lys Thr Asn Leu Glu Ser Ile Leu Ser Tyr Pro Lys Asp Phe Thr 690 695 700 Cys Val His Gln Ala Leu Lys Gly Phe Thr Thr Lys Gly Val Thr Ser 705 710 715 720 Val Ser Gln Ile Phe His Ser Pro Asp Leu Ala Ile Arg Asp Thr Phe 725 730 735 Val Asn Ala Ser Arg Thr Leu Tyr Ser Ser Ser Pro Arg Val Leu Ser 740 745 750 Asn Asn Ser Asp Ala Asn Leu Glu Leu Ile Asn Thr Trp Val Ala Lys 755 760 765 Asn Thr Asn Asn Lys Ile Ser Arg Leu Leu Asp Ser Leu Pro Ser Asp 770 775 780 Thr Arg Leu Val Leu Leu Asn Ala Ile Tyr Leu Ser Ala Lys Trp Lys 785 790 795 800 Thr Thr Phe Asp Pro Lys Lys Thr Arg Met Glu Pro Phe His Phe Lys 805 810 815 Asn Ser Val Ile Lys Val Pro Met Met Asn Ser Lys Lys Tyr Pro Val 820 825 830 Ala His Phe Ile Asp Gln Thr Leu Lys Ala Lys Val Gly Gln Leu Gln 835 840 845 Leu Ser His Asn Leu Ser Leu Val Ile Leu Val Pro Gln Asn Leu Lys 850 855 860 His Arg Leu Glu Asp Met Glu Gln Ala Leu Ser Pro Ser Val Phe Lys 865 870 875 880 Ala Ile Met Glu Lys Leu Glu Met Ser Lys Phe Gln Pro Thr Leu Leu 885 890 895 Thr Leu Pro Arg Ile Lys Val Thr Thr Ser Gln Asp Met Leu Ser Ile 900 905 910 Met Glu Lys Leu Glu Phe Phe Asp Phe Ser Tyr Asp Leu Asn Leu Cys 915 920 925 Gly Leu Thr Glu Asp Pro Asp Leu Gln Val Ser Ala Met Gln His Gln 930 935 940 Thr Val Leu Glu Leu Thr Glu Thr Gly Val Glu Ala Ala Ala Ala Ser 945 950 955 960 Ala Ile Ser Val Ala Arg Thr Leu Leu Val Phe Glu Val Gln Gln Pro 965 970 975 Phe Leu Phe Val Leu Trp Asp Gln Gln His Lys Phe Pro Val Phe Met 980 985 990 Gly Arg Val Tyr Asp Pro Arg Ala 995 1000 <210> 30 <211> 713 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic polypeptide <400> 30 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 20 25 30 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 35 40 45 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 50 55 60 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 65 70 75 80 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 85 90 95 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 100 105 110 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 115 120 125 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 130 135 140 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 145 150 155 160 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 165 170 175 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 180 185 190 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 195 200 205 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Arg Ala Ala Leu Gly 210 215 220 Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Pro Asn Ala Thr 225 230 235 240 Ser Ser Ser Ser Gln Asp Pro Glu Ser Leu Gln Asp Arg Gly Glu Gly 245 250 255 Lys Val Ala Thr Thr Val Ile Ser Lys Met Leu Phe Val Glu Pro Ile 260 265 270 Leu Glu Val Ser Ser Leu Pro Thr Thr Asn Ser Thr Thr Asn Ser Ala 275 280 285 Thr Lys Ile Thr Ala Asn Thr Thr Asp Glu Pro Thr Thr Gln Pro Thr 290 295 300 Thr Glu Pro Thr Thr Gln Pro Thr Ile Gln Pro Thr Gln Pro Thr Thr 305 310 315 320 Gln Leu Pro Thr Asp Ser Pro Thr Gln Pro Thr Thr Gly Ser Phe Cys 325 330 335 Pro Gly Pro Val Thr Leu Cys Ser Asp Leu Glu Ser His Ser Thr Glu 340 345 350 Ala Val Leu Gly Asp Ala Leu Val Asp Phe Ser Leu Lys Leu Tyr His 355 360 365 Ala Phe Ser Ala Met Lys Lys Val Glu Thr Asn Met Ala Phe Ser Pro 370 375 380 Phe Ser Ile Ala Ser Leu Leu Thr Gln Val Leu Leu Gly Ala Gly Glu 385 390 395 400 Asn Thr Lys Thr Asn Leu Glu Ser Ile Leu Ser Tyr Pro Lys Asp Phe 405 410 415 Thr Cys Val His Gln Ala Leu Lys Gly Phe Thr Thr Lys Gly Val Thr 420 425 430 Ser Val Ser Gln Ile Phe His Ser Pro Asp Leu Ala Ile Arg Asp Thr 435 440 445 Phe Val Asn Ala Ser Arg Thr Leu Tyr Ser Ser Ser Pro Arg Val Leu 450 455 460 Ser Asn Asn Ser Asp Ala Asn Leu Glu Leu Ile Asn Thr Trp Val Ala 465 470 475 480 Lys Asn Thr Asn Asn Lys Ile Ser Arg Leu Leu Asp Ser Leu Pro Ser 485 490 495 Asp Thr Arg Leu Val Leu Leu Asn Ala Ile Tyr Leu Ser Ala Lys Trp 500 505 510 Lys Thr Thr Phe Asp Pro Lys Lys Thr Arg Met Glu Pro Phe His Phe 515 520 525 Lys Asn Ser Val Ile Lys Val Pro Met Asn Ser Lys Lys Tyr Pro 530 535 540 Val Ala His Phe Ile Asp Gln Thr Leu Lys Ala Lys Val Gly Gln Leu 545 550 555 560 Gln Leu Ser His Asn Leu Ser Leu Val Ile Leu Val Pro Gln Asn Leu 565 570 575 Lys His Arg Leu Glu Asp Met Glu Gln Ala Leu Ser Pro Ser Val Phe 580 585 590 Lys Ala Ile Met Glu Lys Leu Glu Met Ser Lys Phe Gln Pro Thr Leu 595 600 605 Leu Thr Leu Pro Arg Ile Lys Val Thr Thr Ser Gln Asp Met Leu Ser 610 615 620 Ile Met Glu Lys Leu Glu Phe Phe Asp Phe Ser Tyr Asp Leu Asn Leu 625 630 635 640 Cys Gly Leu Thr Glu Asp Pro Asp Leu Gln Val Ser Ala Met Gln His 645 650 655 Gln Thr Val Leu Glu Leu Thr Glu Thr Gly Val Glu Ala Ala Ala Ala 660 665 670 Ser Ala Ile Ser Val Ala Arg Thr Leu Leu Val Phe Glu Val Gln Gln 675 680 685 Pro Phe Leu Phe Val Leu Trp Asp Gln Gln His Lys Phe Pro Val Phe 690 695 700 Met Gly Arg Val Tyr Asp Pro Arg Ala 705 710 <210> 31 <211> 616 <212> PRT <213> Synthetic sequence <220> <223> Synthetic sequence description: Synthetic polypeptide <400> 31 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 20 25 30 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 35 40 45 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 50 55 60 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Lys His 65 70 75 80 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 85 90 95 Your Asn Gln Your Cys Will Your Glu Lys Thr Pro Will Be Asp Arg 100 105 110 Val Thr Lys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 115 120 125 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 130 135 140 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 145 150 155 160 Arg Gln Ile Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 165 170 175 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 180 185 190 Ala Phe Val Glu Lys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 195 200 205 Glu Glu Gly Lys Lys Leu Val Val Ser Arg Ala Leu Gly 210 215 220 Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser Phe Cys Pro 225 230 235 240 Gly Pro Val Thr Leu Cys Ser Asp Leu Glu Ser His Ser Thr Glu Ala 245 250 255 Val Leu Gly Asp Ala Leu Val Asp Phe Ser Leu Lys Leu Tyr His Ala 260 265 270 Phe Ser Ala Met Lys Lys Val Glu Thr Asn Met Ala Phe Ser Pro Phe 275 280 285 Ser Ile Ala Ser Leu Leu Thr Gln Val Leu Leu Gly Ala Gly Glu Asn 290 295 300 Thr Lys Thr Asn Leu Glu Ser Ile Leu Ser Tyr Pro Lys Asp Phe Thr 305 310 315 320 Cys Val His Gln Ala Leu Lys Gly Phe Thr Thr Lys Gly Val Thr Ser 325 330 335 Val Ser Gln Ile Phe His Ser Pro Asp Leu Ala Ile Arg Asp Thr Phe 340 345 350 Val Asn Ala Ser Arg Thr Leu Tyr Ser Ser Ser Pro Arg Val Leu Ser 355 360 365 Asn Asn Ser Asp Ala Asn Leu Glu Leu Ile Asn Thr Trp Val Ala Lys 370 375 380 Asn Thr Asn Asn Lys Ile Ser Arg Leu Leu Asp Ser Leu Pro Ser Asp 385 390 395 400 Thr Arg Leu Val Leu Leu Asn Ala Ile Tyr Leu Ser Ala Lys Trp Lys 405 410 415 Thr Thr Phe Asp Pro Lys Lys Thr Arg Met Glu Pro Phe His Phe Lys 420 425 430 Asn Ser Val Ile Lys Val Pro Met Met Asn Ser Lys Lys Tyr Pro Val 435 440 445 Ala His Phe Ile Asp Gln Thr Leu Lys Ala Lys Val Gly Gln Leu Gln 450 455 460 Leu Ser His Asn Leu Ser Leu Val Ile Leu Val Pro Gln Asn Leu Lys 465 470 475 480 His Arg Leu Glu Asp Met Glu Gln Ala Leu Ser Pro Ser Val Phe Lys 485 490 495 Ala Ile Met Glu Lys Leu Glu Met Ser Lys Phe Gln Pro Thr Leu Leu 500 505 510 Thr Leu Pro Arg Ile Lys Val Thr Thr Ser Gln Asp Met Leu Ser Ile 515 520 525 Met Glu Lys Leu Glu Phe Phe Asp Phe Ser Tyr Asp Leu Asn Leu Cys 530 535 540 Gly Leu Thr Glu Asp Pro Asp Leu Gln Val Ser Ala Met Gln His Gln 545 550 555 560 Thr Val Leu Glu Leu Thr Glu Thr Gly Val Glu Ala Ala Ala Ala Ser 565 570 575 Ala Ile Ser Val Ala Arg Thr Leu Leu Val Phe Glu Val Gln Gln Pro 580 585 590 Phe Leu Phe Val Leu Trp Asp Gln Gln His Lys Phe Pro Val Phe Met 595 600 605 Gly Arg Val Tyr Asp Pro Arg Ala 610 615 <210> 32 <211> 707 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 32 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 Asn Pro Asn Ala Thr Ser Ser Ser Ser Gln Asp 225 230 235 240 Pro Glu Ser Leu Gln Asp Arg Gly Glu Gly Lys Val Ala Thr Thr Val 245 250 255 Ile Ser Lys Met Leu Phe Val Glu Pro Ile Leu Glu Val Ser Ser Leu 260 265 270 Pro Thr Thr Asn Ser Thr Thr Asn Ser Ala Thr Lys Ile Thr Ala Asn 275 280 285 Thr Thr Asp Glu Pro Thr Thr Gln Pro Thr Thr Glu Pro Thr Thr Gln 290 295 300 Pro Thr Ile Gln Pro Thr Gln Pro Thr Thr Gln Leu Pro Thr Asp Ser 305 310 315 320 Pro Thr Gln Pro Thr Thr Gly Ser Phe Cys Pro Gly Pro Val Thr Leu 325 330 335 Cys Ser Asp Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala 340 345 350 Leu Val Asp Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys 355 360 365 Lys Val Glu Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu 370 375 380 Leu Thr Gln Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu 385 390 395 400 Glu Ser Ile Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala 405 410 415 Leu Lys Gly Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe 420 425 430 His Ser Pro Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg 435 440 445 Thr Leu Tyr Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala 450 455 460 Asn Leu Glu Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys 465 470 475 480 Ile Ser Arg Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu 485 490 495 Leu Asn Ala Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro 500 505 510 Lys Lys Thr Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys 515 520 525 Val Pro Met Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp 530 535 540 Gln Thr Leu Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu 545 550 555 560 Ser Leu Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp 565 570 575 Met Glu Gln Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys 580 585 590 Leu Glu Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile 595 600 605 Lys Val Thr Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu 610 615 620 Phe Phe Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp 625 630 635 640 Pro Asp Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu 645 650 655 Thr Glu Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala 660 665 670 Arg Thr Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu 675 680 685 Trp Asp Gln Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp 690 695 700 Pro Arg Ala 705 <210> 33 <211> 610 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 33 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 Gly Ser Phe Cys Pro Gly Pro Val Thr Leu Cys 225 230 235 240 Ser Asp Leu Glu Ser His Ser Thr Glu Ala Val Leu Gly Asp Ala Leu 245 250 255 Val Asp Phe Ser Leu Lys Leu Tyr His Ala Phe Ser Ala Met Lys Lys 260 265 270 Val Glu Thr Asn Met Ala Phe Ser Pro Phe Ser Ile Ala Ser Leu Leu 275 280 285 Thr Gln Val Leu Leu Gly Ala Gly Glu Asn Thr Lys Thr Asn Leu Glu 290 295 300 Ser Ile Leu Ser Tyr Pro Lys Asp Phe Thr Cys Val His Gln Ala Leu 305 310 315 320 Lys Gly Phe Thr Thr Lys Gly Val Thr Ser Val Ser Gln Ile Phe His 325 330 335 Ser Pro Asp Leu Ala Ile Arg Asp Thr Phe Val Asn Ala Ser Arg Thr 340 345 350 Leu Tyr Ser Ser Ser Pro Arg Val Leu Ser Asn Asn Ser Asp Ala Asn 355 360 365 Leu Glu Leu Ile Asn Thr Trp Val Ala Lys Asn Thr Asn Asn Lys Ile 370 375 380 Ser Arg Leu Leu Asp Ser Leu Pro Ser Asp Thr Arg Leu Val Leu Leu 385 390 395 400 Asn Ala Ile Tyr Leu Ser Ala Lys Trp Lys Thr Thr Phe Asp Pro Lys 405 410 415 Lys Thr Arg Met Glu Pro Phe His Phe Lys Asn Ser Val Ile Lys Val 420 425 430 Pro Met Met Asn Ser Lys Lys Tyr Pro Val Ala His Phe Ile Asp Gln 435 440 445 Thr Leu Lys Ala Lys Val Gly Gln Leu Gln Leu Ser His Asn Leu Ser 450 455 460 Leu Val Ile Leu Val Pro Gln Asn Leu Lys His Arg Leu Glu Asp Met 465 470 475 480 Glu Gln Ala Leu Ser Pro Ser Val Phe Lys Ala Ile Met Glu Lys Leu 485 490 495 Glu Met Ser Lys Phe Gln Pro Thr Leu Leu Thr Leu Pro Arg Ile Lys 500 505 510 Val Thr Thr Ser Gln Asp Met Leu Ser Ile Met Glu Lys Leu Glu Phe 515 520 525 Phe Asp Phe Ser Tyr Asp Leu Asn Leu Cys Gly Leu Thr Glu Asp Pro 530 535 540 Asp Leu Gln Val Ser Ala Met Gln His Gln Thr Val Leu Glu Leu Thr 545 550 555 560 Glu Thr Gly Val Glu Ala Ala Ala Ala Ser Ala Ile Ser Val Ala Arg 565 570 575 Thr Leu Leu Val Phe Glu Val Gln Gln Pro Phe Leu Phe Val Leu Trp 580 585 590 Asp Gln Gln His Lys Phe Pro Val Phe Met Gly Arg Val Tyr Asp Pro 595 600 605 Arg Ala 610 <210> 34 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 34 Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly Gly 1 5 10 15 Gly Gly Gly Ala Pro 20 <210> 35 <211> 39 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 35 Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly Gly 1 5 10 15 Gly Gly Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly 20 25 30 Gly Gly Gly Gly Gly Ala Pro 35 <210> 36 <211> 57 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 36 Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly Gly 1 5 10 15 Gly Gly Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly 20 25 30 Gly Gly Gly Gly Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala 35 40 45 Ala Gly Gly Gly Gly Gly Gly Ala Pro 50 55 <210> 37 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 37 Ala Leu Glu Val Leu Phe Gln Gly Pro 1 5 <210> 38 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Artificial sequence description: synthetic peptides <400> 38 Gly Gly Gly Gly Ser 1 5 BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The drawings are for illustration purposes only and are not intended to be limiting.

[0063] Figure 1 A is a diagram of C1-INH. From right to left, the three domains are a signal peptide, the N-terminal, also known as the N-terminal domain, and a serine protease inhibitor domain. The N-connected glycans are shown as long vertical lines with diamond-shaped heads, and the O-connected glycans are shown as short vertical lines. Figure 1 B is a diagrammatic representation of an exemplary dimeric C1-INH Fc fusion protein. The C1-INH polypeptide portion is indicated by a rectangle, and the Fc portion is indicated by an oval segment. Figure 1 C is a schematic representation of an exemplary monomeric C1-INH Fc fusion protein.

[0064] Figure 2 A-2D are schematic representations of exemplary C1-INH fusion proteins. Figure 2 A is a representation of an IgG1 Fc with the LALA mutation fused to the full-length C1-INH polypeptide (shown as a shaded rectangle). Figure 2 B is a representation of an IgG1 Fc with the LALA mutation fused to a truncated C1-INH polypeptide (shown as a shaded rectangle). Figure 2 C is a representation of an IgG4 Fc with the S241P mutation fused to the full-length C1-INH polypeptide (shown as a shaded rectangle). Figure 2 D is a representation of an IgG4 Fc with the S241P mutation fused to a truncated C1-INH polypeptide (shown as a shaded rectangle).

[0065] Figure 3 Results of an exemplary Protein A purification of a full length C1-INH human Fc (hFc) IgG1 LALA fusion protein are shown. The graph shows the results of UV 280nm, UV 260nm, concentration and pH over time during capture and elution of the fusion protein during the purification process.

[0066] Figure 4 is a graph of the concentration and total protein amount collected during the purification of the following four exemplary Fc fusion constructs: full length (FL) C1-INH hFc IgG1 LALA fusion protein; truncated (Tr) C1-INH hFc IgG1 LALA fusion protein; full length (FL) C1-INH hFc IgG4m(IgG4 S241P) fusion protein; truncated (Tr) C1-INH hFc IgG4m(IgG4S241P) fusion protein.

[0067] Figure 5is a graph depicting the amount of endotoxin detected in purified samples of full-length (FL) C1-INH hFc IgG1 LALA fusion protein; truncated (Tr) C1-INH hFc IgG1 LALA fusion protein; full-length (FL) C1-INH hFc IgG4m(IgG4 S241P) fusion protein; truncated (Tr) C1-INH hFc IgG4m(IgG4S241P) fusion protein.

[0068] Figure 6 Shown are the results of an exemplary C1q binding ELISA for hFc IgG1-C1-INH, hFc LALA IgG1-C1-INH, and hFc IgG4m-C1-INH fusions, recombinant human C1-INH (rhC1-INH) (expressed in 1080 cells), as well as IgG1 Fc-human follistatin (hFc IgG1-hFst-XTEN) fusion as a positive control and human follistatin-Xten (hFst-XTEN) fusion as a negative control.

[0069] Figure 7 is a schematic diagram of the surface plasmon resonance (SPR) Biacore capture method used to measure binding of exemplary fusion constructs to the extracellular domain of FcγR1.

[0070] Figure 8 The results of SPR analysis of the following exemplary effector dead Fc fusion constructs are shown: full length (FL) C1-INH hFc IgG1 LALA fusion protein; truncated (Tr) C1-INHhFc IgG1 LALA fusion protein; full length (FL) C1-INH hFc IgG4m (IgG4 S241P) fusion protein; truncated (Tr) C1-INH hFc IgG4m (IgG4 S241P) fusion protein. Plasma-derived C1-INH was included as a positive control.

[0071] Fig. 9 A and 9B present the results of assays used to measure the ability of effector-neutral constructs to inhibit C1s cleavage of a colorimetric peptide. Fig. 9 A shows the titration curve for each construct obtained using the mass spectrometry calculated molecular weight for each exemplary effector-neutralizing construct tested. Fig. 9 B shows the titration curve for each exemplary effector-disabled construct tested using the gel-estimated molecular weight obtained for each construct.

[0072] Fig.10A depicts a schematic overview of a hemolytic assay to measure activation of the alternative complement pathway (APC). Fig.10 B depicts a schematic overview of the hemolytic assay used to measure activation of the classical complement pathway.

[0073] Fig.11 A, 11B, and 11C show the results of a hemolytic assay to measure activation of the alternative complement pathway. Fig.11 A shows the results comparing truncated (Tr) C1-INH hFc IgG1 LALA fusion protein, full length (FL) C1-INH hFc IgG1 LALA fusion protein, plasma-derived C1-INH preparations, and CalBiochem commercially available plasma-derived C1-INH. The absorbance readings were plotted as a percentage of the control according to the concentration of the test protein. Fig.11 B shows a graph of the raw data collected for each sample. Fig.11 C shows a graph of controls run in the assay.

[0074] Fig.12 Exemplary results of a rabbit PK study of two formulations of effector-neutral fusion constructs compared to plasma-derived C1-inhibitor and HT1080 expressed recombinant C1-INH are shown. Intravenous administration of plasma-derived C1-INH exhibited a monophasic serum concentration-time profile.

[0075] Fig.13 A-13F is a schematic representation of an exemplary albumin fusion construct generated. Fig.13 A is a schematic representation of human serum albumin (HSA) fused to C1-INH. Fig.13 B is a diagram showing HSA conjugated to C1-INH via a GGG linker. Fig.13 C is a diagram showing HSA conjugated to C1-INH via a (GGGGS)2 linker. Fig.13 D is a schematic representation of the D3 domain of HSA fused to C1-INH. Fig.13 E is a diagram showing the D3 domain of HSA joined to C1-INH via a GGG linker. Fig.13 F is a diagram showing the D3 domain of HSA joined to C1-INH via a (GGGGS)2 linker.

[0076] Fig.14 Results of assays to measure the ability of some exemplary albumin C1-INH fusion constructs to inhibit C1s cleavage of a colorimetric peptide are presented, including plasma-derived C1-INH and HT1080-expressed recombinant C1-INH for comparison.

[0077] definition

[0078] To make the present invention easier to understand, some terms are first defined below. The following terms and additional definitions of other terms are set forth throughout the specification.

[0079] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a person at any stage of development. In some embodiments, "animal" refers to a non-human animal at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, a cow, a primate, and / or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, animals may be transgenic animals, genetically engineered animals, and / or clones.

[0080] Approximately or approximately: as used in the present application, the terms "approximately" and "approximately" are used as equivalents. Any number used in the present application with or without about / approximately is intended to cover any normal fluctuations understood by those of ordinary skill in the relevant art. As used herein, the term "approximately" or "approximately" as applied to one or more target values ​​refers to a numerical value similar to the reference value. In certain embodiments, unless otherwise stated or otherwise apparent according to context, the term "approximately" or "approximately" refers to a certain range of numerical values ​​falling on the reference value in either direction (greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less (exceptions are when such numerical values ​​will exceed 100% of possible values).

[0081] Bioavailability: As used herein, the term "bioavailability" generally refers to the percentage of an administered dose that reaches the bloodstream of a subject.

[0082] Biological activity: As used herein, the phrase "biological activity" refers to the characteristic of any agent that is active in a biological system, and in particular in an organism. For example, an agent that has a biological effect on an organism when administered to the organism is considered to be biologically active. In specific embodiments, when a peptide has biological activity, the portion of the peptide that shares at least one biological activity of the peptide is generally referred to as a "biologically active" portion.

[0083] Carrier or diluent: As used herein, the terms "carrier" and "diluent" refer to a pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) carrier or diluent substance suitable for preparing a pharmaceutical formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

[0084] C1-inhibitor or C1 esterase inhibitor or C1-INH: As used herein, the terms "C1-inhibitor" or "C1 esterase inhibitor" or "C1-INH" are all used interchangeably and, unless otherwise specified, refer to any wild-type or modified C1-INH polypeptide (e.g., C1-INH protein and / or fusion protein having amino acid mutations, deletions, insertions) that retains substantial C1-INH biological activity. C1-INH can be recombinantly expressed in cells. In certain embodiments, C1-INH is expressed in mammalian cells, preferably CHO cells or human cells.

[0085] Functional equivalents or derivatives: As used herein, in the context of functional derivatives of an amino acid sequence, the term "functional equivalent" or "functional derivative" means a molecule that retains a biological activity (function or structure) that is substantially similar to the biological activity of the original sequence. Functional derivatives or equivalents may be natural derivatives, or prepared synthetically. Exemplary functional derivatives include amino acid sequences with substitutions, deletions or additions of one or more amino acids, provided that the biological activity of the protein is maintained. Substituting amino acids desirably have chemical and physical properties similar to those of the substituted amino acids. Required similar chemical and physical properties include similarities in charge, size, hydrophobicity, hydrophilicity, etc.

[0086] Fusion protein: As used herein, the term "fusion protein" or "chimeric protein" refers to a protein produced by joining two or more initially separate proteins or portions thereof. In some embodiments, a linker or spacer will be present between the proteins.

[0087] Half-life: As used herein, the term "half-life" is the time required for a quantity such as protein concentration or activity to fall to half of its value as measured at the beginning of a certain period.

[0088] Hereditary angioedema or HAE: As used herein, the term "hereditary angioedema" or "HAE" refers to a blood disorder characterized by unpredictable and recurring episodes of inflammation. HAE is often associated with C1-INH deficiency, which may be the result of low levels of C1-INH or impaired or reduced activity of C1-INH. Symptoms include, but are not limited to, swelling that may occur in any part of the body such as the face, limbs, genitals, gastrointestinal tract, and upper airway.

[0089] Improvement, increase or decrease: As used herein, the terms "improvement," "increase," or "decrease," or grammatical equivalents indicate that a numerical value is relative to a baseline measurement, such as a measurement in the same individual prior to initiation of a treatment described herein, or a measurement in a control subject (or multiple control subjects) in the absence of a treatment described herein. A "control subject" is a subject afflicted with the same form of disease as the subject being treated and who is about the same age as the subject being treated.

[0090] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as a test tube or reaction vessel, cell culture, etc., rather than in a multicellular organism.

[0091] In vivo: As used herein, the term "in vivo" refers to events occurring within a multicellular organism such as a human and non-human animal. In the context of a cell-based system, the term can be used to refer to events occurring within living cells (as opposed to, for example, an in vitro system).

[0092] Linker: As used herein, the term "linker" refers to an amino acid sequence in a fusion protein other than the amino acid sequence that appears at a specific position in the native protein, and is generally designed to have flexibility or to insert a structure such as an α-helix between two protein parts. Linkers are also called spacers. Linkers or spacers generally do not have biological functions themselves.

[0093] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked together by peptide bonds. The term is used to refer to a chain of amino acids of any length, but one of ordinary skill in the art will appreciate that the term is not limited to long chains and may refer to a minimal chain comprising two amino acids linked together by a peptide bond. As is known to those skilled in the art, polypeptides may be processed and / or modified. As used herein, the terms "polypeptide" and "peptide" are used interchangeably.

[0094] Prevention: As used herein, the terms "prevent" and "prevention" when used in connection with the occurrence of a disease, disorder, and / or condition refer to reducing the risk of developing the disease, disorder, and / or condition. See definition of "risk".

[0095] Protein: As used herein, the term "protein" refers to one or more polypeptides that act as discrete units. If a single polypeptide is a discrete functional unit and does not require permanent or temporary physical association with other polypeptides to form a discrete functional unit, then the terms "polypeptide" and "protein" are used interchangeably. If the discrete functional unit comprises more than one polypeptide physically associated with each other, then the term "protein" refers to multiple polypeptides that are physically coupled and together act as a discrete unit.

[0096] Risk: As will be understood from the context, the "risk" of a disease, illness and / or condition includes the possibility that a particular individual will develop a disease, illness and / or condition (e.g., muscular dystrophy). In some embodiments, risk is expressed as a percentage. In some embodiments, risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 until 100%. In some embodiments, risk is expressed as a risk relative to a certain reference sample or a group of reference samples. In some embodiments, a certain reference sample or a group of reference samples has a known risk of a disease, illness, condition and / or event (e.g., muscular dystrophy). In some embodiments, a certain reference sample or a group of reference samples is from an individual similar to a specific individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater.

[0097] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, which refers to a person presented to a medical provider for diagnosis or treatment of a disease. The term "subject" may be used interchangeably herein with "individual" or "patient". A subject may be afflicted with or susceptible to a disease or condition, but may or may not show symptoms of the disease or condition.

[0098] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting a characteristic or property of interest in total or near total extent or degree. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, reach perfection and / or proceed to completeness or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to reflect the potential lack of perfection inherent in many biological and chemical phenomena.

[0099] Substantially homologous: The phrase "substantially homologous" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues in corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues that will have appropriately similar structural and / or functional characteristics. For example, as is well known to one of ordinary skill in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and / or have "polar" or "non-polar" side chains. Substituting one amino acid for another amino acid of the same type can often be considered a "homologous" substitution.

[0100] As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, Gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above-mentioned programs also generally provide an indication of the degree of homology. In some embodiments, two sequences are considered substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the corresponding residues in the two sequences are homologous over a stretch of related residues. In some embodiments, the related segments are complete sequences. In some embodiments, the cognate stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more residues.

[0101] Substantial identity: The phrase "substantial identity" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered to be "substantially identical" if they contain the same residues in corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, GAP BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul, ed., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above-mentioned programs also typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the corresponding residues of the two sequences are identical over a stretch of related residues. In some embodiments, the related segments are complete sequences. In some embodiments, the related segments are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0102] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with or displays one or more symptoms of the disease, disorder, and / or condition.

[0103] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition has not been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, condition, or event (e.g., DMD) may have one or more of the following characteristics: (1) a genetic mutation associated with developing the disease, disorder, and / or condition; (2) a genetic polymorphism associated with developing the disease, disorder, and / or condition; (3) an increase and / or decrease in the expression and / or activity of a protein associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with developing the disease, disorder, condition, and / or event; (5) has undergone, plans to undergo, or requires a transplant. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0104] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent symptoms of, and / or delay the onset of the disease, disorder, and / or condition. One of ordinary skill in the art will appreciate that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0105] Treatment: As used herein, the term "treat / treatment / treating" refers to any method used to partially or completely alleviate, improve, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a specific disease, disorder, and / or condition. Treatment can be administered to subjects who do not show signs of the disease and / or only show early signs of the disease to achieve the purpose of reducing the risk of developing pathology associated with the disease.

[0106] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0107] In particular, the present invention provides methods and compositions based on C1-INH as a protein therapeutic for treating complement-mediated disorders including hereditary angioedema (HAE).

[0108] The purpose of the fusion is to extend the half-life. The result is a reduction in the frequency of dosing and an increase in preventive and therapeutic efficacy.

[0109] Various aspects of the present invention are described in detail in the following sections. The use of sections is not intended to limit the present invention. Each section may be applicable to any aspect of the present invention. In this application, unless otherwise stated, the use of "or" means "and / or". The disclosure of all technologies cited herein is incorporated herein by reference in its entirety.

[0110] C1-INH

[0111] Human C1-INH is an important anti-inflammatory plasma protein with a wide range of inhibitory and non-inhibitory biological activities. In terms of sequence homology, the structure of its C-terminal domain and the mechanism of protease inhibition, it belongs to the serine protease inhibitor superfamily (i.e., the largest class of plasma protease inhibitors), which also includes antithrombin, α1-proteinase inhibitor, plasminogen activator inhibitor and many other structurally similar proteins that regulate different physiological systems. C1-INH is an inhibitor of proteases in the complement system, the kinin-generating contact system and the intrinsic coagulation pathway. Cai, S. and Davis, AE, Complement Regulatory Protein C1 Inhibitor Binds to Selectins and Interferes with Endothelial-Leukocyte Adhesion, J Immunol, 171: 4786-4791 (2003). Specifically, C1-INH has been shown to inhibit C1r and C1s of the complement system. C1-INH is also a major regulator of coagulation factors XI and XII, as well as kallikrein and other serine proteases of the coagulation and fibrinolytic systems, including tissue-type plasminogen activator and plasmin.

[0112] Low plasma levels of C1-INH or its dysfunction leads to activation of both the complement cascade and the contact plasma cascade, and may also affect other systems. It has been shown that a decrease in C1-INH plasma levels to levels below 55 μg / mL (approximately 25% of normal) induces spontaneous activation of C1.

[0113] A schematic diagram depicting the structure of C1-INH is provided in Figure 1In A. Signal peptide, N-terminal domain and serine protease inhibitor domain are shown. The 22 amino acid signal peptide is required for secretion and is cleaved from the rest of the C1-INH protein. C1-INH has two domains: a C-terminal domain with 365 amino acids, which is a typical serine protease inhibitor domain, and an N-terminal domain with 113 amino acids. The protein is stabilized by two disulfide bridges connecting each domain. These disulfide bridges are formed by Cys101 of the N-terminal domain and Cys406 of the C-terminal (serine protease inhibitor) domain, and Cys108 of the N-terminal domain and Cys183 of the C-terminal domain. The serine protease inhibitor domain is responsible for the protease activity of C1-INH. P1-P1' represents the Arg444-Thr445 easy scission bond.

[0114] More than 26% of the weight of the glycosylated protein is carbohydrate. Glycans are unevenly distributed in human C1-INH. The N-terminus is heavily glycosylated, with three N-linked (shown as long vertical lines with diamond-shaped heads) and at least seven O-linked (shown as short vertical lines) carbohydrate groups. The three N-linked glycans are connected to asparagine residues Asn216, Asn231, and Asn330 (shown as long vertical lines with diamond-shaped heads) in the serine protease inhibitor domain. Although the functional role of the extraordinarily long and heavily glycosylated N-terminal domain remains unclear, it may be necessary for the conformational stability, recognition, affinity for endotoxins and selectins, and clearance of the protein. The intrinsic heterogeneity of the carbohydrate portion greatly contributes to the heterogeneity of the entire C1-INH, which is one reason why it is difficult to produce a recombinant C1-INH that mimics the properties of plasma-derived C1-INH.

[0115] As used herein, C1-INH fusion proteins suitable for the present invention include any wild-type and modified C1-INH polypeptides (e.g., C1-INH proteins with amino acid mutations, deletions, truncations and / or insertions) that retain substantial C1-INH biological activity. Typically, C1-INH fusion proteins are produced using recombinant technology.

[0116] Typically, a suitable recombinant C1-INH fusion protein has an in vivo half-life of about or greater than 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, or 10 days. In some embodiments, the recombinant C1-INH fusion protein has an in vivo half-life of between 0.5 and 10 days, between 1 day and 10 days, between 1 day and 9 days, between 1 day and 8 days, between 1 day and 7 days, between 1 day and 6 days, between 1 day and 5 days, between 1 day and 4 days, between 1 day and 3 days, between 2 days and 10 days, between 2 days and 9 days, between 2 days and 8 days, between 2 days and 7 days, between 2 days and 6 days, between 2 days and 5 days, between 2 days and 4 days. days, between 2 days and 3 days, between 2.5 days and 10 days, between 2.5 days and 9 days, between 2.5 days and 8 days, between 2.5 days and 7 days, between 2.5 days and 6 days, between 2.5 days and 5 days, between 2.5 days and 4 days, between 3 days and 10 days, between 3 days and 9 days, between 3 days and 8 days, between 3 days and 7 days, between 3 days and 6 days, between 3 days and 5 days, between 3 days and 4 days, between 3.5 days and 10 days, between Between 3.5 days and 9 days, Between 3.5 days and 8 days, Between 3.5 days and 7 days, Between 3.5 days and 6 days, Between 3.5 days and 5 days, Between 3.5 days and 4 days, Between 4 days and 10 days, Between 4 days and 9 days, Between 4 days and 8 days, Between 4 days and 7 days, Between 4 days and 6 days, Between 4 days and 5 days, Between 4.5 days and 10 days, Between 4.5 days and 9 days, Between 4.5 days and 8 days, Between 4.5 days and 7 days, Between In vivo half-life of between 4.5 days and 6 days, between 4.5 days and 5 days, between 5 days and 10 days, between 5 days and 9 days, between 5 days and 8 days, between 5 days and 7 days, between 5 days and 6 days, between 5.5 days and 10 days, between 5.5 days and 9 days, between 5.5 days and 8 days, between 5.5 days and 7 days, between 5.5 days and 6 days, between 6 days and 10 days, between 7 days and 10 days, between 8 days and 10 days, and between 9 days and 10 days.

[0117] Typically, a suitable recombinant C1-INH fusion protein has an in vivo half-life of or greater than about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 days. In some embodiments, the recombinant C1-INH fusion protein has an in vivo half-life of between 4 days and 9 days, between 4 days and 8 days, between 4 days and 7 days, between 4 days and 6 days, between 4 days and 5 days, between 4.5 days and 10 days, between 4.5 days and 9 days, between 4.5 days and 8 days, between 4.5 days and 7 days, between 4.5 days and 6 days, between 4.5 days and 5 days, between 5 days and 10 days, between 5 days and 9 days, between 5 days and 8 days, between 5 days and 7 days, between 5 days and 6 days, between 5.5 days and 10 days, between 5.5 days and 9 days, between 5.5 days and 8 days, between 5.5 days and 7 days, between 5.5 days and 6 days, between 6 days and 10 days, between 7 days and 10 days, between 8 days and 10 days, and between 9 days and 10 days.

[0118] In some embodiments, a recombinant C1-INH polypeptide suitable for the present invention comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following wild-type human C1-INH protein (amino acids 1-478) (amino acids 1-97 are underlined):

[0119]

[0120] In some embodiments, a recombinant C1-INH polypeptide suitable for the present invention comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following wild-type human C1-INH protein (amino acids 98-478):

[0121]

[0122] As disclosed herein, SEQ ID NO: 1 represents a typical amino acid sequence of a human C1-INH protein. In some embodiments, a C1-INH polypeptide may be a truncated C1-INH, such as SEQ ID NO: 2. In some embodiments, a suitable recombinant C1-INH polypeptide may be a homolog or analog of a wild-type or naturally occurring protein. For example, a homolog or analog of a human wild-type or naturally occurring C1-INH polypeptide may contain one or more amino acid or domain substitutions, deletions and / or insertions compared to a wild-type or naturally occurring C1-INH protein (e.g., SEQ ID NO: 1), while retaining substantially C1-INH protein activity. Therefore, in some embodiments, a recombinant C1-INH polypeptide suitable for the present invention is substantially homologous to a human C1-INH protein (SEQ ID NO: 1). In some embodiments, a recombinant C1-INH polypeptide suitable for the present invention has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO: 1. In some embodiments, a recombinant C1-INH polypeptide suitable for the present invention is substantially identical to human C1-INH protein (SEQ ID NO: 1). In some embodiments, a recombinant C1-INH polypeptide suitable for the present invention has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1.

[0123] Homologs or analogs of human C1-INH protein can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those found in references compiling such methods. As will be appreciated by those of ordinary skill in the art, if two sequences contain homologous residues in corresponding positions, they are generally considered to be "substantially homologous". Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues that will have appropriately similar structural and / or functional characteristics. For example, as is well known to those of ordinary skill in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and / or have "polar" or "non-polar" side chains. Substituting one amino acid for another amino acid of the same type can often be considered a "homologous" substitution. In some embodiments, conservative amino acid substitutions include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. In some embodiments, "conservative amino acid substitutions" refer to amino acid substitutions that do not change the relative charge or size characteristics of the protein in which the amino acid substitution is made.

[0124] As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, Gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above-mentioned programs also generally provide an indication of the degree of homology.

[0125] In some embodiments, the recombinant C1-INH polypeptide suitable for the present invention contains one or more amino acid deletions, insertions or substitutions compared to the wild-type human C1-INH protein. For example, a suitable recombinant C1-INH polypeptide may be a truncated polypeptide, such as a polypeptide having SEQ ID NO: 2.

[0126] In some embodiments, a recombinant C1-INH polypeptide suitable for the present invention comprises an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following truncated wild-type human C1-INH protein (amino acids 98-478):

[0127]

[0128] In some embodiments, the C1-INH polypeptide may be a truncated C1-INH while retaining substantial C1-INH protein activity, such as SEQ ID NO: 2. Thus, in some embodiments, the recombinant C1-INH polypeptide suitable for the present invention is substantially homologous to the human C1-INH protein (SEQ ID NO: 2). In some embodiments, the recombinant C1-INH polypeptide suitable for the present invention has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to SEQ ID NO: 2. In some embodiments, the recombinant C1-INH polypeptide suitable for the present invention is substantially identical to the human C1-INH protein (SEQ ID NO: 2). In some embodiments, a recombinant C1-INH polypeptide suitable for the present invention has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2.

[0129] Recombinant C1-INH fusion proteins suitable for the present invention can be fusion proteins between a C1-INH domain and another domain or portion that can generally promote the therapeutic effect of C1-INH by, for example, enhancing or increasing the half-life, stability, efficacy and / or delivery of the C1-INH protein, or reducing or eliminating immunogenicity, clearance or toxicity. Such suitable domains or portions of C1-INH fusion proteins include, but are not limited to, Fc domains and albumin domains.

[0130] Fc domain

[0131] In some embodiments, suitable C1-INH fusion proteins contain an Fc domain or a portion thereof that binds an FcRn receptor. As a non-limiting example, suitable Fc domains may be derived from immunoglobulin subclasses such as IgG. In some embodiments, suitable Fc domains are derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, suitable Fc domains are derived from IgM, IgA, IgD, or IgE. Particularly suitable Fc domains include those derived from human antibodies or humanized antibodies. In some embodiments, suitable Fc domains are modified Fc portions, such as modified human Fc portions.

[0132] A.IgG1

[0133] C1-inhibitor Fc fusion protein can exist in dimer form, such as Figure 1 As shown in A.

[0134] In some embodiments, Fc domains suitable for the invention include an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following wild-type human IgG1 Fc domain:

[0135]

[0136] i.LALA

[0137] Wild-type IgG1 has low levels of complement cascade activation, any of which may be undesirable for patients suffering from complement-mediated disorders. The selection of IgGs with effector-neutral constructs that reduce or eliminate complement activation and antibody-dependent cell-mediated cytotoxicity (ADCC) activity may be important. Suitable Fc domains include IgG1 with mutations L234A and L235A (LALA).

[0138] In some embodiments, Fc domains suitable for the present invention include amino acid sequences that are at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following human IgG1 Fc domain with a LALA mutation (the mutated residue is underlined):

[0139]

[0140] ii.NHance

[0141] It is expected that the improved binding between the Fc domain and the FcRn receptor will lead to an extended serum half-life. Therefore, in some embodiments, the suitable Fc domain comprises one or more amino acid mutations that result in improved binding to FcRn. Various mutations that achieve improved binding to FcRn in the Fc domain are known in the art and may be suitable for implementing the present invention. In some embodiments, the suitable Fc domain comprises one or more mutations at the position of one or more Thr250, Met252, Ser254, Thr256, Thr 307, Glu380, Met428, His 433 and / or Asn434 corresponding to human IgG1.

[0142] For example, a suitable Fc domain may contain the mutations H433K (His433Lys) and / or N434F (Asn434Phe). As a non-limiting example, a suitable Fc domain may contain the mutations H433K (His433Lys) and N434F (Asn434Phe) (Nhance). Additional amino acid substitutions that may be included in the Fc domain include, for example, those described in U.S. Pat. Nos. 6,277,375; 8,012,476; and 8,163,881, which are incorporated herein by reference.

[0143] In some embodiments, Fc domains suitable for the present invention include amino acid sequences that are at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following human IgG1 Fc domain with a Nhance mutation (the mutated residues are underlined):

[0144]

[0145] In some embodiments, Fc domains suitable for the present invention include amino acid sequences that are at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following human IgG1 Fc domain with both the LALA mutation and the Nhance mutation (the mutated residues are underlined):

[0146]

[0147] In some embodiments, Fc domains suitable for the present invention include amino acid sequences that are at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following human IgG1 Fc domain with a signal peptide and with a Nhance mutation (signal peptide and mutated residues are underlined):

[0148]

[0149] In some embodiments, Fc domains suitable for the present invention include amino acid sequences that are at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following human IgG1 Fc domain with a signal peptide and having both a LALA mutation and a Nhance mutation (the mutated residues are underlined):

[0150]

[0151] In some embodiments, Fc domains suitable for the invention include an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following wild-type human IgG4 Fc domain:

[0152]

[0153] In some embodiments, Fc domains suitable for the present invention include amino acid sequences that are at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the following human IgG4 Fc domain with an S241P mutation (the mutated residue is underlined):

[0154]

[0155] In some embodiments, a suitable Fc domain comprises an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0156] B.IgG4

[0157] In some embodiments, in the case of HAE, IgG4 IgG selection of "effector-ineffective" Fc fusion proteins is important (eliminating complement activation and ADCC). Specifically, IgG4 is reported to have lower complement activation than wild-type IgG1. Expression of native IgG4 will produce a mixed species of construct size classification. Therefore, based on the prior art, IgG4S241P was selected for this procedure due to the increased stability and dimeric structure maintenance exhibited by the IgG4S241P mutant.

[0158] The human IgG4 core hinge region sequence according to the present invention preferably comprises a S228P substitution according to the EU index as per Kabat. According to Kabat et al. (1987 Sequences of proteins of immunological interest. United States Department of Health and Human Services, Washington DC.), this substitution has also been referred to as S241P. The substitution has the effect of making the sequence of the core of the hinge region identical to the hinge region core sequence of a wild-type IgG1 or IgG2 isotype antibody. With regard to IgG4 isotype antibodies, it results in the generation of a homologous form of IgG4 antibodies, and therefore eliminates the dissociation and reassociation of the heavy chains that often result in the generation of heterodimeric IgG4 antibodies. In some embodiments, IgG4 is preferred for stability at high concentrations.

[0159] C. Exemplary C1-INH Fusion Proteins

[0160] In certain embodiments, a suitable recombinant C1-INH fusion protein comprises a C1-INH polypeptide, an Fc domain, wherein the C1-INH polypeptide comprises an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to a wild-type human C1-INH polypeptide (SEQ ID NO: 1) or a truncated C1-INH polypeptide (SEQ ID NO: 2). In some embodiments, there is a linker that associates the C1-INH polypeptide with the Fc domain.

[0161] In some embodiments, a suitable recombinant C1-INH fusion protein has an in vivo half-life in the range of about 0.5-10 days (e.g., about 0.5-5.5 days, about 0.5-5 days, about 1-5 days, about 1.5-5 days, about 1.5-4.5 days, about 1.5-4.0 days, about 1.5-3.5 days, about 1.5-3 days, about 1.5-2.5 days, about 2-6 days, about 2-5.5 days, about 2-5 days, about 2-4.5 days, about 2-4 days, about 2-3.5 days, about 2-3 days). In some embodiments, a suitable recombinant C1-INH fusion protein has an in vivo half-life in the range of about 2-10 days (e.g., in the range of about 2.5-10 days, about 3-10 days, about 3.5-10 days, about 4-10 days, about 4.5-10 days, about 5-10 days, about 3-8 days, about 3.5-8 days, about 4-8 days, about 4.5-8 days, about 5-8 days, about 3-6 days, about 3.5-6 days, about 4-6 days, about 4.5-6 days, about 5-6 days).

[0162] Typically, a suitable recombinant C1-INH fusion protein has an in vivo half-life of about or greater than 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 2.5 days, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, 8 days, 8.5 days, 9 days, 9.5 days, or 10 days. In some embodiments, the recombinant C1-INH fusion protein has an in vivo half-life of between 0.5 and 10 days, between 1 day and 10 days, between 1 day and 9 days, between 1 day and 8 days, between 1 day and 7 days, between 1 day and 6 days, between 1 day and 5 days, between 1 day and 4 days, between 1 day and 3 days, between 2 days and 10 days, between 2 days and 9 days, between 2 days and 8 days, between 2 days and 7 days, between 2 days and 6 days, between 2 days and 5 days, between 2 days ... Between 2 days and 3 days, Between 2.5 days and 10 days, Between 2.5 days and 9 days, Between 2.5 days and 8 days, Between 2.5 days and 7 days, Between 2.5 days and 6 days, Between 2.5 days and 5 days, Between 2.5 days and 4 days, Between 3 days and 10 days, Between 3 days and 9 days, Between 3 days and 8 days, Between 3 days and 7 days, Between 3 days and 6 days, Between 3 days and 5 days, Between 3 days and 4 days, Between 3.5 days and 10 days, Between 3.5 between 3.5 days and 9 days, between 3.5 days and 8 days, between 3.5 days and 7 days, between 3.5 days and 6 days, between 3.5 days and 5 days, between 3.5 days and 4 days, between 4 days and 10 days, between 4 days and 9 days, between 4 days and 8 days, between 4 days and 7 days, between 4 days and 6 days, between 4 days and 5 days, between 4.5 days and 10 days, between 4.5 days and 9 days, between 4.5 days and 8 days, between 4.5 days and 7 days, between 4. An in vivo half-life of between 5 days and 6 days, between 4.5 days and 5 days, between 5 days and 10 days, between 5 days and 9 days, between 5 days and 8 days, between 5 days and 7 days, between 5 days and 6 days, between 5.5 days and 10 days, between 5.5 days and 9 days, between 5.5 days and 8 days, between 5.5 days and 7 days, between 5.5 days and 6 days, between 6 days and 10 days, between 7 days and 10 days, between 8 days and 10 days, and between 9 days and 10 days.

[0163] In certain embodiments, such as Figure 2 A and Figure 2As shown in B, the Fc portion can be directly fused to the N-terminal region of the full-length (1-478 amino acids) as well as the truncated (98-478) C1-inhibitor. As a non-limiting example, a suitable C1-INH Fc fusion protein can have the amino acid sequence shown below:

[0164]

[0165] or

[0166]

[0167] or

[0168]

[0169] or

[0170]

[0171] or

[0172]

[0173] or

[0174]

[0175] or

[0176]

[0177] or

[0178]

[0179] In some embodiments, a suitable recombinant C1-INH Fc fusion protein has an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 32, or SEQ ID NO: 33.

[0180] It is contemplated that the C1-INH-Fc fusion protein can be provided in various configurations, including homodimeric or monomeric configurations. For example, a suitable homodimeric configuration can be designed so that the C-terminus of the fusion partner (e.g., C1-INH polypeptide plus a linker) is connected to the N-termini of two Fc polypeptide chains. A suitable monomeric configuration can be designed so that the C-terminus of the fusion partner (e.g., C1-INH polypeptide plus a linker) is fused to an Fc dimer.

[0181] For certain applications and routes of administration, such as subcutaneous administration, monomeric (also referred to herein as monovalent) forms may be preferred. The monomeric configuration may reduce steric hindrance, increase half-life, and / or may increase bioavailability.

[0182] For C1-INH Fc fusion constructs, a monovalent format may also be preferred because C1-INH is a suicide inhibitor. Because it is a suicide inhibitor, binding of one C1-INH "arm" of the dimeric Fc fusion will result in increased clearance of the bound C1-INH fusion protein, even when the second arm remains unbound.

[0183] An advantage of both monomeric and dimeric Fc fusion proteins is that expression in the case of Fc was found to occur at higher levels compared to expression of C1-INH alone. Activity assays comparing dimeric C1-INH Fc constructs to recombinant C1-INH have been shown to have similar C1q binding activity. Inclusion of a linker was also tested and found to not affect the ability of the Fc-C1-INH fusion protein to bind its target.

[0184] Methods for preparing monomeric antibody fusion proteins include, for example, those described in PCT Publication Nos. WO2011 / 063348; WO2012 / 020096; WO2013 / 138643; WO2014087299; Dumont, J. et al., Monomeric Fc Fusions: Impact on Pharmacokinetic and Biological Activity of Protein Therapeutics, Biodrugs, 20(3): 151-160 (2006); Ishino, T. et al., Protein Structure and Folding: Half-life Extension of Biotherapeutics Modality by N-Glycosylation for the Engineering a Monomeric Fc Domain, J. Biol. Chem., 288: 16529-16537 (2013), the disclosures of which are incorporated herein by reference.

[0185] A monovalent C1-inhibitor can be prepared by using a plasmid containing Fc-C1 co-transfected with a plasmid expressing Fc alone. In addition, it can be prepared by using a dual promoter plasmid, where one promoter produces Fc-C1 and the other promoter produces Fc alone. Monovalent Fc can also be prepared using bispecific technology, where specific amino acids in the hinge region of Fc are mutated to confer stability to the Fc region (e.g., Knob and hole technology or other stabilizing mutations that drive the formation of monovalent C1).

[0186] As used herein, "percent (%) amino acid sequence identity" with respect to a reference protein sequence identified herein (e.g., with reference to the C1-INH protein sequence) is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences, introducing gaps if necessary to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences. Preferably, WU-BLAST-2 software is used to determine amino acid sequence identity (Altschul et al., Methods in Enzymology266, 460-480 (1996); http: / / blast.wustl / edu / blast / README.html). WU-BLAST-2 uses several search parameters, most of which are set to default values. Adjustable parameters are set to the following values: overlap spacing = 1, overlap fraction = 0.125, word threshold (worldthreshold) (T) = 11. The HSP score (S) and HSP S2 parameters are dynamic values ​​and are determined by the program itself depending on the composition of the particular sequence, however, the minimum value can be adjusted and is set as indicated above.

[0187] Albumin domain

[0188] Albumin is a soluble monomeric protein that accounts for about half of serum proteins. Albumin mainly acts as a carrier protein for steroids, fatty acids and thyroid hormones, and plays a role in stabilizing the volume of extracellular fluid. Albumin appears as a globular, unglycosylated serum protein with a molecular weight of 66,500. Albumin is synthesized in the liver in the form of preproalbumin with an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product proalbumin is in turn cleaved in the Golgi vesicles to produce secretory albumin.

[0189] Albumin consists of three homology domains (I-III), and each of these domains contains two subdomains (A and B). The main ligand binding region of human serum albumin is located in a cavity in subdomains IIA and IIIA, which is mainly formed by hydrophobic and positively charged residues and exhibits similar chemical properties. Human serum albumin has 585 amino acids and a molecular mass of 66,500 Da. The amino acids include 35 cysteines, all of which except one are involved in the formation of 17 stabilizing disulfide bonds.

[0190] Albumin has an extended serum half-life of 19 days. FcRn controls the long serum half-life of albumin. FcRn is a dual binding receptor that binds IgG in addition to albumin and protects both proteins from intracellular degradation. The C-terminal domain of the albumin molecule has been shown to be essential for binding to FcRn. The lack of domain IIIB or mutations 464His, 510His and 535His almost completely eliminate FcRn binding.

[0191] The albumin fusion proteins of the invention are monomeric. In some embodiments, this feature may be an advantage over dimeric Fc fusion embodiments for the reasons described above with respect to monomeric Fc fusion embodiments.

[0192] In some embodiments, albumin polypeptides suitable for the invention include an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the following wild-type human serum albumin:

[0193]

[0194] In some embodiments, albumin polypeptides suitable for the invention include an amino acid sequence that is at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the D3 domain of wild-type human serum albumin:

[0195]

[0196] Linker or spacer

[0197] The C1-INH domain can be directly or indirectly connected to the Fc domain or the albumin domain. In some embodiments, a suitable recombinant C1-INH fusion protein contains a linker or spacer that joins the C1-INH polypeptide and the Fc domain. In some embodiments, a suitable recombinant C1-INH fusion protein contains a linker or spacer that joins the C1-INH polypeptide and the albumin polypeptide. The amino acid linker or spacer is generally designed to have flexibility or a structure such as an alpha-helix inserted between two protein parts. The linker or spacer can be relatively short, or can be longer. Typically, in terms of length, the linker or spacer contains, for example, 3-100 (e.g., 5-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 5-55, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20) amino acids. In some embodiments, in terms of length, the linker or spacer is equal to or longer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids. Typically, longer linkers can reduce steric hindrance. In some embodiments, the joint comprises a mixture of glycine and serine residues. In some embodiments, the joint may additionally comprise threonine, proline and / or alanine residues. Therefore, in some embodiments, the joint comprises 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 10-15 between amino acids. In some embodiments, the joint comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95 amino acids. In some embodiments, the joint is not a joint consisting of ALEVLFQGP.

[0198] As non-limiting examples, linkers or spacers suitable for the present invention include, but are not limited to, a GGG linker and a GGGGSGGGGS ((GGGGS)2 linker SEQ ID NO: 27). In some embodiments, the linker comprises the sequence GGG and / or the sequence SEQ ID NO: 27.

[0199] Other suitable connectors include GAP GGGGGAAAAAGGGGG GAP (GAG linker, SEQ ID NO: 34); GAP GGGGGAAAAAGGGGG GAP GGGGGAAAAAGGGGG GAP(GAG2 linker, SEQ ID NO:35); and GAP GGGGGAAAAAGGGGG GAP GGGGGAAAAAGGGGG GAP GGGGGAAAAAGGGGG GAP (GAG3 linker, SEQ ID NO:36).

[0200] Suitable linkers or spacers also include those having an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology or identity to the above exemplary linkers, such as GGG linker, GGGGSGGGGS ((GGGGS)2 linker SEQ ID NO: 27), GAG linker (SEQ ID NO: 34), GAG2 linker (SEQ ID NO: 35), or GAG3 linker (SEQ ID NO: 36). Additional linkers suitable for use with some embodiments can be found in US2012 / 0232021, filed March 2, 2012, the disclosure of which is hereby incorporated by reference in its entirety.

[0201] In some embodiments, a linker is provided that allows the C1-INH polypeptide to associate with the albumin domain without substantially affecting the ability of the C1-INH polypeptide to bind to any of its cognate ligands (e.g., C1s, etc.). In some embodiments, a linker is provided such that the binding of the C1-INH peptide to one or more of its cognate ligands is not altered compared to the C1-INH polypeptide alone. In some embodiments, a linker is provided such that the binding of the C1-INH peptide to one or more of its cognate ligands is not reduced or diminished compared to the C1-INH polypeptide alone.

[0202] Production of recombinant C1-INH fusion protein

[0203] Recombinant C1-INH fusion proteins suitable for the present invention can be produced by any available means. For example, recombinant C1-INH fusion proteins can be recombinantly produced by utilizing a host cell system engineered to express a nucleic acid encoding a recombinant C1-INH fusion protein. Alternatively or additionally, recombinant C1-INH fusion proteins can be produced by activating an endogenous gene. Alternatively or additionally, recombinant C1-INH fusion proteins can be prepared partially or completely by chemical synthesis.

[0204] When the protein is produced recombinantly, any expression system can be used. Known expression systems include, for example, E. coli, egg, baculovirus, plant, yeast, or mammalian cells, to name a few.

[0205] In some embodiments, recombinant C1-INH fusion proteins suitable for the present invention are produced in mammalian cells. Non-limiting examples of mammalian cells that can be used according to the present invention include BALB / c mouse myeloma line (NSO / 1, ECACC No.: 85110503); human retinoblastoma cells (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or 293 cells subcloned for suspension culture growth, Graham et al., J. Gen Virol., 36:59, 1977); human fibrosarcoma cell line (e.g., HT1080); baby hamster kidney cells (BHK21, ATCC CCL 10); Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse Sertoli cells (Sertoli cells). cell) (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2).

[0206] In some embodiments, the present invention provides a recombinant C1-INH fusion protein produced by human cells. In some embodiments, the present invention provides a recombinant C1-INH fusion protein produced by CHO cells or HT1080 cells.

[0207] Typically, cells engineered to express a recombinant C1-INH fusion protein may contain a transgene encoding a recombinant C1-INH fusion protein as described herein. It should be understood that the nucleic acid encoding the recombinant C1-INH fusion protein may contain regulatory sequences, gene control sequences, promoters, non-coding sequences, and / or other appropriate sequences for expressing the recombinant C1-INH fusion protein. Typically, the coding region is operably linked to one or more of these nucleic acid components.

[0208] The coding region of the transgene may include one or more silent mutations to optimize codon usage for a specific cell type. For example, the codons of the C1-INH fusion transgene may be optimized for expression in vertebrate cells. In some embodiments, the codons of the C1-INH fusion transgene may be optimized for expression in mammalian cells. In some embodiments, the codons of the C1-INH fusion transgene may be optimized for expression in human cells. In some embodiments, the codons of the C1-INH fusion transgene may be optimized for expression in CHO cells.

[0209] Nucleic acid encoding C1-inhibitor Fc fusion protein and / or C1-inhibitor albumin fusion protein

[0210] In some embodiments, a nucleic acid molecule comprising a nucleic acid sequence encoding a recombinant target gene (referred to herein as a transgene) such as a C1-inhibitor Fc fusion protein and / or a C1-inhibitor albumin fusion protein as described in various embodiments herein is provided. In some embodiments, the nucleic acid encoding the transgene may be modified to provide increased expression of the encoded C1-inhibitor Fc fusion protein and / or C1-inhibitor albumin fusion protein, which is also referred to as codon optimization. For example, the nucleic acid encoding the transgene may be modified by altering the open reading frame of the coding sequence. As used herein, the term "open reading frame" is synonymous with "ORF" and means any nucleotide sequence that is potentially capable of encoding a protein or a portion of a protein. An open reading frame typically begins with a start codon (represented, for example, as AUG for RNA molecules and ATG for DNA molecules in the case of a standard code) and is read as a codon triplet until the end of the frame with a stop codon (represented, for example, as UAA, UGA or UAG for RNA molecules and TAA, TGA or TAG for DNA molecules in the case of a standard code). As used herein, the term "codon" means a trinucleotide sequence in a nucleic acid molecule that specifies a specific amino acid during protein synthesis; also referred to as a triplet or codon triplet. For example, among the 64 possible codons in the case of the standard genetic code, two codons GAA and GAG encode the amino acid glutamine, while codons AAA and AAG specify the amino acid lysine. In the case of the standard genetic code, three codons are stop codons that do not specify amino acids. As used herein, the term "synonymous codon" means any and all codons encoding a single amino acid. Except for methionine and tryptophan, amino acids are encoded by two to six synonymous codons. For example, in the case of the standard genetic code, the four synonymous codons encoding the amino acid alanine are GCA, GCC, GCG and GCU, the two synonymous codons specifying glutamine are GAA and GAG, and the two synonymous codons encoding lysine are AAA and AAG.

[0211] In some embodiments, nucleic acids encoding the open reading frames of the C1-inhibitor Fc fusion protein and / or the C1-inhibitor albumin fusion protein can be modified using standard codon optimization methods. Various commercial algorithms for codon optimization are available and can be used to practice the present invention. Generally, codon optimization does not change the encoded amino acid sequence. In some embodiments, codon optimization may result in amino acid changes such as substitutions, deletions, or insertions. Generally, such amino acid changes do not substantially alter protein activity.

[0212] The exemplary nucleic acid sequence encodes a nucleic acid having the sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: NO:33 C1-inhibitor Fc fusion proteins and C1-inhibitor albumin fusion proteins having amino acid sequences of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity or homology.

[0213] In some embodiments, nucleotide changes may alter synonymous codons within the open reading frame to be consistent with endogenous codon usage found in a particular heterologous cell selected to express the C1-inhibitor Fc fusion protein and / or the C1-inhibitor albumin fusion protein. Alternatively or additionally, nucleotide changes may alter the G+C content within the open reading frame to better match the average G+C content of open reading frames found in endogenous nucleic acid sequences present in the heterologous host cell. Nucleotide changes may also alter polysingle nucleotide regions or internal regulatory or structural sites found within the C1-inhibitor Fc fusion protein or C1-inhibitor albumin fusion sequence. Thus, a variety of modified or optimized nucleotide sequences are contemplated, including, but not limited to, nucleic acid sequences that provide increased expression of the C1-inhibitor Fc fusion protein and / or the C1-inhibitor albumin fusion protein in prokaryotes; yeast cells; insect cells; and in mammalian cells.

[0214] Typically, the modified nucleic acid encodes a C1-inhibitor Fc fusion protein and / or a C1-inhibitor albumin fusion protein with or without amino acid sequence changes. Where there are amino acid changes, such changes typically do not substantially reduce the activity of the C1-inhibitor Fc fusion protein or the C1-inhibitor albumin fusion protein. In some embodiments, such changes increase and / or enhance the activity of the C1-inhibitor Fc fusion protein or the C1-inhibitor albumin fusion protein. Activity may refer to the following non-limiting list of parameters: increased half-life, increased / elevated protein expression by the host cell, increased stability of the expressed protein, increased solubility of the expressed protein, reduced aggregation of the expressed protein, simpler formulation of the expressed protein, simpler purification of the expressed protein, increased tolerance of the expressed protein to pH changes, increased ability of the protein to tolerate high pH and low pH conditions, and the expressed protein is suitable for formulation at high concentrations.

[0215] Expression vector

[0216] The nucleic acid sequence encoding the C1-inhibitor Fc fusion protein and / or the C1-inhibitor albumin fusion protein as described in this application can be molecularly cloned (inserted) into a suitable vector for propagation or expression in a host cell. A wide variety of expression vectors can be used to implement the present invention, including but not limited to prokaryotic expression vectors; yeast expression vectors; insect expression vectors and mammalian expression vectors. Exemplary vectors suitable for the present invention include, but are not limited to, viral-based vectors (e.g., AAV-based vectors, retroviral-based vectors, plasmid-based vectors). In some embodiments, the nucleic acid sequence encoding the C1-inhibitor Fc fusion protein can be inserted into a suitable vector. In some embodiments, the nucleic acid sequence encoding the C1-inhibitor albumin fusion protein can be inserted into a suitable vector. Typically, the nucleic acid encoding the C1-inhibitor Fc fusion protein or the C1-inhibitor albumin fusion protein is operably linked to various regulatory sequences or elements.

[0217] Regulatory sequences or elements

[0218] Various regulatory sequences or elements may be incorporated into expression vectors suitable for the present invention. Exemplary regulatory sequences or elements include, but are not limited to, promoters, enhancers, repressors or inhibitors, 5' untranslated (or non-coding) sequences, introns, 3' untranslated (or non-coding) sequences.

[0219] As used herein, "promoter" or "promoter sequence" is a DNA regulatory region that can bind to (e.g., directly or through proteins or substances bound by other promoters) RNA polymerase in cells and initiate transcription of coding sequences. In general, the promoter sequence is bound by the transcription initiation site at its 3' end, and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at any level. The promoter can be operably associated with or operably connected to the following: expression control sequences (including enhancer and repressor sequences) or nucleic acids to be expressed. In some embodiments, the promoter can be an inducible promoter. In some embodiments, the inducible promoter can be a unidirectional or bidirectional (bio-directional) promoter. In some embodiments, the promoter can be a constitutive promoter. In some embodiments, the promoter can be a hybrid promoter, in which the sequence containing the transcriptional regulatory region is obtained from one source, and the sequence containing the transcriptional initiation region is obtained from a second source. Systems for joining control elements to coding sequences within a transgene are well known in the art (general molecular biology and recombinant DNA techniques are described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, which is incorporated herein by reference). Commercial vectors suitable for inserting transgenes for expression in a variety of host cells under a variety of growth and induction conditions are also well known in the art.

[0220] In some embodiments, specific promoters can be used to control expression of transgenes in mammalian host cells, such as, but not limited to, the SRα promoter (Takebe et al., Molec. and Cell. Bio. 8:466-472 (1988)), the human CMV immediate early promoter (Boshart et al., Cell 41:521-530 (1985); Foecking et al., Gene 45:101-105 (1986)), the human CMV promoter, the human CMV5 promoter, the murine CMV immediate early promoter, the EF1-α-promoter, a hybrid CMV promoter for liver-specific expression (e.g., prepared by conjugating the CMV immediate early promoter to the transcriptional promoter elements of the human α-1-antitrypsin (HAT) or albumin (HAL) promoter), or a promoter for hepatoma-specific expression (e.g., in which the transcriptional promoter elements of human albumin ( HAL; about 1000 bp) or the transcriptional promoter element of human alpha-1-antitrypsin (HAT, about 2000 bp) combined with a 145-length enhancer element of the human alpha-1-microglobulin and bikunin precursor gene (AMBP); HAL-AMBP and HAT-AMBP); the SV40 early promoter region (Benoist et al., Nature 290:304-310 (1981)), the immediate early promoter of the yellow pine moth (Orgyia pseudotsugata), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)); or the regulatory sequence of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)). In some embodiments, the mammalian promoter is a constitutive promoter, such as, but not limited to, the hypoxanthine phosphoribosyltransferase (HPTR) promoter, the adenosine deaminase promoter, the pyruvate kinase promoter, the β-actin promoter, and other constitutive promoters known to those of ordinary skill in the art.

[0221] In some embodiments, specific promoters can be used to control expression of transgenes in prokaryotic host cells, such as, but not limited to, the β-lactamase promoter (Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978)); the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 75:3727-3731 (1978)); 80:21-25 (1983)); T7 promoter, T3 promoter, M13 promoter or M16 promoter; control the expression of transgenes in yeast host cells, such as but not limited to GAL1, GAL4 or GAL10 promoter, ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, glyceraldehyde-3-phosphate dehydrogenase III (TDH3) promoter, glyceraldehyde-3-phosphate dehydrogenase II (TDH2) promoter, glyceraldehyde-3-phosphate dehydrogenase I (TDH1) promoter, pyruvate kinase (PYK) promoter, enolase (ENO) promoter or triosephosphate isomerase (TPI) promoter.

[0222] In some embodiments, the promoter may be a viral promoter, many of which are capable of regulating expression of transgenes in several host cell types, including mammalian cells. Viral promoters that have been shown to drive constitutive expression of coding sequences in eukaryotic cells include, for example, simian virus promoters, herpes simplex virus promoters, papilloma virus promoters, adenovirus promoters, human immunodeficiency virus (HIV) promoters, Rous sarcoma virus promoters, cytomegalovirus (CMV) promoters, the long terminal repeats (LTRs) of Moloney murine leukemia virus and other retroviruses, the thymidine kinase promoter of herpes simplex virus, and other viral promoters known to those of ordinary skill in the art.

[0223] In some embodiments, the genetic control elements of the expression vector may also include 5' non-transcribed sequences and 5' non-translated sequences involved in initiating transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, Kozak sequences, etc. Enhancer elements can be optionally used to increase the expression level of the polypeptide or protein to be expressed. Examples of enhancer elements that have been shown to work in mammalian cells include the SV40 early gene enhancer as described in Dijkema et al., EMBO J. (1985) 4: 761, and the enhancer / promoter derived from the long terminal repeat sequence (LTR) of Rous sarcoma virus (RSV) (such as Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79: 6777) and human cytomegalovirus (such as Boshart et al., Cell (1985) 41: 521). The genetic control elements of the expression vector will also include 3' non-transcribed sequences and 3' non-translated sequences involved in terminating transcription and translation. Respectively, such as a polyadenylation (polyA) signal for stabilizing and processing the 3' end of mRNA transcribed from the promoter. Poly A signals include, for example, rabbit β globin polyA signal, bovine growth hormone polyA signal, chicken β globin terminator / polyA signal or SV40 late polyA region.

[0224] Selectable marker

[0225] The expression vector will preferably but optionally include at least one selectable marker. In some embodiments, a selectable marker is a nucleic acid sequence encoding a resistance gene that is operably connected to one or more genetic control elements to give the host cell the ability to maintain vigor when growing in the presence of cytotoxic chemicals and / or drugs. In some embodiments, a selectable agent can be used to maintain that the expression vector is retained in the host cell. In some embodiments, a selectable agent can be used to prevent modification (i.e., methylation) and / or silencing of the transgenic sequence in the expression vector. In some embodiments, a selectable agent can be used to maintain episomal expression of the vector in the host cell. In some embodiments, a selectable agent can be used to promote the stable integration of the transgenic sequence into the host cell genome. In some embodiments, the agents and / or resistance genes may include, but are not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017), ampicillin, neomycin (G418), zeomycin, mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) for eukaryotic host cells; tetracycline, ampicillin, kanamycin, or chloramphenicol for prokaryotic host cells; and URA3, LEU2, HIS3, LYS2, HIS4, ADE8, CUP1, or TRP1 for yeast host cells.

[0226] The expression vector may be transfected, transformed, or transduced into a host cell. As used herein, the terms "transfection," "transformation," and "transduction" all refer to the introduction of an exogenous nucleic acid sequence into a host cell. In some embodiments, expression vectors containing nucleic acid sequences encoding a C1-inhibitor Fc fusion protein and / or a C1-inhibitor albumin fusion protein are transfected, transformed, or transduced into a host cell simultaneously. In some embodiments, expression vectors containing nucleic acid sequences encoding a C1-inhibitor Fc fusion protein and / or a C1-inhibitor albumin fusion protein are sequentially transfected, transformed, or transduced into a host cell. For example, a vector encoding a C1-inhibitor fusion protein may be transfected, transformed, or transduced into a host cell first, followed by a vector encoding one or more proteins that enhance glycosylation of the expressed C1-inhibitor fusion protein, preferably by increasing sialylation, and vice versa.

[0227] Examples of transformation, transfection and transduction methods well known in the art include liposome delivery, i.e., lipofectamine 5, Hawley-Nelson, Focus 15:73(1193). TM(Gibco BRL) method, electroporation, CaPO by Graham and van der Erb, Virology, 52: 456-457 (1978) 4 Delivery methods, DEAE-dextran mediated delivery, microinjection, gene gun particle delivery, polybrene mediated delivery, cation mediated lipid delivery, transduction and viral infection, such as retrovirus, lentivirus, adenovirus adeno-associated virus and baculovirus (insect cells). General aspects of cell host transformation have been described in the art, such as by Axel in U.S. Patent No. 4,399,216; Sambrook (supra), Chapters 1-4 and 16-18; Ausubel (supra), Chapters 1, 9, 13, 15 and 16. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology (1989), Keown et al., Methods in Enzymology, 185: 527-537 (1990), and Mansour et al., Nature, 336: 348-352 (1988).

[0228] Once introduced into the cell, the expression vector can be stably integrated into the genome or exist as an extrachromosomal construct. The vector can also be amplified, and multiple copies can exist or be integrated into the genome. In some embodiments, the cells of the present invention may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more copies of a nucleic acid encoding a C1-inhibitor fusion protein. In some embodiments, the cells of the present invention may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more copies of a nucleic acid encoding a C1-inhibitor fusion protein. In some embodiments, the cells of the present invention may contain multiple copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more) of nucleic acids encoding both the C1-inhibitor Fc fusion protein and one or more proteins that preferably enhance the glycosylation of the expressed C1-inhibitor fusion protein by increasing sialylation. Sialylation can also be manipulated by the various methods described herein. Without wishing to be bound by any theory, it was found that reduced sialylation correlated with increased heparin binding of the disclosed constructs, thereby preventing the C1-INH binding site from binding to the target. Heparin binding also increases internalization into lysosomes, thereby increasing the possibility of clearance.

[0229] Host cells

[0230] As used herein, the term "host cell" refers to a cell that can be used to produce a recombinant C1-inhibitor fusion protein. Specifically, the host cell is suitable for producing a recombinant C1-inhibitor fusion protein on a large scale. Suitable host cells can be derived from a variety of organisms, including but not limited to mammals, plants, birds (e.g., avian systems), insects, yeast, and bacteria. In some embodiments, the host cell is a mammalian cell. In some embodiments, a suitable host cell is engineered to improve the glycosylation characteristics of the expressed recombinant C1-inhibitor fusion protein. In some embodiments of the present invention, the C1-INH polypeptide has the same or similar glycosylation characteristics as a similar portion of natural plasma-derived C1-INH. In some embodiments, the C1-INH polypeptide has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% isoglycans with natural plasma-derived C1-INH. In some embodiments, the glycosylation profile of the C1-INH fusion protein has a humanized glycosylation profile. In some embodiments, the C1-INH fusion protein has increased sialylation compared to native plasma-derived C1-INH.

[0231] Improving glycosylation characteristics can refer to increasing sialylation and / or humanizing glycosylation characteristics, such as expressing a recombinant C1-inhibitor fusion protein comprising a C1-INH polypeptide in an engineered cell, thereby producing a glycosylation characteristic that is more similar to native human C1-INH than a C1-INH polypeptide expressed in a non-engineered cell. Improving can also refer to increasing, enhancing and / or optimizing the glycosylation characteristics of the C1-INH fusion compared to Ruconest.

[0232] Various methods of altering, controlling, manipulating, improving, enhancing and / or optimizing the glycosylation characteristics of proteins are known in the art. Glycosylation characteristics that can be optimized include the number of glycan residues, the position of glycan residue attachment, the glycan attachment mode (e.g., the type of bond), the glycan attachment process, and the identity of the glycan residue attached to the protein or polypeptide. The glycosylation characteristics of any portion of the fusion protein of the present invention are contemplated as targets suitable for glycosylation optimization. Portions of the fusion protein of the present invention that can be optimized for glycosylation include, but are not limited to, the C1-INH polypeptides, Fc domains, albumin polypeptides, and / or linkers disclosed herein. These methods of controlling glycosylation characteristics are encompassed, as well as other methods yet to be discovered that the skilled person will understand in light of the present disclosure to be useful in optimizing the glycosylation of the fusion protein of the present invention. Methods of altering, controlling, manipulating, improving, enhancing and / or optimizing the glycosylation characteristics of the C1-INH proteins and polypeptides of the present invention include in vitro, in situ, and in vivo methods.

[0233] In some embodiments, the glycosylation characteristics of an expressed protein or polypeptide are altered by post-translational and / or chemical modification of the expressed protein or polypeptide.

[0234] In some embodiments, the C1-INH fusion protein that has been subjected to the post-translational and / or chemical modification has an altered, improved, enhanced, humanized and / or optimized glycosylation profile compared to a C1-INH fusion protein with the same sequence that has not been subjected to the post-translational and / or chemical modification. In some embodiments, the C1-INH fusion protein that has been subjected to the post-translational and / or chemical modification has an altered, improved, enhanced, humanized and / or optimized sialylation profile compared to a C1-INH fusion protein with the same sequence that has not been subjected to the post-translational and / or chemical modification.

[0235] In some embodiments, the C1-INH fusion protein expressed by a cell line engineered to enhance glycosylation has an altered, improved, enhanced, humanized, and / or optimized glycosylation profile compared to a C1-INH fusion protein with the same sequence expressed by the same cell line that has not been engineered to enhance glycosylation. In some embodiments, the C1-INH fusion protein expressed by a cell line engineered to enhance sialylation has an altered, improved, enhanced, humanized, and / or optimized sialylation profile compared to a C1-INH fusion protein with the same sequence expressed by the same cell line that has not been engineered to enhance sialylation.

[0236] In some embodiments, the C1-INH fusion protein expressed by a cell line cultured under various conditions to enhance glycosylation has an altered, improved, enhanced, humanized, and / or optimized glycosylation profile compared to a C1-INH fusion protein with the same sequence expressed by a cell line cultured under various conditions to enhance glycosylation. In some embodiments, the C1-INH fusion protein expressed by a cell line cultured under various conditions to enhance sialylation has an altered, improved, enhanced, humanized, and / or optimized sialylation profile compared to a C1-INH fusion protein with the same sequence expressed by a cell line cultured under various conditions to enhance sialylation.

[0237] In some embodiments, the C1-INH fusion protein has an altered, improved, enhanced, humanized and / or optimized glycosylation profile compared to Ruconest. In some embodiments, the C1-INH fusion protein has an altered, improved, enhanced, humanized and / or optimized glycosylation profile compared to human plasma-derived C1-INH.

[0238] In some embodiments, cell culture conditions are manipulated to achieve expression of proteins with desired glycosylation characteristics. These cell culture conditions include controlling the production and culture process (including the length of culture), adding additives to the culture medium, co-expressing genes for enhancing glycosylation by increasing glycosylation, and / or engineering cells to eliminate, prevent expression, inactivate, or destroy enzymes associated with glycan degradation (e.g., sialidase). Methods suitable for manipulating glycosylation include, but are not limited to, those described in, for example, U.S. Patent Nos. 5,047,335; 5,096,816; 5,705,364; 7,645,609; 8,273,723; 8,524,477; 8,617,878; 8,871,723; PCT Publication Nos. WO2006 / 106348; WO2007 / 095506; WO2008 / 025856; WO2010 / 007214; WO2010 / 099394; and WO2013 / 093760, the disclosures of which are incorporated herein by reference.

[0239] Selection of specific clones of host cells and transfected host cells can also be used to enhance glycosylation.Other methods of enhancing glycosylation include the development of purification methods to enrich for proteins or polypeptides having the desired glycosylation characteristics.

[0240] Various methods for manipulating the sialylation characteristics of proteins are known in the art. The present invention encompasses these methods as well as other methods yet to be discovered. Methods for manipulating the sialylation characteristics of the C1-INH proteins and polypeptides of the present invention include in vitro, in situ, and in vivo methods. In some embodiments, the sialylation characteristics of the expressed proteins or polypeptides are changed by chemically modifying the expressed proteins or polypeptides after expression. In some embodiments, cell culture conditions are manipulated to achieve expression of proteins with desired sialylation characteristics. These cell culture conditions include controlling the production and culture processes (including the length of culture), adding additives to the culture medium, and / or co-expressing genes to enhance sialylation. Selection of host cells and specific clones of transfected host cells can also be used to enhance sialylation. Other methods for enhancing sialylation include developing purification methods to enrich for proteins or polypeptides with desired sialylation characteristics.

[0241] Sialylation can also be manipulated by various methods described herein. Without wishing to be bound by any theory, it was found that reduced sialylation was associated with increased heparin binding of the disclosed constructs, thereby preventing the C1-INH binding site from binding to the target. Heparin binding also increases internalization into lysosomes, thereby increasing the possibility of increased clearance.

[0242] Mammalian cell lines

[0243] Any mammalian cell or cell type that is susceptible to cell culture and polypeptide expression can be used as a host cell according to the present invention. Non-limiting examples of mammalian cells that can be used according to the present invention include human embryonic kidney 293 cells (HEK293), HeLa cells; BALB / c mouse myeloma strain (NSO / 1, ECACC No.: 85110503); human retinoblastoma (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CV1 strain transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney strain (293 or 293 cells subcloned to grow in suspension culture, Graham et al., J. Gen Virol., 36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells + / - DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In some embodiments, the suitable mammalian cell is not an endosomal acidification-deficient cell.

[0244] In addition, many commercially available and non-commercially available hybridoma cell lines expressing polypeptides or proteins can be utilized in accordance with the present invention. Those skilled in the art will appreciate that hybridoma cell lines may have different nutritional requirements and / or may require different culture conditions to achieve optimal growth and polypeptide or protein expression, and will be able to modify the conditions as needed.

[0245] Non-mammalian cell lines

[0246] Any cell or cell type of non-mammalian origin that is amenable to cell culture and polypeptide expression may be used as a host cell according to the present invention. Non-limiting examples of non-mammalian host cells and cell lines that can be used in accordance with the present invention include cells and cell lines derived from Pichia pastoris, Pichia methanolica, Pichia angusta, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Yarrowia lipolytica for yeast; Sodoptera frugiperda, Trichoplusis ni, Drosophila melangoster, and Manducasexta for insects; and Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Bacillus licheniformis for bacteria. lichenifonnis, Bacteroides fragilis, Clostridia perfringens, Clostridia difficile; and Xenopus laevis from amphibians.

[0247] Suitable for growth on the wall rather than in suspension

[0248] In certain embodiments, host cells for producing cell lines are selected based on certain preferred properties or growth under specific conditions selected for culturing cells. It will be appreciated by those skilled in the art that the properties may be determined based on known features and / or traits of a defined strain (i.e., a commercially available characterized cell line) or by empirical evaluation. In some embodiments, a cell line may be selected because it is able to grow on a cell trophoblast. In some embodiments, a cell line may be selected because it is able to grow in suspension. In some embodiments, a cell line may be selected because it is able to grow in the form of an adherent cell monolayer. In some embodiments, such cells may be used with any tissue culture container or any container treated with a suitable adherent matrix. In some embodiments, a suitable adherent matrix is ​​selected from the group consisting of: collagen (e.g., collagen I, II, II, or IV), gelatin, fibronectin, laminin, vitronectin, fibrinogen, BD Matrigel TM , basement membrane matrix, dermatan sulfate proteoglycans, poly-D-lysine and / or combinations thereof. In some embodiments, adherent host cells may be selected and modified for suspension growth under specific growth conditions. Such methods for modifying adherent cells for suspension growth are known in the art. For example, cells may be adapted to suspension culture growth by gradually removing animal serum from the growth medium over time.

[0249] Cell line selection and evaluation

[0250] According to the present invention, the cell that is engineered to express recombinant C1-INH fusion protein is selected because it can produce recombinant C1-INH fusion protein on a commercially viable scale. Specifically, the engineered cell according to the present invention can produce recombinant C1-INH fusion protein at a high level and / or with a high degree of enzyme activity. In some embodiments, once cultivated under cell culture conditions (e.g., standard large-scale suspension or adherent culture conditions), the desired cell can be or greater than about 5 pg / cell / day (e.g., greater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 pg / cell / day) to produce C1-INH fusion protein. In some embodiments, once cultivated under cell culture conditions (e.g., standard large-scale suspension or adherent culture conditions), the desired cells can be grown at about 5-100 pg / cell / day (e.g., about 5-90 pg / cell / day, about 5-80 pg / cell / day, about 5-70 pg / cell / day, about 5-60 pg / cell / day, about 5-50 pg / cell / day, about 5-40 pg / cell / day, about 5-30 pg / cell / day, about 10-90 pg / cell / day, about 10-80 pg / cell / day, about The C1-INH fusion protein is produced in an amount within the range of about 10-70 picograms / cell / day, about 10-60 picograms / cell / day, about 10-50 picograms / cell / day, about 10-40 picograms / cell / day, about 10-30 picograms / cell / day, about 20-90 picograms / cell / day, about 20-80 picograms / cell / day, about 20-70 picograms / cell / day, about 20-60 picograms / cell / day, about 20-50 picograms / cell / day, about 20-40 picograms / cell / day, about 20-30 picograms / cell / day).

[0251] The half-life of C1-INH can be affected by the glycosylation profile. As discussed above, the cells of the invention can be engineered to improve the glycosylation profile of the expressed C1-INH fusion protein. For example, it has been shown that (Pharming N.V.) As a recombinant C1-INH polypeptide with a lesser degree of sialylation and / or a different sialylation distribution from plasma-derived human C1-INH, it has a shorter half-life than plasma-derived human C1-INH. Davis, B. and Bernstein, JA, Conestat alfa for the treatment of angioedema attacks, Ther Clin Risk Manag. 7:265–273 (2011); Koles, K. et al., Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits, Glycobiology, 14(11):979-986 (2004); Koles, K. et al., N-and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits, Glycobiology, 14(1):51-64 (2004).

[0252] Cell culture media and culture conditions

[0253] Various cell culture media and culture conditions can be used to produce recombinant C1-INH fusion proteins using engineered cells according to the present invention. For example, recombinant C1-INH fusion proteins can be produced in serum-containing or serum-free medium. In some embodiments, recombinant C1-INH fusion proteins are produced in serum-free medium. In some embodiments, recombinant C1-INH fusion proteins are produced in animal-free medium, i.e., medium lacking animal-derived components. In some embodiments, recombinant C1-INH fusion proteins are produced in chemically defined medium. As used herein, the term "chemically defined nutrient medium" refers to a medium in which substantially all chemical components are known. In some embodiments, the chemically defined nutrient medium does not contain animal-derived components such as serum, serum-derived proteins (e.g., albumin or fetuin) and other components. In some cases, the chemically defined medium comprises one or more proteins (e.g., protein growth factors or cytokines.) In some cases, the chemically defined nutrient medium comprises one or more protein hydrolysates. In other cases, the chemically defined nutrient medium is a protein-free medium, i.e., a serum-free medium that does not contain proteins, hydrolysates, or components of unknown composition.

[0254] In some embodiments, the chemically defined medium may be supplemented with one or more animal-derived components. Such animal-derived components include, but are not limited to, fetal bovine serum, horse serum, goat serum, donkey serum, human serum, and serum-derived proteins such as albumin (e.g., bovine serum albumin or human serum albumin).

[0255] Various cell culture conditions can be used to produce recombinant C1-INH fusion proteins on a large scale, including but not limited to roller bottle culture, bioreactor batch culture, perfusion culture, and bioreactor fed-batch culture. In some embodiments, recombinant C1-INH fusion proteins are produced by cells in suspension culture. In some embodiments, recombinant C1-INH fusion proteins are produced by adherent cells. In some embodiments, perfusion culture is used to control the glycosylation of expressed proteins. Exemplary perfusion culture methods include but are not limited to those described in, for example, U.S. Patent No. 6,528,286 and PCT Publication No. WO1996 / 039488A1, the disclosure of which is incorporated herein by reference.

[0256] Exemplary cell culture media and culture conditions are described in the Examples section. The examples are not intended to be limiting.

[0257] Purification of expressed C1-INH fusion protein

[0258] Various methods can be used for purifying or separating the C1-INH fusion protein produced according to the various methods described herein. In some embodiments, the expressed C1-INH fusion protein is secreted into the culture medium, and therefore can be such as, for example, by centrifugation or filtration to remove cells and other solids, as the first step in the purification process. Alternatively or in addition, the expressed C1-INH fusion protein is bound to the surface of the host cell. In this embodiment, the host cell expressing the polypeptide or protein is dissolved for purification. Dissolving mammalian host cells can be achieved by many means known to those of ordinary skill in the art, including physical destruction with glass beads and exposure to high pH conditions.

[0259] The C1-INH fusion protein can be isolated and purified by standard methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or solubility differences, ethanol precipitation, or by any other available technique for purifying proteins (see, e.g., Scopes, Protein Purification Principles and Practice 2nd ed., Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, MP, Simon, MI, Abelson, JN (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol. 182), Academic Press, 1997, all of which are incorporated herein by reference). In particular, for immunoaffinity chromatography, it can be separated by binding the protein to an affinity column, which comprises antibodies produced for the protein and fixed to a fixed carrier. Alternatively, affinity tags such as influenza capsid sequences, polyhistidine or glutathione-S-transferase can be attached to the protein by standard recombinant techniques to allow simple purification to be achieved by passing through an appropriate affinity column. Protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), leupeptin, peptidomimetics or aprotinin can be added at any or all stages to reduce or eliminate the degradation of polypeptides or proteins during the purification process. When cells must be dissolved to separate and purify expressed polypeptides or proteins, protease inhibitors are particularly desired.

[0260] Protein A HP purification of IgG1 LALA Fc full length C1-inhibitor is shown in Figure 3 In some embodiments, the purification step does not involve a low pH step. In some embodiments, the fusion protein is mutated to stabilize the protein at low pH. In other embodiments, additives are used to protect the C1-INH fusion protein from aggregation during the low pH elution step. In other embodiments, non-protein A resins are used to purify the protein.

[0261] Exemplary purification methods that avoid the problems associated with protein A purification of C1-INH Fc fusion proteins are described in the following Examples section. Resins suitable for purification include, but are not limited to, anion exchange resins, albumin affinity resins, C1 inhibitor affinity resins, and protein A resins. In some embodiments, purification methods that do not require a pH drop are preferred. In some embodiments, purification methods that do not require an acidic pH for elution are preferred. In other embodiments, stabilizers that prevent aggregation are utilized. In some embodiments, stabilizers that prevent aggregation are used in methods that require a pH drop. Non-limiting examples of suitable stabilizers include EDTA.

[0262] Methods suitable for purifying the C1-INH fusion proteins of the present invention include, but are not limited to, those described in, for example, U.S. Pat. Nos. 5,276,141; US7384754; 8,802,816; PCT Publication Nos. WO2012107572; and WO2013009526, the disclosures of which are incorporated herein by reference.

[0263] Pharmaceutical composition

[0264] The present invention further provides a pharmaceutical composition containing the recombinant C1-INH fusion protein described herein and a physiologically acceptable carrier or excipient. The carrier and the recombinant C1-INH fusion protein may be sterile. The formulation should be suitable for the mode of administration.

[0265] Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline solutions (e.g., NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (e.g., lactose, amylose or starch), sugars (e.g., mannitol, sucrose or other sugars), dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, aromatic oils, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like, and combinations thereof. If necessary, the pharmaceutical preparations may be mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavoring agents, and / or aromatic substances, etc.) that do not adversely react with the active compounds or interfere with their activity. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.

[0266] If necessary, suitable pharmaceutical compositions or medicaments may also contain a small amount of wetting agents or emulsifiers or pH buffers. The composition may be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder. The composition may also be formulated into a suppository with traditional adhesives and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0267] The pharmaceutical composition or medicament can be formulated into a pharmaceutical composition suitable for human administration according to conventional procedures. For example, in some embodiments, the composition for intravenous administration is generally a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic to relieve pain at the injection site. Usually, the composition is supplied to an airtight container such as an ampoule or a sachet indicating the amount of the active agent, either alone or mixed together in a unit dosage form, in the form of a dry lyophilized powder or anhydrous concentrate. When the composition is to be administered by infusion, it can be distributed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose / water. When the composition is administered by injection, an ampoule containing sterile water for injection or saline may be provided so that the composition can be mixed before administration.

[0268] The recombinant C1-INH fusion protein described herein can be formulated into a neutral form or a salt form. Pharmaceutically acceptable salts include salts formed with free amino groups, such as those obtained by hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; and salts formed with free carboxyl groups, such as those obtained by sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0269] A preferred formulation comprises 50 mM NaPO4 (pH 7.2), 50 mM sorbitol and 150 mM glycine. The formulation may be liquid, or may be lyophilized and reconstituted prior to administration.

[0270] Route of administration

[0271] The recombinant C1-INH fusion protein described herein (or a composition or medicament containing the recombinant C1-INH fusion protein described herein) is administered by any appropriate route. In some embodiments, the recombinant C1-INH fusion protein or a pharmaceutical composition containing it is administered systemically. Systemic administration may be intravenous, intradermal, intracranial, intrathecal, inhaled, transdermal (topical), intraocular, intramuscular, subcutaneous, intramuscular, oral and / or transmucosal administration. In some embodiments, the recombinant C1-INH fusion protein or a pharmaceutical composition containing it is administered subcutaneously. As used herein, the term "subcutaneous tissue" is defined as a layer of loose, irregular connective tissue just below the skin. For example, subcutaneous administration can be performed by injecting the composition into an area including, but not limited to, the thigh area, the abdominal area, the buttocks area, or the scapular area. In some embodiments, the recombinant C1-INH fusion protein or a pharmaceutical composition containing it is administered intravenously. In some embodiments, the recombinant C1-INH fusion protein or a pharmaceutical composition containing it is administered orally. In some embodiments, the recombinant C1-INH fusion protein or a pharmaceutical composition containing the same is administered intracranially. In some embodiments, the recombinant C1-INH fusion protein or a pharmaceutical composition containing the same is administered intrathecally. If necessary, more than one route can be used in parallel.

[0272] In some embodiments, the recombinant C1-INH fusion protein or a pharmaceutical composition containing it is administered to a subject by intrathecal administration. As used herein, the term "intrathecal administration" or "intrathecal injection" refers to injection in the spinal canal (the intrathecal space around the spinal cord). Various techniques can be used, including but not limited to lateral ventricular injection by drilling or cistern or lumbar puncture. In some embodiments, "intrathecal administration" or "intrathecal delivery" according to the present invention refers to intrathecal administration or delivery through the lumbar area or region, i.e., lumbar intrathecal administration or delivery. As used herein, the term "lumbar region" or "lumbar region" refers to the region between the third and fourth lumbar (lower back) vertebrae, and more inclusively, refers to the L2-S1 region of the spine.

[0273] In some embodiments, the recombinant C1-INH fusion protein or a pharmaceutical composition containing the same is administered to a subject by subcutaneous (i.e., under the skin) administration. For such purposes, the formulation may be injected using a syringe. However, other devices for administering the formulation are available, such as an injection device (e.g., Inject-ease TM and Genject TM devices); injection pens (such as the GenPen TM ); needle-free devices (e.g. MediJector TM and BioJector TM ); and subcutaneous patch delivery systems.

[0274] In some embodiments, intrathecal administration can be used in combination with other routes of administration, such as intravenous, subcutaneous, intramuscular, parenteral, transdermal, or transmucosal (eg, oral or nasal).

[0275] The present invention encompasses single as well as multiple administrations of a therapeutically effective amount of a recombinant C1-INH fusion protein as described herein or a pharmaceutical composition containing the same. Depending on the nature, severity, and extent of the subject's condition (e.g., lysosomal storage disease), the recombinant C1-INH fusion protein or a pharmaceutical composition containing the same may be administered at regular intervals. In some embodiments, a therapeutically effective amount of a recombinant C1-INH fusion protein or a pharmaceutical composition containing the same may be administered at regular intervals (e.g., once a year, once every six months, once every five months, once every three months, every two months (once every two months), monthly (once every month), every two weeks (once every two weeks), weekly, daily, or continuously).

[0276] In some embodiments, administration produces only local effects in individuals, while in other embodiments, administration produces effects throughout multiple parts of individuals, such as systemic effects. Typically, administration results in delivery of recombinant C1-INH fusion protein to one or more target tissues. In some embodiments, delivery of recombinant C1-INH fusion protein to one or more target tissues, the tissues include but are not limited to heart, brain, skin, blood, spinal cord, striated muscle (e.g., skeletal muscle), smooth muscle, kidney, liver, lung and / or spleen. In some embodiments, delivery of recombinant C1-INH fusion protein to the heart. In some embodiments, delivery of recombinant C1-INH fusion protein to the central nervous system, particularly brain and / or spinal cord. In some embodiments, delivery of recombinant C1-INH fusion protein to triceps, tibialis anterior, soleus, gastrocnemius, biceps, trapezius, deltoid, quadriceps and / or diaphragm.

[0277] Dosage form and dosing regimen

[0278] In some embodiments, the composition is administered in a therapeutically effective amount and / or according to a dosing regimen associated with a particular desired outcome (eg, associated with preventing or treating a complement-mediated chronic disease such as HAE).

[0279] The specific dose or amount to be administered according to the present invention may vary, for example, depending on the nature and / or extent of the desired result, the details of the route of administration and / or timing, and / or one or more characteristics (e.g., weight, age, personal history, genetic characteristics, lifestyle parameters, severity of heart defect and / or risk level of heart defect, etc. or a combination thereof). The dose or amount may be determined by a person of ordinary skill. In some embodiments, the appropriate dose or amount is determined according to standard clinical techniques. Alternatively or additionally, in some embodiments, the appropriate dose or amount is determined by using one or more in vitro or in vivo assays to help determine the desired or optimal dose range or amount to be administered.

[0280] In various embodiments, the recombinant C1-INH fusion protein is administered in a therapeutically effective amount. Typically, the therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., prevention, treatment, regulation, cure, prevention and / or improvement of a latent disease or condition). Typically, the amount of therapeutic agent (e.g., recombinant C1-INH fusion protein) administered to a subject in need thereof will depend on the characteristics of the subject. The characteristics include the subject's condition, severity of disease, overall health, age, sex, and body weight. One of ordinary skill in the art will be readily able to determine the appropriate dosage depending on these and other relevant factors. In addition, both objective and subjective determinations may be optionally used to determine the optimal dosage range. In some specific embodiments, the appropriate dosage or amount to be administered may be extrapolated based on a dose-response curve obtained by an in vitro or animal model test system.

[0281] In some embodiments, a composition is provided in the form of a pharmaceutical formulation. In some embodiments, the pharmaceutical formulation is or comprises a unit dose for administration according to a dosing regimen associated with achieving a reduction in the incidence or risk of HAE attacks.

[0282] In some embodiments, a preparation comprising a recombinant C1-INH fusion protein as described herein is administered in a single dose form. In some embodiments, a preparation comprising a recombinant C1-INH fusion protein as described herein is administered at regular intervals. As used herein, administration at a certain "interval" indicates that a therapeutically effective amount (different from a one-time administration) is administered regularly. Intervals can be determined by standard clinical techniques. In some embodiments, a preparation comprising a recombinant C1-INH fusion protein as described herein is administered every two months, every month, twice a month, every three weeks, every two weeks, every week, twice a week, three times a week, every day, twice a day, or every six hours. Depending on individual needs, the administration interval for a single individual need not be a fixed interval, but may change over time.

[0283] The therapeutically effective amount is usually administered in a dosing regimen that can include multiple unit doses. For any particular therapeutic protein, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration, the combination with other pharmaceutical preparations. In addition, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend on a variety of factors, including the severity of the condition being treated and the condition; the activity of the specific pharmaceutical preparation used; the specific composition used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and / or the excretion or metabolic rate of the specific fusion protein used; the duration of treatment; and similar factors as are well known in the medical field.

[0284] As used herein, the term "every two months" means administration once every two months (i.e., once every two months); the term "monthly" means administration once every month; the term "every three weeks" means administration once every three weeks (i.e., once every three weeks); the term "every two weeks" means administration once every two weeks (i.e., once every two weeks); the term "weekly" means administration once a week; and the term "daily" means administration once a day.

[0285] In some embodiments, a formulation comprising a recombinant C1-INH fusion protein described herein is administered at regular intervals indefinitely. In some embodiments, a formulation comprising a recombinant C1-INH fusion protein described herein is administered at regular intervals for a determined period of time.

[0286] It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the enzyme replacement therapy, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed invention.

[0287] Combination therapy

[0288] In some embodiments, the recombinant C1-INH fusion protein is administered in combination with one or more known therapeutic agents (e.g., corticosteroids) currently used to treat complement-mediated diseases. In some embodiments, known therapeutic agents are administered according to its standard or approved dosing regimen and / or time course. In some embodiments, known therapeutic agents are administered according to a scheme that is changed compared to its standard or approved dosing regimen and / or time course. In some embodiments, the scheme of this change is different from the standard or approved dosing regimen in the following aspects: the amount of one or more unit doses is changed (e.g., reduced or increased), and / or the frequency of administration is changed (e.g., one or more intervals between unit doses are enlarged, resulting in a lower frequency, or the interval is reduced, resulting in a higher frequency).

[0289] A. Symptoms

[0290] A preferred embodiment is the treatment of chronic conditions.

[0291] In some embodiments, the fusion protein provided by the present invention is suitable for acute attacks associated with complement-mediated disorders, such as NMOSD AMR and HAE events. These attacks can be long-term or short-term. In some embodiments, the disease or disorder is chronic. In some embodiments, the compositions and methods of the present invention are used in a preventive and therapeutic manner. Exemplary complement-mediated diseases that can be treated using the compositions and methods disclosed herein include, but are not limited to, hereditary angioedema, antibody-mediated rejection, neuromyelitis optica spectrum disorders, traumatic brain injury, spinal cord injury, ischemic brain injury, burn injury, toxic epidermal necrolysis, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, stroke, chronic inflammatory demyelinating polyneuropathy (CIDP), myasthenia gravis, multifocal motor neuropathy.

[0292] The present invention provides the following implementation modes:

[0293] Embodiment 1. A fusion protein comprising:

[0294] Human C1-inhibitor polypeptide; and

[0295] Fc domain.

[0296] Embodiment 2. A fusion protein as described in embodiment 1, wherein the human C1-inhibitor polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to the full-length human C1-inhibitor having SEQ ID NO:1.

[0297] Embodiment 3. The fusion protein of embodiment 1, wherein the human C1-inhibitor polypeptide comprises SEQ ID NO: 1.

[0298] Embodiment 4. The fusion protein of embodiment 1, wherein the human C1-inhibitor polypeptide is truncated compared to SEQ ID NO: 1.

[0299] Embodiment 5. The fusion protein of embodiment 4, wherein the human C1-inhibitor polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:2.

[0300] Embodiment 6. The fusion protein of embodiment 4, wherein the human C1-inhibitor polypeptide comprises the amino acid sequence of SEQ ID NO:2.

[0301] Embodiment 7. The fusion protein of any of the preceding embodiments, wherein the Fc domain is linked to the N-terminus of the human C1-inhibitor polypeptide.

[0302] Embodiment 8. The fusion protein of any of the preceding embodiments, wherein the Fc domain is a human IgG1 Fc domain.

[0303] Embodiment 9. A fusion protein as described in embodiment 8, wherein the Fc domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:3.

[0304] Embodiment 10. A fusion protein as described in any of the preceding embodiments, wherein the Fc domain comprises L234A and / or L235A mutations.

[0305] Embodiment 11. The fusion protein of embodiment 10, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:4.

[0306] Embodiment 12. A fusion protein as described in any of the preceding embodiments, wherein the Fc domain comprises one or more mutations that extend the half-life of the fusion protein.

[0307] Embodiment 13. A fusion protein as described in embodiment 12, wherein the one or more mutations are selected from one or more positions corresponding to Thr250, Met 252, Ser 254, Thr256, Thr 307, Glu 380, Met428, His433 and / or Asn 434 of human IgG1.

[0308] Embodiment 14. A fusion protein as described in embodiment 12, wherein the one or more mutations are selected from H433K and / or N434F.

[0309] Embodiment 15. A fusion protein as described in any of embodiments 12-14, wherein the Fc domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOs: 5-8.

[0310] Embodiment 16. The fusion protein of embodiment 14, wherein the Fc domain comprises the amino acid sequence of any one of SEQ ID NOs: 5-8.

[0311] Embodiment 17. A fusion protein as described in any of embodiments 1-7, wherein the Fc domain is derived from human IgG4 Fc.

[0312] Embodiment 18. A fusion protein as described in embodiment 17, wherein the Fc domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:9.

[0313] Embodiment 19. A fusion protein as described in embodiment 18, wherein the Fc domain comprises an S241P mutation.

[0314] Embodiment 20. The fusion protein of embodiment 19, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:10.

[0315] Embodiment 21. A fusion protein as described in embodiment 1, which comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:11.

[0316] Embodiment 22. A fusion protein as described in embodiment 1, which comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:12.

[0317] Embodiment 23. A fusion protein as described in embodiment 1, which comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:13.

[0318] Embodiment 24. The fusion protein of embodiment 1, comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:14.

[0319] Embodiment 25. The fusion protein of embodiment 1, comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:15.

[0320] Embodiment 26. The fusion protein of embodiment 1, comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:16.

[0321] Embodiment 27. The fusion protein of embodiment 1, comprising an amino acid sequence selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.

[0322] Embodiment 28. A fusion protein as described in any of the preceding embodiments, wherein the Fc domain comprises a mutation that reduces or eliminates ADCC activity.

[0323] Embodiment 29. A fusion protein as described in any of the preceding embodiments, wherein the Fc domain comprises a mutation that reduces or eliminates CDC activity.

[0324] Embodiment 30. The fusion protein of any of the preceding embodiments, wherein the Fc domain comprises a mutation that reduces or eliminates FcγR binding.

[0325] Embodiment 31. The fusion protein of any of the preceding embodiments, wherein the Fc domain comprises a mutation that reduces or eliminates FcγR effector function.

[0326] Embodiment 32. The fusion protein of any of the preceding embodiments, wherein the recombinant human C1-inhibitor Fc fusion protein comprises a mutation that reduces or eliminates C1q binding.

[0327] Embodiment 33. A fusion protein as described in embodiment 32, wherein the mutation that reduces or eliminates C1q binding is in the Fc domain.

[0328] Embodiment 34. The fusion protein of any of the preceding embodiments, further comprising a linker between the human C1-inhibitor polypeptide and the Fc domain.

[0329] Embodiment 35. A fusion protein as described in embodiment 34, wherein the linker is a peptide comprising 3-100 amino acids.

[0330] Embodiment 36. The fusion protein of any of the preceding embodiments, wherein the fusion protein inhibits and / or inactivates C1r and / or C1s protease activity.

[0331] Embodiment 37. The fusion protein of any of the preceding embodiments, wherein the fusion protein inhibits lysis of red blood cells in vitro.

[0332] Embodiment 38. The fusion protein of any of the preceding embodiments, wherein the fusion protein has a longer half-life than plasma-derived human C1-inhibitor.

[0333] Embodiment 39. A fusion protein as described in any of the preceding embodiments, wherein the fusion protein has a half-life of at least 4 days.

[0334] Embodiment 40. The fusion protein of any of the preceding embodiments, wherein the fusion protein has a half-life of at least 5 days.

[0335] Embodiment 41. A fusion protein as described in any of the preceding embodiments, wherein the fusion protein has a half-life of at least 6 days.

[0336] Embodiment 42. A fusion protein as described in any of the preceding embodiments, wherein the fusion protein has a half-life of at least 7 days.

[0337] Embodiment 43. A fusion protein as described in any of the preceding embodiments, wherein the fusion protein is monovalent.

[0338] Embodiment 44. A fusion protein as described in any of embodiments 1-42, wherein the fusion protein is dimeric.

[0339] Embodiment 45. A fusion protein comprising:

[0340] Human C1-inhibitor polypeptide; and

[0341] Albumin polypeptide.

[0342] Embodiment 46. A fusion protein as described in embodiment 45, wherein the human C1-inhibitor polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to the human full-length C1-inhibitor having SEQ ID NO:1.

[0343] Embodiment 47. The fusion protein of embodiment 46, wherein the human C1-inhibitor polypeptide comprises SEQ ID NO:1.

[0344] Embodiment 48. The fusion protein of any one of embodiments 45-47, wherein the human C1-inhibitor polypeptide is truncated compared to SEQ ID NO: 1.

[0345] Embodiment 49. The fusion protein of embodiment 48, wherein the human C1-inhibitor polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:2.

[0346] Embodiment 50. The fusion protein of embodiment 49, wherein the human C1-inhibitor polypeptide comprises the amino acid sequence of SEQ ID NO:2.

[0347] Embodiment 51. A fusion protein as described in any of embodiments 45-50, wherein the albumin is linked to the N-terminus of the human C1-inhibitor polypeptide.

[0348] Embodiment 52. A fusion protein as described in any of embodiments 45-51, wherein the albumin polypeptide comprises one or more domains of human serum albumin.

[0349] Embodiment 53. A fusion protein as described in embodiment 52, wherein the albumin polypeptide comprises the D3 domain of human serum albumin.

[0350] Embodiment 54. A fusion protein as described in embodiment 53, wherein the albumin polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:20.

[0351] Embodiment 55. The fusion protein of embodiment 54, wherein the albumin polypeptide comprises the amino acid sequence of SEQ ID NO:20.

[0352] Embodiment 56. A fusion protein as described in any of embodiments 45-52, wherein the albumin polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:17.

[0353] Embodiment 57. The fusion protein of embodiment 56, wherein the albumin polypeptide comprises the amino acid sequence of SEQ ID NO:17.

[0354] Embodiment 58. A fusion protein as described in any of embodiments 45-57, which comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:18.

[0355] Embodiment 59. A fusion protein as described in any of embodiments 45-57, which comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:19.

[0356] Embodiment 60. A fusion protein as described in any of embodiments 45-57, which comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:21.

[0357] Embodiment 61. A fusion protein as described in any of embodiments 45-57, which comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% identical to SEQ ID NO:22.

[0358] Embodiment 62. A fusion protein as described in any one of embodiments 44-56, which comprises an amino acid sequence identical to SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:22.

[0359] Embodiment 63. A fusion protein as described in any of embodiments 45-62, wherein the fusion protein binds to FcRN.

[0360] Embodiment 64. The fusion protein of any one of embodiments 45-63, wherein the fusion protein inhibits C1 esterase activity.

[0361] Embodiment 65. A fusion protein as described in any of embodiments 45-64, wherein the fusion protein inhibits lysis of red blood cells in vitro.

[0362] Embodiment 66. The fusion protein of any one of embodiments 45-65, further comprising a linker between the human C1-inhibitor polypeptide and the albumin polypeptide.

[0363] Embodiment 67. A fusion protein as described in embodiment 66, wherein the linker is a peptide comprising 3-100 amino acids.

[0364] Embodiment 68. A fusion protein as described in embodiment 67, wherein the linker comprises the sequence GGG.

[0365] Embodiment 69. The fusion protein of embodiment 67, wherein the linker comprises the sequence of SEQ ID NO:27.

[0366] Embodiment 70. A fusion protein as described in any of embodiments 45-69, wherein the albumin polypeptide does not contain one or more mutations selected from the group consisting of 464His, 510His, 535His and combinations the...

Claims

1. A fusion protein comprising: Human C1-inhibitor polypeptide; and Fc domain, The fusion protein consists of the amino acid sequence of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:

16.

2. The fusion protein according to claim 1, in: (a) the fusion protein inhibits the activity of C1r and / or C1s protease and / or inactivates the activity of C1r and / or C1s protease; (b) the fusion protein inhibits lysis of red blood cells in vitro; (c) the fusion protein has a longer half-life compared to plasma-derived human C1-inhibitor; and / or (d) The fusion protein is monovalent or dimeric.

3. A nucleic acid encoding the fusion protein according to claim 1 or 2.

4. A cell comprising the nucleic acid of claim 3, wherein the cell is a mammalian cell.

5. The cell of claim 4, wherein the mammalian cell is a human cell or a Chinese Hamster Ovary (CHO) cell.

6. The cell of claim 4 or 5, wherein the cell is engineered to modify glycosylation.

7. The cell of claim 4 or 5, wherein the cell is engineered for improved sialylation.

8. A method for producing a fusion protein, comprising the step of culturing the cell according to any one of claims 4 to 7.

9. The fusion protein of claim 1 or 2, wherein the fusion protein is produced by cells engineered to improve sialylation.

10. A pharmaceutical composition comprising the fusion protein according to any one of claims 1, 2 or 9 and a pharmaceutically acceptable carrier.

11. Use of the fusion protein according to any one of claims 1, 2 or 9 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating a hemolytic disorder.

12. Use of the fusion protein according to any one of claims 1, 2 or 9 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating hereditary angioedema.

13. The method of claim 11 or 12, wherein: (a) the fusion protein inhibits the activity of C1r and / or C1s protease and / or inactivates the activity of C1r and / or C1s protease; (b) the fusion protein inhibits lysis of red blood cells in vitro; (c) the fusion protein has a longer half-life compared to plasma-derived human C1-inhibitor; and / or (d) The fusion protein is monovalent or dimeric.

14. The method of claim 11 or 12, wherein the drug is administered every two months, monthly, twice a month, every three weeks, every two weeks, weekly, twice a week, three times a week, daily, twice a day, or every six hours.

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