Compositions and methods for use in alpha-1-antitrypsin disorders

Abstract

Disclosed herein are compositions and methods that can be used to treat alpha-1-antitrypsin deficiency.

Description

Background Art

[0001] This application claims priority to 62 / 752,182, filed October 29, 2018, which is incorporated herein by reference.

[0002] Serpins (serine protease inhibitors) are a superfamily of proteins with similar structures that were first identified for their ability to inhibit proteases. The name serpin was originally coined because the first identified serine protease inhibitor was found to act on chymotrypsin-like serine proteases. Therefore, the acronym serpin was chosen to combine the first two letters of the words serine protease inhibitor.

[0003] Serine protease inhibitors are of interest due to their unusual mechanism of action, in which serine protease inhibitors irreversibly inhibit target proteases by undergoing large conformational changes to destroy the active site on the target protease. The inhibition of proteases by serine protease inhibitors controls a range of biological processes, including coagulation and inflammation. The mechanism of protease inhibition has certain advantages, but also has disadvantages. A major disadvantage is that serine protease inhibitors are particularly susceptible to mutations, which may result in serpinopathy such as protein misfolding and formation of inactive long-chain polymers.

[0004] Alpha-1-antitrypsin or alpha 1-antitrypsin ("A1AT", "A1A" or "AAT") is a protein belonging to the superfamily of serine protease inhibitors. It is encoded by the SERPINA1 gene in humans, which has 4 coding exons and 3 untranslated exons and is located at q31-31.2 on chromosome 14. It is also called alpha 1-proteinase inhibitor or alpha 1-antiproteinase because it not only inhibits trypsin, but also inhibits a variety of proteases. It is a 52kD glycoprotein with antiprotease function. It has a single chain of 394 amino acids, which is connected to a signal peptide with a length of 24 amino acids. As an enzyme inhibitor, it protects tissues from the enzymes of inflammatory cells, particularly neutrophil elastase and protease 3 ("PR3"), and the normal reference range in the blood is 1.5-3.5g / L. In acute inflammation, the concentration of alpha-1-antitrypsin can increase many times.

[0005] Neutrophil elastase is a serine protease that breaks down elastase, a rubbery macromolecule that provides elastic recoil to the lungs. When the blood contains insufficient amounts of alpha-1-antitrypsin or contains functionally defective alpha-1-antitrypsin, neutrophil elastase breaks down elastin too freely, thereby reducing the elasticity of the lungs. Reduced elasticity can lead to respiratory complications such as chronic obstructive pulmonary disease.

[0006] PR3 is a serine protease expressed primarily in neutrophils. Its exact role in neutrophil function is unclear, but in human neutrophils, PR3 contributes to the proteolytic generation of antimicrobial peptides.

[0007] Alpha-1-antitrypsin deficiency ("AATD") is a genetic disorder that causes defective production of alpha-1-antitrypsin. AATD is an autosomal recessive disorder caused by decreased serum levels of AATD. The disease results in a decrease in alpha-1-antitrypsin. The disease typically causes lung and liver disease and is known as hereditary emphysema. AATD is one of the most common fatal genetic disorders in Caucasians of European descent.

[0008] There are several forms and degrees of alpha-1-antitrypsin deficiency, and the forms and degrees depend on whether the patient has one or two copies of the defective allele. Severe alpha-1-antitrypsin deficiency can cause panacinar emphysema or COPD-like symptoms in adult life for many people with the condition, especially if they are exposed to cigarette smoke. The condition can also cause various liver diseases in children and adults, and sometimes even rarer problems. Alpha-1-antitrypsin deficiency usually causes some degree of disability and shortens life expectancy.

[0009] Alpha-1-antitrypsin deficiency is treated by avoiding the destruction of inhalants and by intravenous infusion of alpha-1-antitrypsin or by transplantation of the liver or lung. Recombinant alpha-1-antitrypsin is also known, but is currently used more in medical research than for treatment. The approved alpha-1-antitrypsin deficiency products are purified from human plasma and require frequent infusions with high doses (60-120 mg / kg) per week and have limited efficacy. Although high concentrations of plasma-derived AAT are used, peaks and troughs occur in circulating AAT levels due to poor pharmacokinetics, so alpha-1-antitrypsin enhancement therapy is not ideal.

[0010] Some current research focuses on the development of long-acting α-1-antitrypsin fusions through protein engineering. For example, AAT Fc was expressed and developed by two groups: Soohyun Kim (Konkuk University, Seoul, Korea); licensed to OmniBio ("Kim / OmniBio molecules") (see Lee S, Lee Y, Hong K, Hong J, Bae S, Choi J, Jhun H, Kwak A, Kim E, Jo S, Dinarello CA, Kim S. Effect of recombinant α1-antitrypsin Fc-fused (AAT-Fc) protein on the inhibition of inflammatory cytokine production and streptozotocin-induced diabetes. Mol Medicine Med. 2013 May 20;19:65-71. doi:10.2119 / molmed.2012.00308. PubMed PMID:23552726; PubMed Central PMCID:PMC3667213, which is incorporated herein by reference in its entirety, including any drawings), and InhibRx ("InhibRx molecule") (see, e.g., US 8,980,266 B2, Serpin Fusion Polypeptides and Methods and Use Thereof), which is incorporated herein by reference in its entirety, including any drawings. Both molecules are N-terminal AAT fusions to IgG1 Fc. The Kim / OmniBio molecule does not inhibit human neutrophil elastase as well as plasma-derived AAT, which may be due to low pH instability during elution from protein A chromatography during purification. The InhibRx molecule is reported to inhibit human neutrophil elastase, such as plasma-derived AAT.

[0011] Other groups have modified the glycosylation pattern of AAT by increasing the number of N-glycans (see, e.g., Chung HS, Kim JS, Lee SM, Park SJ. Additional N-glycosylation in the N-terminal region of recombinant human alpha-1 antitrypsin enhances the circulatory half-life in Sprague-Dawley rats. Glycoconj J. 2016 Apr;33(2):201-8. doi:10.1007 / s10719-016-9657-3. Epub 2016 Mar 7. PubMed PMID: 26947874, which is incorporated herein by reference in its entirety, including any drawings); or increasing polysialylation (see, e.g., Lindhout T, Iqbal U, Willis LM, Reid AN, Li J, Liu X, Moreno M, Wakarchuk WW. Site-specific enzymatic polysialylation of therapeutic proteins using bacterial enzymes. Proc Natl Acad Sci USA. 2011 May 3; 108(18):7397-402. doi: 10.1073 / pnas.1019266108. Epub 2011 Apr 18. PubMed PMID: 21502532; PubMed Central PMCID: PMC3088639, which are incorporated herein by reference in their entirety, including any drawings).Several groups have produced aglycosylated AAT (see, e.g., Cantin AM, Woods DE, Cloutier D, Héroux J, Dufour EK, Leduc R. Leukocyte elastase inhibition therapy in cystic fibrosis: role of glycosylation on the distribution of alpha-1-proteinase inhibitor in blood versus lung. J Aerosol Med. Summer 2002; 15(2): 141-8. PubMed PMID: 12184864, which is incorporated herein by reference in its entirety, including any drawings). Other groups have also developed PEGylated aglycosylated AAT to extend half-life (see, e.g., Cantin AM, Woods DE, Cloutier D, Dufour EK, Leduc R. Polyethylene glycol conjugation at Cys232 prolongs the half-life of alpha1 proteinase inhibitor. Am J Respir Cell Mol Biol. 2002 Dec; 27(6): 659-65. PubMed PMID: 12444025, which is incorporated herein by reference in its entirety, including any drawings).

[0012] AAT is prone to Met oxidation in the lung, and therefore several groups have constructed mutant AAT variants that are resistant to methionine oxidation but retain activity. Specifically, the M351V / M358V mutant is resistant to oxidation (see, e.g., Taggart C, Cervantes-Laurean D, Kim G, McElvaney NG, Wehr N, Moss J, Levine RL. Oxidation of either methionine 351 or methionine 358 in alpha 1-antitrypsin causes loss of anti-neutrophil elastase activity. J Biol Chem. 2000 Sep 1; 275(35): 27258-65. PubMed PMID: 10867014, which is incorporated herein by reference in its entirety, including any drawings). Baek et al. have introduced disulfide bonds (K168C-F189C) in AAT and significantly improved the resistance to loop-sheet polymerization (see Baek JH, Im H, Kang UB, Seong KM, Lee C, Kim J, Yu MH. Probing the local conformational change of alpha 1-antitrypsin. Protein Sci. 2007 Sep;16(9):1842-50. Epub 2007 Jul 27. PubMed PMID: 17660256; PubMed Central PMCID: PMC2206966m, which is incorporated herein by reference in its entirety, including any drawings).Single point mutations in AAT (F51L, G117F, K331F, K335A) were also created and showed increased stability and resistance to ring-sheet polymerization (see Dafforn TR, Mahadeva R, Elliott PR, Sivasothy P, Lomas DA. A kinetic mechanism for the polymerization of alpha 1-antitrypsin. J Biol Chem 1999 Apr 2;274(14):9548-55. PubMed PMID: 10092640; Parfrey H, Mahadeva R, Ravenhill NA, Zhou A, Dafforn TR, Foreman RC, Lomas DA. Targeting a surface cavity of alpha 1-antitrypsin to prevent conformational disease). J Biol Chem. 2003 Aug 29;278(35):33060-6. Epub 2003 Jun 13. PubMed PMID:12807889; Gilis D, McLennan HR, Dehouck Y, Cabrita LD, Rooman M, Bottomley SP. In vitro and in silico design of alpha1-antitrypsin mutants with different conformational stabilities. J Mol Biol. 2003 Jan 17;325(3):581-9. PubMed PMID:12498804; and Im H, Seo EJ, Yu MH. Metastability in the inhibitory mechanism of human alpha1-antitrypsin. alpha1-antitrypsin). Journal of Biol. Chem. 1999 Apr 16;274(16):11072-7. PubMed PMID:10196190, each of which is incorporated herein by reference, including any figures).

[0013] However, despite the above progress, there remains a need for additional treatment options. Summary of the invention

[0014] The invention described herein is based, in part, on a recombinantly engineered alpha-1 -antitrypsin serpin domain fused to a human serum albumin binding domain or a human serum albumin domain.

[0015] Some embodiments include a recombinant protein comprising an alpha-1-antitrypsin serine protease inhibitor domain and a human serum albumin binding domain or a human serum albumin domain. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has a sequence comprising all or part of SEQ ID NO: 1. In some embodiments, the recombinant protein comprises one or more linkers. In some embodiments, the C-terminus of the alpha-1-antitrypsin serine protease inhibitor domain in the recombinant protein is fused to the N-terminus of the human serum albumin or human serum albumin binding domain. Some embodiments include a recombinant protein comprising an alpha-1-antitrypsin serine protease inhibitor domain and a human serum albumin domain, wherein the alpha-1-antitrypsin serine protease inhibitor domain and the human serum albumin domain are not both wild-type alpha-1-antitrypsin and wild-type human serum albumin.

[0016] In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain retains activity compared to the wild-type alpha-1-antitrypsin serine protease inhibitor domain. In some embodiments, the one or more mutations cause resistance to methionine oxidation compared to wild-type alpha-1-antitrypsin. In some embodiments, the one or more mutations include M351V and / or M358V, wherein the residues are numbered according to SEQ ID NO: 1. In some embodiments, the one or more mutations include K168C and F189C, wherein the residues are numbered according to SEQ ID NO: 1. In some embodiments, the one or more mutations confer a substantial increase in resistance to ring-sheet polymerization compared to wild-type alpha-1-antitrypsin. In some embodiments, the one or more mutations are selected from one or more point mutations selected from the following: F51L, G117F, K331F or K335A, wherein the residues are numbered according to SEQ ID NO: 1. In some embodiments, the alpha-1 -antitrypsin serpin domain retains activity against human neutrophil elastase and / or PR3 compared to a wild-type alpha-1 -antitrypsin serpin domain.

[0017] In some embodiments, the one or more mutations include a mutation in at least one of the following residue positions according to SEQ ID NO 1: 51, 100, 114, 117, 163, 164, 165, 168, 169, 172, 173, 174, 183, 189, 232, 283, 300, 302, 303, 304, 306, 330, 331, 333, 335, 336, 337, 338, 339, 340, 351, 356, 358, or 361. ... 1 one or more of the following mutations: F51L, L100F, L100C, T114F, G117F, K163T, G164V, T165S, K168C, K168I, K168A, K169V, L172V, L172A, L172C, V173C, K174T, A183V, F189C, F189I, F189V, C232S, S283C, K300A, V302A, L303A, G 304A, L306A, S330R, K331F, K331I, K331V, K331T, K331C, V333C, V333A, K335A, K331F, K335G, K335T, A336G, V337C, V337A, L338A, T339S, I340V, M351V, I356M, M358I, M358P, M358A, M358R, M358L, M358V, or P361C. In some embodiments, the one or more mutations include F51L, G117F, K168C, F189C, C232S, S283C, K331F, K335A, M351V, M358V, M358L, or P361C according to SEQ ID NO: 1. In some embodiments, the one or more mutations are both K168C and F189C according to SEQ ID NO: 1. In some embodiments, the one or more mutations are both P361C and S283C according to SEQ ID NO: 1.

[0018] In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including M351V and M358V according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including M351V and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, M351V, and M358V according to SEQ ID NO: 1.

[0019] In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including F51L, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including G117F, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, K331F, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, K335A, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including K168C, F189C, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, S283C, M351V, M358L, and P361C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including F51L, G117F, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including F51L, C232S, K331F, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including F51L, C232S, K335A, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including K51L, K168C, F189C, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including F51L, C232S, S283C, M351V, M358L, and P361C according to SEQ ID NO: 1.In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including G117F, C232S, K331F, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including G117F, K335A, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including G117F, K168C, F189C, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including G117F, C232S, S283C, M351V, M358L, and P361C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, K331F, K335A, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including K168C, F189C, C232S, K331F, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, S283C, K331F, M351V, M358L, and P361C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including K168C, F189C, C232S, K335A, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, S283C, K335A, M351V, M358L, and P361C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including K168C, F189C, C232S, S283C, M351V, M358L, and P361C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain includes the point mutation K335A according to SEQ ID NO: 1.In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain comprises a point mutation K331F according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain comprises a point mutation G117F according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain comprises a point mutation S283C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain comprises a point mutation P361C according to SEQ ID NO: 1.

[0020] In some embodiments, the human serum albumin is at least a portion of SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, the human serum albumin binding domain is one of SEQ ID NO:22-36. In some embodiments, the human serum albumin binding domain comprises one or more CDRs selected from SEQ ID NO:6-21. In some embodiments, the human serum albumin or human serum albumin binding domain increases the plasma half-life of the alpha-1-antitrypsin serine protease inhibitor domain compared to wild-type alpha-1-antitrypsin. In some embodiments, the number of N-glycans or polysialylation of the recombinant protein in the alpha-1-antitrypsin serine protease inhibitor domain is greater than that of the wild-type alpha-1-antitrypsin serine protease inhibitor domain.

[0021] In some embodiments, the human serum albumin comprises one or more mutations. In some embodiments, the one or more mutations are one or more of residues 407, 408, 409, 410, 413, and 414, wherein the one or more residues are numbered according to SEQ ID NO: 5. In some embodiments, the one or more mutations are one or more of L407A, L408V, V409A, R410A, L413Q, and L414Q, wherein the one or more residues are numbered according to SEQ ID NO. 5. In some embodiments, the human serum albumin comprises a portion or variant of human serum albumin.

[0022] In some embodiments, the human serum albumin binding protein comprises an amino acid sequence capable of binding to all or a portion of human serum albumin having the sequence shown in SEQ ID NO:3 or SEQ ID NO:5.

[0023] In some embodiments, the recombinant protein comprises or consists of a protein having any one of SEQ ID Nos: 64-68 or SEQ ID NO: 71. In some embodiments, the recombinant protein comprises or consists of a protein having SEQ ID NO: 64. In some embodiments, the recombinant protein comprises or consists of a protein having SEQ ID NO: 65. In some embodiments, the recombinant protein comprises or consists of a protein having SEQ ID NO: 66. In some embodiments, the recombinant protein comprises or consists of a protein having SEQ ID NO: 67. In some embodiments, the recombinant protein comprises or consists of a protein having SEQ ID NO: 68. In some embodiments, the recombinant protein comprises or consists of a protein having SEQ ID NO: 71.

[0024] In some embodiments, a nucleic acid is provided, the nucleic acid encoding a recombinant protein as provided herein. In some embodiments, the nucleic acid comprises a vector. In some embodiments, the vector comprises a host cell.

[0025] Some embodiments relate to a method for preparing a recombinant protein, the method comprising culturing a host cell with one of the nucleic acids provided herein and collecting the recombinant protein. In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the host cell is a mammalian host cell. In some embodiments, the mammalian host is a CHO cell. In some embodiments, the recombinant protein is collected at a neutral pH using α-1-antitrypsin-Fc capture selection medium.

[0026] Some embodiments relate to a method for treating alpha-1-antitrypsin deficiency in a patient in need thereof, the method comprising administering a recombinant protein provided herein to the patient. In some embodiments, the recombinant protein comprises a pharmaceutical composition. In some embodiments, the recombinant protein is administered about once a week or at intervals longer than about once a week, about once every 10 days or at intervals longer than about once every ten days, about once every 15 days or at intervals longer than about once every 15 days, about once every 20 days or at intervals longer than about once every 20 days, about once every 25 days or at intervals longer than about once every 25 days, about once a month or at intervals longer than about once a month, or about once every two months or at intervals longer than about once every two months.

[0027] Some embodiments relate to a method of improving the pharmacokinetics of a protein comprising an alpha-1-antitrypsin serine protease inhibitor domain. In some embodiments, the protein is any of the recombinant proteins described herein. In some embodiments, improved pharmacokinetics means an improvement relative to Prolastin. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Depicted are the structures of alpha-1-antitrypsin protein and gene. The top shows the normal protein in linear form, which is a single-chain protein of 394 amino acids, wherein at residues 46, 83 and 247 according to SEQ ID NO:1 there are three complex carbohydrate side chains connected by asparagine groups. The bottom shows the gene (SEQ ID NO:69) and is composed of three non-coding exons (IA, IB and IC) and four coding exons (II-V). The alpha helix is ​​shown as having a box and the beta sheet is shown as having a wavy curve. The start codon is in exon II, followed by a signal peptide of 24 residues. The carbohydrate attachment site is shown as CHO in the figure.

[0029] Figure 2 Shown are the locations of mutations associated with the alpha-1 -antitrypsin gene, which is associated with alpha-1 -antitrypsin deficiency.

[0030] Figure 3 The alpha-1-antitrypsin secondary structure is shown.

[0031] Figure 4 depicts CE-SDS purification of constructs of the invention. Figure 4A CE-SDS of the purification of PP11288 is depicted. Figure 4B CE-SDS of the purification of PP11289 is depicted. Figure 4C CE-SDS of the purification of PP11290 is depicted. Figure 4D CE-SDS of the purification of PP11291 is depicted. Figure 4E CE-SDS of the purification of PP11292 is depicted. Figure 4F CE-SDS of the purified samples is depicted (reduced overlap). Figure 4G CE-SDS (non-reducing conditions) of the purified samples is depicted.

[0032] Figure 5 Stability analysis of five AAT-albumin fusions is depicted.

[0033] Figure 6 Depicted are the results of hNE inhibition assays of five AAT-albumin fusions.

[0034] Figure 7 depicts CE-SDS purification of constructs of the invention. Fig. 7A CE-SDS of the purification of PP13579 is depicted. Figure 7B CE-SDS of the purification of PP13580 is depicted. Figure 7C CE-SDS of the purification of PP13581 is depicted. Fig.7D CE-SDS of the purification of PP13582 is depicted. Fig. 7E CE-SDS of the purification of PP13583 is depicted. Figure 7F CE-SDS of the purification of PP13584 is depicted. Figure 7G CE-SDS of the purification of PP13585 is depicted. Figure 7H CE-SDS of the purification of PP13586 is depicted. Fig.7I CE-SDS of the purification of PP13587 is depicted. Figure 7J CE-SDS of the purification of PP13588 is depicted. Figure 7K CE-SDS of the purification of PP13589 is depicted. Figure 7L CE-SDS of the purification of PP13590 is depicted. Figure 7M CE-SDS of the purification of PP13592 is depicted. Figure 7N CE-SDS of the purification of PP13593 is depicted. Fig.7O CE-SDS of the purification of PP13594 is depicted. Figure 7P CE-SDS of the purification of PP13595 is depicted. Figure 7Q CE-SDS of the purification of PP13596 is depicted. Figure 7R CE-SDS of the purification of PP13597 is depicted. Figure 7S CE-SDS of the purification of PP13598 is depicted. Figure 7T CE-SDS of the purification of PP13599 is depicted. Figure 7U CE-SDS of the purification of PP13600 is depicted.

[0035] Figure 8 Stability analysis of 21 AAT-albumin fusions is depicted.

[0036] FIG. 9 depicts IC50 analysis of AAT mutants. Fig. 9A IC50 analysis of AAT mutants is depicted. Fig. 9B IC50 analysis of AAT mutants is depicted. Fig. 9C IC50 analysis of AAT mutants is depicted.

[0037] Fig.10EpiMatrix protein scores normalized and plotted on a standardized scale are depicted.

[0038] Fig.11 Stability analysis of AAT variants (K335A, S283C / P361C) of the base (C232S, M351V, M351L) is depicted.

[0039] Fig.12 Cynomolgus monkey PK data are depicted.

[0040] Fig.13 The PK of AAT-HSA and prolastin in monkeys after 10 mg / kg is depicted as 3 compartments. DETAILED DESCRIPTION

[0041] the term

[0042] As used herein, the term "about" shall refer to an approximate mathematical quantity and may be used relative to integer values ​​as well as percentages. When an upper or lower limit includes an integer value, the term "about" shall refer to the approximate boundary, and the term shall also include all values ​​above the boundary (relative to the lower boundary) or below the boundary (relative to the upper boundary). For example, a portion of a sequence of "about" 20 residues in a sequence of 393 residues shall include a boundary of approximately 20 residues and all integer values ​​above the value.

[0043] As used herein, "albumin" shall refer to an albumin protein or an albumin fragment, portion or variant having one or more functional activities of albumin, or to a nucleic acid sequence encoding an albumin protein, a fragment, portion or variant thereof having one or more functional activities of albumin. Albumin may be derived from any vertebrate, particularly any mammal. In some embodiments, albumin is derived from humans, cattle, sheep or pigs. Non-mammalian albumins include but are not limited to hens and salmon. In some embodiments, albumin derived from any vertebrate may replace human albumin or human serum albumin.

[0044] As used herein, the term "antibody" refers to an immunoglobulin molecule and is used in its broadest sense. Antibodies specifically include complete antibodies (e.g., complete immunoglobulins) and antibody fragments. Antibodies include at least one antigen binding domain. An example of an antigen binding domain is a V H -V L The antigen binding domain formed by the dimer. Other examples of antibodies are described in the following sections.

[0045] V H and V LThe V regions can be further subdivided into regions of high variability ("hypervariable regions (HVRs" as defined herein; also referred to as CDRs) intercalated with more conserved regions. The more conserved regions are called framework regions (FRs). Each V H and V L Typically includes three CDRs and four FRs arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. CDRs participate in antigen binding and confer antigen specificity and binding affinity to antibodies. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition (1991), Public Health Service, National Institutes of Health, Bethesda, Maryland, which is incorporated by reference in its entirety.

[0046] The light chains from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of the constant domains.

[0047] Heavy chains from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also referred to as α, δ, ε, γ, and μ, respectively. Based on differences in sequence and function, the IgG and IgA classes are further divided into subclasses. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0048] "Antibody fragments" include a portion of an intact antibody, such as the antigen binding region or variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0049] An "Fv" fragment comprises a non-covalently linked dimer of one heavy-chain and one light-chain variable domain.

[0050] In addition to the heavy and light chain variable domains, the "Fab" fragment also includes the constant domain of the light chain and the first constant domain of the heavy chain (C H1 ). Fab fragments can be produced, for example, by papain digestion of full-length antibodies.

[0051] "F(ab')2" fragments contain two Fab' fragments linked by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by pepsin digestion of intact antibodies. F(ab') fragments can be dissociated, for example, by treatment with β-mercaptoethanol.

[0052] "Single-chain Fv" or "sFv" or "scFv" antibody fragments comprise V and V in a single polypeptide chain connected by a peptide linker. H Domain and V L domain. See Plückthun A. (1994). A "scFv-Fc" fragment comprises a scFv linked to an Fc domain. For example, the Fc domain can be linked to the C-terminus of the scFv. Depending on the orientation of the variable domains in the scFv (i.e., V H -V L or V L -V H ), the Fc domain can follow the V H or V L Any suitable Fc domain known in the art or described herein may be used.

[0053] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from non-human antibodies. Humanized antibodies are typically human immunoglobulins (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody).

[0054] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody library or human antibody encoding sequence (e.g., obtained from a human source or designed de novo). Human antibodies specifically do not include humanized antibodies.

[0055] As used herein, "single domain antibody" shall refer to an antibody whose CDR is part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from conventional four-chain antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies can be derived from any species, including, but not limited to, mice, humans, camels, llamas, goats, rabbits, sharks, and cattle. As an example, single domain antibodies can be produced in camelid species such as camels, dromedaries, alpacas, and guanacos. In some embodiments, the single domain antibody comprises the sequence shown in any one of SEQ ID NOs: 22-28 and SEQ ID NOs: 29-36.

[0056] As used herein, a "complementarity determining region" or "CDR" is a portion of a variable chain in an antibody. The CDR is responsible for the specificity of an antibody. One skilled in the art will understand how to determine the location of a CDR within an antibody for a specific antibody, and there are several systems for determining the location of a CDR. For clarity, in this application, the CDRs of a single domain antibody shall be determined according to US2010 / 011339, which is incorporated herein by reference in its entirety.

[0057] The term "epitope" means a portion of an antigen that is capable of specific binding to a binding protein (such as, but not limited to, an antibody). An epitope is typically composed of surface accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural properties as well as specific charge properties. An epitope may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding. The epitope that binds to an antibody may be determined using known techniques for epitope determination.

[0058] As used herein, "human albumin" refers to a human albumin protein or a human albumin fragment or variant or portion having one or more functional activities of human albumin, or a nucleic acid sequence encoding a human albumin, a fragment, a portion or a variant thereof having one or more functional activities of human albumin. Human albumin is a polypeptide sequence having 609 amino acids, the first 18 amino acids of which constitute a leader sequence that is not found in the final blood-borne product. In some embodiments, human albumin is shown in SEQ ID NO: 3.

[0059] As used herein, "human serum albumin" shall refer to human serum albumin found in human blood, which is generally without the initial 18 amino acid leader sequence. In some embodiments, human serum albumin has the sequence shown in SEQ ID NO: 5. A human serum albumin domain shall also refer to any human serum albumin protein or human serum albumin fragment or variant or portion having one or more functional activities of human serum albumin.

[0060] As used herein, "human serum albumin binding protein" or "human albumin binding domain" shall refer to any protein or protein fragment or variant having one or more binding activities to human albumin or human serum albumin.

[0061] As used herein, "alpha-1-antitrypsin" or "alpha 1-antitrypsin" (also referred to as "A1AT", "A1A" or "AAT") shall refer to an alpha-1-antitrypsin protein or an alpha-1-antitrypsin fragment or variant having one or more functional activities of alpha-1-antitrypsin, or a nucleic acid sequence encoding an alpha-1-antitrypsin protein, a fragment or variant thereof having one or more functional activities of alpha-1-antitrypsin. In some embodiments, the alpha-1-antitrypsin protein has the sequence shown in SEQ ID NO: 1.

[0062] As used herein, "α-1-antitrypsin serine protease inhibitor domain" or "α1-antitrypsin serine protease inhibitor domain" (also referred to as "A1AT domain", "A1A domain" or "AAT domain") shall refer to an α-1-antitrypsin serine protease inhibitor domain or an α-1-antitrypsin serine protease inhibitor domain fragment or variant having one or more functional activities of an α-1-antitrypsin protein or fragment, or a nucleic acid sequence encoding an α-1-antitrypsin serine protease inhibitor domain or a fragment or variant thereof having one or more functional activities of an α-1-antitrypsin protein.

[0063] As used herein, "alpha-1-antitrypsin deficiency" or "AATD" is a genetic disorder that causes a defect in the production of alpha-1-antitrypsin. The disease causes a decrease in alpha-1-antitrypsin and is also known as hereditary emphysema, and causes lung and liver disease. The deficiency has several forms and degrees. The form and degree depend on whether the patient has one or two copies of the defective allele. Severe alpha-1-antitrypsin deficiency can cause pan-alveolar emphysema or COPD in the adult life of many people with the condition, especially if they are exposed to cigarette smoke. The condition can also cause a small number of children and adults to suffer from various liver diseases, and sometimes even rarer problems. Alpha-1-antitrypsin deficiency usually causes a certain degree of disability and shortens life expectancy.

[0064] An "effective amount" of a recombinant protein or a functional fragment or variant thereof as described herein refers to an amount of the polypeptide or a functional fragment or variant thereof that produces a measurable statistical improvement in outcome or prevention when administered in a total of multiple doses or as part of any other type of defined treatment regimen, as demonstrated by at least one clinical parameter associated with a complication.

[0065] As used herein, "neutrophil elastase" shall refer to a serine protease that destroys elastase, a rubbery macromolecule that provides elastic recoil to the lungs. When the amount of AAT contained in the blood is insufficient, neutrophil elastase breaks down elastin, thereby reducing the elasticity of the lungs, which can lead to respiratory complications such as chronic obstructive pulmonary disease.

[0066] As used herein, the term "portion" shall mean a portion of a whole or a segment of something such as a protein or polypeptide.

[0067] As used herein, "recombinant" in reference to a protein or polypeptide molecule refers to a protein or polypeptide molecule that is expressed using an isolated nucleic acid molecule or a recombinant nucleic acid molecule.

[0068] As used herein, the term "variant(s)" shall mean different sequences of a protein, including, for example, but not limited to, conservative or non-conservative insertions, deletions and substitutions, wherein such changes do not significantly alter one or more of the expandability, useful ligand binding and non-immunogenicity of the albumin or albumin binding domain or do not alter the serine protease inhibitor domain.

[0069] Variants of the polypeptides described herein include polypeptides having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of a human wild-type polypeptide provided herein. For example, variants of alpha-1-antitrypsin include polypeptides having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 1. For example, variants of human albumin include polypeptides having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5. The calculation of "identity" or "sequence homology" (the terms are used interchangeably herein) between two sequences is performed by alignment for the purpose of optimal comparison (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for the purpose of comparison). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at the position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percentage identity between two sequences is a function of the number of identical positions shared by the sequences. Variants include, but are not limited to, polypeptides that have been chemically modified and / or contain one or more amino acid sequence changes relative to human wild-type polypeptides.

[0070] Variants of the polypeptides described herein may be variants having amino acid modifications (e.g., deletions, additions or substitutions, such as conservative substitutions) from the wild-type amino acid sequence of the polypeptide. In some embodiments, variants of AAT may differ from alpha-1-antitrypsin (SEQ ID NO: 1) by at least 1, 2, 3, 4, 5 or more residues, and variants of human serum albumin may differ from human serum albumin (SEQ ID NO: 5) by at least 1, 2, 3, 4 or 5 residues.

[0071] "Conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Recombinant proteins with conservative substitutions are intended to be within the scope of the present invention.

[0072] Depending on the context, the term "fusion" or "fusion molecule" may refer to a polypeptide or nucleic acid fusion. It may comprise a full-length sequence or a protein or a nucleic acid or a fragment thereof. It may also comprise a recombinant protein with or without a linker molecule.

[0073] An "isolated" protein is one that is removed from at least about 90% of at least one component of a natural sample from which the isolated protein may be obtained. A protein may have a degree of purity of "at least about" if the purity of the species or population of species of interest is at least about 5%, 25%, 50%, 75%, 80%, 90%, 92%, 95%, 98%, or 99% on a weight-to-weight basis.

[0074] As used herein, the term "swelling" shall refer to swelling pressure or colloid osmotic pressure. It is a form of osmotic pressure exerted by proteins (especially albumin) in vascular plasma that draws water into the circulatory system. It is the force opposing hydrostatic pressure. It has a significant impact on the pressure across the glomerular filter.

[0075] A "patient," "subject," or "host" (these terms are used interchangeably) to be treated by the subject methods may refer to a human or non-human animal. Preferably, the patient, subject, or host refers to a human patient.

[0076] The term "prophylactically treating" a subject for a disease refers to subjecting the subject to a drug treatment, such as drug administration, such that at least one symptom of the disease is treated prophylactically, that is, administered prior to clinical manifestation of the undesirable condition, thereby protecting the host from the undesirable condition. "Prophylactically treating" a disease may also be referred to as "prevention." In some embodiments, prophylactic treatment prevents the condition.

[0077] As used herein, "treating" a subject in need thereof or "treating" a subject suffering from a disorder associated with alpha-1 -antitrypsin deficiency means subjecting the subject to drug therapy, e.g., drug administration, such that at least one symptom of the disease is cured, alleviated, or lessened.

[0078] Any treatment described herein can be administered in combination with another agent or therapy. The term "combination" refers to the use of two or more agents or therapies to treat the same patient, wherein the use or effect of the agents or therapies overlaps in time. The agents or therapies can be administered simultaneously (e.g., as a single formulation administered to a patient or as two separate formulations administered simultaneously) or sequentially in any order.

[0079] Recombinant protein

[0080] Some embodiments provide a recombinant protein comprising an alpha-1-antitrypsin serine protease inhibitor domain or a functional fragment or variant thereof and a human serum albumin domain or a functional fragment or variant thereof, or a human serum albumin binding domain or a functional fragment or variant thereof.

[0081] α-1- Antitrypsin serine protease inhibitor domain

[0082] In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain comprises full-length alpha-1-antitrypsin. In some embodiments, the alpha-1-antitrypsin is a protein comprising the sequence shown in SEQ ID NO:1.

[0083] In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain comprises one or more mutations. In some embodiments, the one or more mutations comprise a mutation in at least one of the following residue positions according to SEQ ID NO 1: 51, 100, 114, 117, 163, 164, 165, 168, 169, 172, 173, 174, 183, 189, 232, 283, 300, 302, 303, 304, 306, 330, 331, 333, 335, 336, 337, 338, 339, 340, 351, 356, 358, or 361 ... 1 one or more of the following mutations: F51L, L100F, L100C, T114F, G117F, K163T, G164V, T165S, K168C, K168I, K168A, K169V, L172V, L172A, L172C, V173C, K174T, A183V, F189C, F189I, F189V, C232S, S283C, K300A, V302A, L303A, G 304A, L306A, S330R, K331F, K331I, K331V, K331T, K331C, V333C, V333A, K335A, K331F, K335G, K335T, A336G, V337C, V337A, L338A, T339S, I340V, M351V, I356M, M358I, M358P, M358A, M358R, M358L, M358V, or P361C. In some embodiments, the one or more mutations include F51L, G117F, K168C, F189C, C232S, S283C, K331F, K335A, M351V, M358V, M358L, or P361C according to SEQ ID NO: 1. In some embodiments, the one or more mutations are both K168C and F189C according to SEQ ID NO: 1. In some embodiments, the one or more mutations are both P361C and S283C according to SEQ ID NO: 1.

[0084] In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including M351V and M358V according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including M351V and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, M351V, and M358V according to SEQ ID NO: 1.

[0085] In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including F51L, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including G117F, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, K331F, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, K335A, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including K168C, F189C, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, S283C, M351V, M358L, and P361C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including F51L, G117F, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including F51L, C232S, K331F, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including F51L, C232S, K335A, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including K51L, K168C, F189C, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including F51L, C232S, S283C, M351V, M358L, and P361C according to SEQ ID NO: 1.In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including G117F, C232S, K331F, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including G117F, K335A, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including G117F, K168C, F189C, C232S, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including G117F, C232S, S283C, M351V, M358L, and P361C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, K331F, K335A, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including K168C, F189C, C232S, K331F, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, S283C, K331F, M351V, M358L, and P361C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including K168C, F189C, C232S, K335A, M351V, and M358L according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including C232S, S283C, K335A, M351V, M358L, and P361C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain has one or more mutations including K168C, F189C, C232S, S283C, M351V, M358L, and P361C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain includes the point mutation K335A according to SEQ ID NO: 1.In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain comprises a point mutation K331F according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain comprises a point mutation G117F according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain comprises a point mutation S283C according to SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain comprises a point mutation P361C according to SEQ ID NO: 1.

[0086] In some embodiments, the glycan site in the alpha-1-antitrypsin serine protease inhibitor domain is truncated and deleted. In some embodiments, the first residue of SEQ ID NO: 1 is mutated such that the glycan site is deleted. In some embodiments, residues 1-46 of SEQ ID NO: 1 are deleted.

[0087] In some embodiments, the one or more mutations provide improved stability (including improved oxidative stability), improved retention of potency against human neutrophil elastase, improved resistance to aggregation, and improved ease of production, wherein the improved stability is measured relative to wild-type.

[0088] In some embodiments, the one or more mutations are one or more mutations listed in Table 1 and Tables 2-5, wherein the residue positions are listed according to SEQ ID NO:1.

[0089]

[0090] AAT mutant

[0091]

[0092] Table 1

[0093] In some embodiments, the alpha-1-antitrypsin serine protease inhibitor domain includes a portion of alpha-1-antitrypsin. In some embodiments, the portion of the alpha-1-antitrypsin serine protease inhibitor domain has an activity equal to or greater than that of full-length human alpha-1-antitrypsin (e.g., but not limited to, an alpha-1-antitrypsin serine protease inhibitor domain having a sequence as shown in SEQ ID NO: 1). In some embodiments, the portion is at least about 20 residues of alpha-1-antitrypsin, at least about 30 residues of alpha-1-antitrypsin, at least about 40 residues of alpha-1-antitrypsin, at least about 50 residues of alpha-1-antitrypsin, at least about 60 residues of alpha-1-antitrypsin, at least about 70 residues of alpha-1-antitrypsin, at least about 80 residues of alpha-1-antitrypsin, at least about 90 residues of alpha-1-antitrypsin, at least about 100 residues of alpha-1-antitrypsin, at least about 150 residues of alpha-1-antitrypsin, at least about 200 residues of alpha-1-antitrypsin, at least about 300 residues of alpha-1-antitrypsin, at least about 400 residues of alpha-1-antitrypsin, at least about 500 residues of alpha-1-antitrypsin, at least about 600 residues of alpha-1-antitrypsin, at least about 700 residues of alpha-1-antitrypsin, at least about 800 residues of alpha-1-antitrypsin, at least about 900 residues of alpha-1-antitrypsin, at least about 1000 residues of alpha-1-antitrypsin, at least about 150 at least about 110 residues of α-1-antitrypsin, at least about 120 residues of α-1-antitrypsin, at least about 130 residues of α-1-antitrypsin, at least about 140 residues of α-1-antitrypsin, at least about 150 residues of α-1-antitrypsin, at least about 160 residues of α-1-antitrypsin, at least about 170 residues of α-1-antitrypsin, at least about 180 residues of α-1-antitrypsin, at least about 190 residues of α-1-antitrypsin, at least about 200 residues of α-1-antitrypsin, residues, at least about 210 residues of alpha-1-antitrypsin, at least about 220 residues of alpha-1-antitrypsin, at least about 230 residues of alpha-1-antitrypsin, at least about 240 residues of alpha-1-antitrypsin, at least about 250 residues of alpha-1-antitrypsin, at least about 260 residues of alpha-1-antitrypsin, at least about 270 residues of alpha-1-antitrypsin, at least about 280 residues of alpha-1-antitrypsin, at least about 290 residues of alpha-1-antitrypsin ... at least about 300 residues of alpha-1-antitrypsin, at least about 310 residues of alpha-1-antitrypsin, at least about 320 residues of alpha-1-antitrypsin, at least about 330 residues of alpha-1-antitrypsin, at least about 340 residues of alpha-1-antitrypsin, at least about 350 residues of alpha-1-antitrypsin, at least about 360 residues of alpha-1-antitrypsin, at least about 370 residues of alpha-1-antitrypsin, at least about 380 residues of alpha-1-antitrypsin, or at least about 390 residues of alpha-1-antitrypsin.

[0094] In some embodiments, the portion is at least about 20 residues of the alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 30 residues of the alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 40 residues of the alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 50 residues of the alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 60 residues of the alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 70 residues of the alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 80 residues of the alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 90 residues of the alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 100 residues of the alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 150 residues of the alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, 1, at least about 110 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 120 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 130 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 140 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 150 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 160 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 170 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 180 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 190 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 110 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1 1, at least about 200 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 210 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 220 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 230 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 240 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 250 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 260 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 11, at least about 260 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 270 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 280 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 290 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 300 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 310 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 320 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 330 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 340 residues of an alpha-1-antitrypsin having the sequence set forth in SEQ ID NO: 1, at least about 350 residues of an alpha-1-antitrypsin having the sequence shown in SEQ ID NO:1, at least about 360 residues of an alpha-1-antitrypsin having the sequence shown in SEQ ID NO:1, at least about 370 residues of an alpha-1-antitrypsin having the sequence shown in SEQ ID NO:1, at least about 380 residues of an alpha-1-antitrypsin having the sequence shown in SEQ ID NO:1, or at least about 390 residues of an alpha-1-antitrypsin having the sequence shown in SEQ ID NO:1.

[0095] In some embodiments, the alpha-1-antitrypsin serpin domain comprises a variant of normal alpha-1-antitrypsin as shown in SEQ ID NO: 1. In some embodiments, the variant comprises an active portion of the alpha-1-antitrypsin serpin domain.

[0096] albumin

[0097] In some embodiments, the human serum albumin domain comprises full-length human albumin. In some embodiments, the human serum albumin domain comprises the sequence shown in SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, the human serum albumin domain has a sequence comprising SEQ ID NO:3 or SEQ NO:5.

[0098] In some embodiments, the human serum albumin domain comprises one or more mutations. In some embodiments, the one or more mutations comprise mutations in one or more of the following positions: 407, 408, 409, 410, 413, and 414, wherein the position numbering is for SEQ ID NO.5. In some embodiments, the one or more mutations comprise at least one of the following mutations: L407A, L408V, V409A, R410A, L413Q, and L414Q, wherein the position numbering is for SEQ ID NO.5 (see, e.g., International Publication No. WO95 / 23857, which is incorporated herein by reference in its entirety). In some embodiments, the one or more mutations comprise L407A, L408V, V409A, and R410A, wherein the position numbering is for SEQ ID NO.5. In some embodiments, the one or more mutations comprise R410A, L413Q, and L414Q, wherein the position numbering is for SEQ ID NO.5. In some embodiments, the one or more mutations improve the stability of the recombinant protein and / or increase the yield of the recombinant protein.

[0099] In some embodiments, the human serum albumin domain comprises a portion of human albumin.The portion is at least about 20 residues of human serum albumin, at least about 30 residues of human serum albumin, at least about 40 residues of human serum albumin, at least about 50 residues of human serum albumin, at least about 60 residues of human serum albumin, at least about 70 residues of human serum albumin, at least about 80 residues of human serum albumin, at least about 90 residues of human serum albumin, at least about 100 residues of human serum albumin, at least about 110 residues of human serum albumin, at least about 120 residues of human serum albumin, at least about 130 residues of human serum albumin, at least about 140 residues of human serum albumin, at least about 150 residues of human serum albumin, at least about 160 residues of human serum albumin, , at least about 170 residues of human serum albumin, at least about 180 residues of human serum albumin, at least about 190 residues of human serum albumin, at least about 200 residues of human serum albumin, at least about 210 residues of human serum albumin, at least about 220 residues of human serum albumin, at least about 230 residues of human serum albumin, at least about 240 residues of human serum albumin, at least about 250 residues of human serum albumin, at least about 260 residues of human serum albumin, at least about 270 residues of human serum albumin, at least about 280 residues of human serum albumin, at least about 290 residues of human serum albumin, at least about 300 residues of human serum albumin, at least about 310 residues of human serum albumin, at least about 320 residues of human serum albumin, at least about 330 residues of human serum albumin, at least about 340 residues of human serum albumin, at least about 350 residues of human serum albumin, at least about 360 residues of human serum albumin, at least about 370 residues of human serum albumin, at least about 380 residues of human serum albumin, at least about 390 residues of human serum albumin, at least about 400 residues of human serum albumin, at least about 410 residues of human serum albumin, at least about 420 residues of human serum albumin, at least about 430 residues of human serum albumin, at least about 440 residues of human serum albumin at least about 310 residues of human serum albumin, at least about 320 residues of human serum albumin, at least about 330 residues of human serum albumin, at least about 340 residues of human serum albumin, at least about 350 residues of human serum albumin, at least about 360 residues of human serum albumin, at least about 370 residues of human serum albumin, at least about 380 residues of human serum albumin, at least about 390 residues of human serum albumin, at least about 400 residues of human serum albumin, at least about 410 residues of human serum albumin, at least about 420 residues of human serum albumin, at least about 430 residues of human serum albumin, at least about 440 residues of human serum albumin, at least about 450 residues of human serum albumin, at least about 460 residues of human serum albumin, at least about 470 residues of human serum albumin, at least about 480 residues of human serum albumin, at least about 490 residues of human serum albumin, at least about 500 residues of human serum albumin, at least about 510 residues of human serum albumin, at least about 520 residues of human serum albumin, at least about 530 residues of human serum albumin, at least about 540 residues of human serum albumin at least about 460 residues of human serum albumin, at least about 470 residues of human serum albumin, at least about 480 residues of human serum albumin, at least about 490 residues of human serum albumin, at least about 500 residues of human serum albumin, at least about 510 residues of human serum albumin, at least about 520 residues of human serum albumin, at least about 530 residues of human serum albumin, at least about 540 residues of human serum albumin, at least about 550 residues of human serum albumin, at least about 560 residues of human serum albumin, at least about 570 residues of human serum albumin, at least about 580 residues of human serum albumin, at least about 590 residues of human serum albumin, or at least about 600 residues of human serum albumin.

[0100] In some embodiments, the portion is at least about 20 residues of human serum albumin having the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, at least about 30 residues of human serum albumin having the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, at least about 40 residues of human serum albumin having the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, at least about 50 residues of human serum albumin having the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, at least about 60 residues of human serum albumin having the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, at least about 70 residues of human serum albumin having the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, at least about 80 residues of human serum albumin having the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, at least about 90 residues of human serum albumin having the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, at least about 100 residues of human serum albumin having the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5, NO:5, at least about 100 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 110 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 120 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 130 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 140 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 150 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 160 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 170 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 180 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 190 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5 NO:3 or SEQ ID NO:5, at least about 190 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 200 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 210 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5,: at least about 220 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 230 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 240 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 250 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 260 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 270 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 280 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 290 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 300 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 310 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5 3 or SEQ ID NO:5, at least about 310 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 320 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 330 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 340 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 350 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 360 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 370 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 380 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 390 residues of human serum albumin having the sequence set forth in SEQ ID NO:5, at least about 400 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 410 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5,: at least about 420 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 430 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 440 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 450 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 460 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 470 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 480 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 490 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 500 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5, at least about 510 residues of human serum albumin having the sequence as shown in SEQ ID NO:3 or SEQ ID NO:5 NO:3 or SEQ ID NO:5, at least about 510 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 520 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 530 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 540 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 550 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 560 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 570 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 580 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 590 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5 At least about 590 residues of human serum albumin having the sequence shown in SEQ ID NO:5 or at least about 600 residues of human serum albumin having the sequence shown in SEQ ID NO:3 or SEQ ID NO:5.

[0101] In some embodiments, the portion of human serum albumin consists of or includes at least one of the amino acid sequences set forth in amino acid residues 1-194 of SEQ ID NO:5, amino acid residues 195-387 of SEQ ID NO:5, amino acid residues 388-585 of SEQ ID NO:5, 1-387 of SEQ ID NO:5, 195-585 of SEQ ID NO:5, or amino acid residues 1-194 of SEQ ID NO:5 and amino acid residues 388-585 of SEQ ID NO:5, or variants thereof. Each domain itself consists of two homologous subdomains (i.e., 1-105, 120-194, 195-291, 316-387, 388-491, and 512-585), with a flexible inter-subdomain linker region (see, e.g., EP 2 090589A1, which is incorporated herein by reference, including any drawings). In some embodiments, the portion consists of or includes at least one subdomain.

[0102] In some embodiments, the activity of the portion of the human serum albumin domain is equal to or greater than the activity of full-length human albumin. In some embodiments, the human serum albumin has a sequence including the sequence shown in SEQ ID NO:5.

[0103] In some embodiments, the human serum albumin domain comprises a variant of wild-type human serum albumin.

[0104] albumin binding protein

[0105] In some embodiments, the human serum albumin binding domain comprises an amino acid sequence capable of binding to human serum albumin. In some embodiments, human serum albumin has the sequence shown in SEQ ID NO:3 or SEQ ID NO:5. In some embodiments, human serum albumin comprises a portion of human serum albumin shown in SEQ ID NO:5. In some embodiments, the portion of human albumin comprises an epitope.

[0106] In some embodiments, the human serum albumin binding domain comprises an antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human immunoglobulin (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4); IgE (e.g., IgE1), IgA (e.g., IgA1, IgA2), IgM (e.g., IgM1), IgD (e.g., IgD1)) or a fragment, portion, or variant of a human immunoglobulin.

[0107] In some embodiments, the human serum albumin binding domain comprises a single domain antibody. In some embodiments, the single domain antibody comprises a CDR1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 6-9. In some embodiments, the single domain antibody comprises a CDR2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 10-16. In some embodiments, the single domain antibody comprises a CDR3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 17-21. In some embodiments, the single domain antibody comprises any one or more framework regions having a framework region comprising a sequence of SEQ ID NOs: 37-62.

[0108] In some embodiments, a single domain antibody comprises a combination of one or more binding domains. In some embodiments, a single domain antibody comprises a combination of one or more of CDR1, CDR2, and CDR3. In some embodiments, a single domain antibody comprises a CDR1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 6-9, and a CDR2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 10-16. In some embodiments, a single domain antibody comprises a CDR1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 6-9, and a CDR3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 17-21. In some embodiments, the single domain antibody comprises a CDR2 sequence comprising, consisting of, or consisting essentially of a sequence selected from the group consisting of SEQ ID NOs: 10-16, and a CDR3 sequence comprising, consisting of, or consisting essentially of a sequence selected from the group consisting of SEQ ID NOs: 17-21. In some embodiments, the single domain antibody comprises a CDR1 sequence comprising, consisting of, or consisting essentially of a sequence selected from the group consisting of SEQ ID NOs: 6-9, a CDR2 sequence comprising, consisting of, or consisting essentially of a sequence selected from the group consisting of SEQ ID NOs: 10-16, and a CDR3 sequence comprising, consisting of, or consisting essentially of a sequence selected from the group consisting of SEQ ID NOs: 17-21.

[0109] In some embodiments, the single domain antibody comprises a sequence selected from SEQ ID NOs: 22-28.

[0110] In some embodiments, the single domain antibody comprises a humanized sequence selected from SEQ ID NOs: 29-36. In some embodiments, the single domain antibody comprises a humanized sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 29. In some embodiments, the single domain antibody comprises a humanized sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 30. In some embodiments, the single domain antibody comprises a humanized sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 31. In some embodiments, the single domain antibody comprises a humanized sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 32. In some embodiments, the single domain antibody comprises a humanized sequence having SEQ ID NO: 33. In some embodiments, the single domain antibody comprises a humanized sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 34. In some embodiments, the single domain antibody comprises a humanized sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 35. In some embodiments, the single domain antibody comprises a humanized sequence comprising, consisting of, or consisting essentially of SEQ ID NO:36.

[0111] In some embodiments, the single domain antibody comprises a variant of a humanized sequence selected from SEQ ID NOs: 29-36. The variants described herein include polypeptides having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any sequence of the polypeptides provided herein. For example, a variant of a humanized single domain antibody will have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of any one of SEQ ID NOs: 29-36.

[0112] Nucleic Acids

[0113] Some embodiments include an isolated nucleic acid molecule encoding a recombinant protein, the isolated nucleic acid molecule comprising a nucleic acid encoding an alpha-1-antitrypsin serine protease inhibitor domain operably linked to a nucleotide sequence encoding human serum albumin or a human serum albumin binding domain. In some embodiments, the human serum albumin binding domain comprises an antibody. In some embodiments, the antibody comprises a single domain antibody.

[0114] In some embodiments, the disclosure features a nucleic acid molecule comprising a fragment of an alpha-1-antitrypsin serine protease inhibitor domain coding sequence and a fragment of a human serum albumin binding domain or a human serum albumin domain coding sequence. In some embodiments, the nucleotide sequence encodes a fragment or variant of alpha-1-antitrypsin of SEQ ID NO: 1 and / or a fragment of a human serum albumin binding domain or a human serum albumin domain coding sequence of any one of SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ NO: 22-36.

[0115] In some embodiments, the nucleic acid comprises a vector as described below.

[0116] Domains, domain order, and linkers

[0117] In some embodiments, the recombinant protein of the present invention has a human serum albumin domain or a human serum albumin binding domain and an α1-antitrypsin serum domain. However, in some embodiments, multiple domains of each protein can be used to prepare the recombinant protein of the present invention. Similarly, more than one α-1-antitrypsin serum domain can be used to prepare the recombinant protein of the present invention. For example, the α-1-antitrypsin serum domain can be fused to both the N-terminus and the C-terminus of the human serum albumin domain or the human serum albumin binding domain.

[0118] In some embodiments, the recombinant protein does not contain a linker between the fusion portions of the polypeptide (e.g., a portion of the alpha-1-antitrypsin serine protease inhibitor domain is fused to a portion of the human serum albumin domain and / or a human serum albumin binding domain). In some embodiments, the recombinant protein comprises a linker peptide between the fusion portions of the polypeptide. The linker can be a peptide linker. The linker can increase the expression yield of the recombinant protein. The linker can improve and / or enhance the biological activity of the alpha-1-antitrypsin serine protease inhibitor domain.

[0119] Those skilled in the art will appreciate that there are many linkers that can be used in the present invention (see, e.g., Chen et al., Fusion Protein Linkers: Property, Design, and Functionality, Adv Drug Deliv Rev. 2013 Oct 15; 65(10): 1357-1369 (2012), which is incorporated herein by reference in its entirety). In some embodiments, the linker is a peptide having a sequence comprising, consisting of, or consisting essentially of the sequence shown in SEQ ID NO: 63.

[0120] Composition

[0121] The present disclosure provides a pharmaceutical composition comprising any recombinant protein in the recombinant protein set forth herein. The pharmaceutical composition can take the form of any acceptable pharmaceutical formulation. The pharmaceutical composition can be formulated in a variety of different forms such as liquid, semisolid and solid dosage forms (such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories). The preferred form can depend on the intended mode of administration and therapeutic application.

[0122] Exemplary pharmaceutical compositions are described below. The pharmaceutical compositions include those suitable for parenteral (including intravenous, subcutaneous, intradermal, intramuscular and intraarticular), topical (including cutaneous, transdermal, transmucosal, buccal, sublingual and intraocular) and rectal administration, although the most appropriate route may depend, for example, on the condition and disorder of the recipient.

[0123] The pharmaceutical compositions described herein can be administered systemically, e.g., parenterally (e.g., intravenously, subcutaneously, intradermally, intramuscularly, and intraarticularly). The pharmaceutical compositions described herein can be administered locally, e.g., to an area affected by the condition or disorder to be treated by administering the pharmaceutical composition.

[0124] Compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that provide compositions that are isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The composition may be present in unit dose or multi-dose containers, such as sealed ampoules and vials, and may be stored under freeze drying (lyophilization) conditions that require only the addition of a sterile liquid carrier (e.g., saline or water for injection) immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Exemplary compositions for parenteral administration include injectable solutions or suspensions, which may contain, for example, suitable nontoxic, parenterally acceptable diluents or solvents (e.g., EDTA, mannitol, 1,3-butanediol, water, Ringer's solution, isotonic sodium chloride solution) or other suitable dispersants or wetting agents and suspending agents. The composition may contain pharmaceutically acceptable substances or adjuvants, including but not limited to EDTA (e.g. 0.5 mM EDTA); pH adjusters and buffers and / or tonicity adjusters (e.g. sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate), small amounts of non-toxic auxiliary substances (such as wetting agents or emulsifiers or preservatives).

[0125] treat

[0126] In some embodiments, the disclosure features a method for treating a condition associated with alpha-1 antitrypsin deficiency, the method comprising administering an effective amount of a recombinant protein or pharmaceutical composition provided herein to a patient or subject in need of a condition associated with alpha-1-antitrypsin deficiency. In some embodiments, the disclosure features a method for prophylactically treating a condition associated with alpha-1 antitrypsin deficiency, the method comprising administering an effective amount of a recombinant protein or pharmaceutical composition described herein to a patient or subject in need of a condition associated with alpha-1-antitrypsin deficiency. In some embodiments, the recombinant protein or pharmaceutical composition may be administered to a patient in need alone or in combination with another compound.

[0127] In some embodiments, the recombinant protein or pharmaceutical composition is administered about once a week or at longer intervals than about once a week, about once every 10 days or at longer intervals than about once every 10 days, about once every 15 days or at longer intervals than about once every 15 days, about once every 20 days or at longer intervals than about once every 20 days, about once every 25 days or at longer intervals than about once every 25 days, about once a month or at longer intervals than about once a month, about once every two months or at longer intervals than about once every two months, or about once every three months or at longer intervals than about once every three months.

[0128] In some embodiments, the subject has been diagnosed with alpha-1-antitrypsin deficiency or is at risk of being diagnosed with alpha-1-antitrypsin deficiency. In some embodiments, alpha-1-antitrypsin deficiency shows evidence of liver disease or evidence of lung disease. In some embodiments, alpha-1-antitrypsin deficiency occurs in people without obvious causes of liver disease or lung disease. In some embodiments, evidence of liver disease or evidence of lung disease is selected from at least one of the following groups consisting of emphysema, liver failure, hepatitis, hepatomology, jaundice, cirrhosis, nephrotic syndrome, autosomal recessive inheritance, COPD-like symptoms, dyspnea, elevated liver transaminases, and hepatocellular carcinoma.

[0129] In certain embodiments, alpha-1-antitrypsin deficiency is caused by deficiency mutation in SERPINA1 gene. More than 120 deficiency mutations have been identified in SERPINA1 gene, and the deficiency mutation can be used for diagnosis according to the present invention. Some deficiency mutations do not affect the production of alpha-1 antitrypsin, while other deficiency mutations cause deficiency or deficiency of the protein. In certain embodiments, the deficiency mutation in SERPINA1 gene causes the amino acid glutamic acid to be replaced with amino acid lysine at protein position 342 according to SEQ IDNO:1.

[0130] Abnormal alpha-1 antitrypsin proteins can bind together to form large molecules that cannot leave the liver. The accumulation of these polymers often leads to liver damage. In addition, lung tissue is destroyed because there is not enough alpha-1 antitrypsin available to protect against neutrophil elastase or PR3. Polymers of alpha-1 antitrypsin may also contribute to excessive inflammation, which may explain some of the other characteristics of alpha-1 antitrypsin deficiency, such as a skin condition called panniculitis. Other SERPINA1 deficiency mutations lead to the production of abnormally small forms of alpha-1 antitrypsin that are quickly broken down in the liver. As a result, there is almost no alpha-1 antitrypsin available in the lungs. Although the liver remains healthy in individuals with these deficiency mutations, the lungs are not protected from neutrophil elastase or PR3.

[0131] In some embodiments, the deficiency mutation is a loss-of-function mutation. In some embodiments, the deficiency mutation produces a stop codon. In some embodiments, the deficiency mutation results in an amino acid substitution. In some embodiments, the deficiency mutation is a point mutation, an insertion, a deletion and / or a substitution. In some embodiments, the deficiency mutation is a point mutation. In some embodiments, the deficiency mutation is a deletion. The deletion may involve only one or a few base pairs, multiple exons or the entire gene. In some embodiments, the deficiency mutation is an entire gene deletion. In some embodiments, the deficiency mutation is a partial gene deletion. In some embodiments, the deficiency mutation results in an intron change that affects splicing. In some embodiments, the deficiency mutation is a change in the 3' untranslated region of a gene. In some embodiments, the deficiency mutation is an overall chromosomal rearrangement. In some embodiments, the deficiency mutation results in a truncation of the SERPINA1 gene product.

[0132] In some embodiments, the expression of alpha-1-antitrypsin protein in the subject is abnormal compared to a reference standard. In some embodiments, the reference standard is the expression level of alpha-1-antitrypsin protein in a subject who does not suffer from a condition associated with alpha-1-antitrypsin deficiency. In some embodiments, the expression of alpha-1-antitrypsin protein in the subject is reduced or substantially absent compared to a reference standard. In some embodiments, the reference standard is the expression level of alpha-1-antitrypsin protein in a subject who does not suffer from a condition associated with alpha-1-antitrypsin deficiency.

[0133] In some embodiments, the alpha-1-antitrypsin serpin domain is a normal alpha-1-antitrypsin domain allele (see Crystal, RG The alpha-1-antitrypsin gene and its deficiency states. Trends Genet. 5:411-417, 1989. [PubMed:2696185], which is incorporated herein by reference in its entirety). In some embodiments, the allele is an allele with valine at position 213 according to SEQ ID NO: 1 (M1V, 107400.0002) and an allele with alanine at position 213 (M1A; 107400.0001) (see Nukiwa, T., Brantly, ML, Ogushi, F., Fells, GA, Crystal, RG Characterization of the gene and protein of the common alpha-1-antitrypsin normal M2 allele. Am. J. Hum. Genet. 43:322-330, 1988. [PubMed: 2901226], which is incorporated herein by reference in its entirety). In some embodiments, the allele is a defective allele or a null allele.

[0134] In some embodiments, the subject has been diagnosed with or is at risk of a condition associated with alpha-1-antitrypsin deficiency. In some embodiments, the subject has a mutation in a gene encoding an alpha-1-antitrypsin domain. In some embodiments, the mutation is a mutation in one of the exons of alpha-1-antitrypsin. In some embodiments, the mutation is a mutation in exon 1 of alpha-1-antitrypsin, the 5' coding region of exon 2 of alpha-1-antitrypsin, or the 3' portion of exon 5 of alpha-1-antitrypsin (see Long, GL, Chandra, T., Woo, SLC, Davie, EW, Kurachi, K. Complete sequence of the cDNA for human alpha-1-antitrypsin and the gene for the S variant. Biochemistry 23:4828-4837, 1984. [PubMed:6093867], which is incorporated herein by reference in its entirety).

[0135] In some embodiments, the recombinant protein or pharmaceutical composition is administered intravenously. In some embodiments, the recombinant protein or pharmaceutical composition is administered intradermally. In some embodiments, the recombinant protein or pharmaceutical composition is administered topically. In some embodiments, the recombinant protein or pharmaceutical composition is administered subcutaneously. In some embodiments, the recombinant protein or pharmaceutical composition is administered intrathecally. In some embodiments, the recombinant protein or pharmaceutical composition is administered intrathecally.

[0136] Vectors and host cells and polypeptide expression

[0137] In some embodiments, the nucleic acid is a vector. In some embodiments, the vector comprises a host cell. Some embodiments relate to a method for preparing a recombinant protein, comprising culturing a host cell with one of the nucleic acids provided herein and collecting the recombinant protein. In some embodiments, the recombinant protein is collected at a neutral pH using α-1-antitrypsin-Fc capture selection medium.

[0138] The recombinant proteins described herein can be produced as recombinant molecules, for example, by secretion from any suitable host cell. Many expression systems are known and can be used, including bacteria (e.g., E. coli and Bacillus subtilis), yeasts (e.g., Saccharomyces cerevisiae, Kluyveromyces lactis, and Pichia pastoris), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells.

[0139] In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is selected from the group consisting of: CHO cells, BHK cells, COS-7 cells, L cells, C127 cells and 3T3 cells. In some embodiments, the host cell is a CHO cell.

[0140] In some embodiments, the host cell is a yeast cell. Exemplary yeast genera that can be used as hosts described herein include, but are not limited to, Pichia, Saccharomyces, Kluyveromyces, Aspergillus, Candida, Tolopopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces, De Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, etc. Examples of Saccharomyces spp. are Saccharomyces cerevisiae, Saccharomyces italicus, and Saccharomyces rouxii. Yeast strains suitable for producing the fusion polypeptides described herein may include, but are not limited to, D88, DXY1, and BXP10.

[0141] In addition to the transformed host cells themselves, cultures of those cells in a nutrient medium are described herein, preferably monoclonal (clonally homogeneous) cultures or cultures derived from monoclonal cultures. If the recombinant protein is secreted, the culture medium will contain the recombinant protein with or without the cells if the cells have been filtered or centrifuged off.

[0142] The recombinant protein can be produced in a conventional manner, for example, by a coding sequence inserted into a host chromosome or on a free plasmid. By any method known in the art, such as electroporation, host cells are transformed with the coding sequence of the recombinant protein. The cells successfully transformed, i.e., cells containing a DNA construct as described herein, can be identified by techniques known to those skilled in the art. For example, cells produced by the introduction of an expression construct can be grown to produce a recombinant protein. Cells can be harvested and the cells can be lysed, and their DNA content can be checked using methods known in the art to determine whether DNA exists. The presence of a recombinant protein in the supernatant can be detected using any method known in the art (e.g., an antibody-based detection method).

[0143] Also disclosed herein are plasmid vectors comprising nucleic acids for expressing the recombinant proteins described herein. In order to express the recombinant proteins described herein, nucleotide sequences encoding suitable recombinant proteins or functional equivalents can be inserted into suitable vectors. Suitable vectors contain necessary and appropriate transcription and translation control sequences for expressing the inserted nucleic acid sequences. Standard methods known to those skilled in the art can be used to construct recombinant expression vectors containing the nucleic acid sequences described herein. These methods include, but are not limited to, in vitro recombinant techniques, synthetic techniques, and in vivo recombination / genetic recombination; the choice of method depends on the properties of the specific nucleotide fragments and can be determined by those skilled in the art.

[0144] Suitable vectors for use herein may contain replication origins and restriction endonuclease sequence sites. Those skilled in the art will know suitable replication origins and restriction endonuclease sequences for use in host cells. Suitable vectors for use herein may contain sequence elements that assist transcription, including but not limited to promoters and enhancer elements. Those skilled in the art will know various transcription control elements that are suitable for use in host cells, including but not limited to promoters, inducible promoters and enhancer elements.

[0145] Vectors suitable for use herein may also contain selectable marker genes that encode products necessary for the growth and survival of host cells under specific conditions, thereby facilitating the selection of host cells into which the vector has been introduced. Typical selection genes may include, but are not limited to, genes encoding proteins that confer resistance to antibiotics, drugs, or toxins (e.g., tetracycline, ampicillin, neomycin, hygromycin, etc.). Those skilled in the art will know the coding sequences of suitable selectable markers and reporter genes for use in host cells.

[0146] Expression vector as described herein can be introduced into host cell by conventional conversion or transfection technique.Conversion and transfection technique include but are not limited to calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofectamine, electroporation, microinjection and virus-mediated transfection (referring to U.S. Patent No. 6,632,637, which is incorporated herein by reference in its entirety). Those skilled in the art will know suitable conversion and transfection methods based on host cell / vector combination.In order to produce recombinant protein in high yield for a long time, the stable expression of recombinant protein can be preferred.The host cell of stably expressing recombinant protein can be engineered.Yeast plasmid vector can include but are not limited to pRS403-406, pRS413-416, pRS403, pRS404, pRS405, pRS406, pRS413-416, pPPC0005, pScCHSA, pScNHSA and pC4.

[0147] In another embodiment, the recombinant mammalian expression vector is capable of directing the polypeptide to be preferentially expressed in a specific cell type (e.g., a tissue-specific regulatory element is used to express the polypeptide). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), T cell promoters (Winoto and Baltimore (1989) EMBO Journal (EMBO Journal)). J.) 8:729-733) and immunoglobulin promoters (Banerji et al. (1983) Cell 33:729-740, and Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916) and mammary gland-specific promoters (e.g., whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also contemplated, for example, the murine homeobox protein (hox) promoter (Kessel and Gruss (1990) Science 249:374-379) and the alpha-fetoprotein promoter (Campes and Tilghman (1989) Genes & Development 3:537-546). Each of the above references, including any drawings, is incorporated by reference in its entirety.

[0148] The recombinant proteins described herein can be recovered and purified from recombinant cell cultures by well-known methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, hydrophobic charge interaction chromatography, and lectin chromatography. In some embodiments, high performance liquid chromatography ("HPLC") is used for purification. The polypeptides produced and recovered by the recombinant and molecular biology methods described herein can be purified according to standard protocols known in the art (e.g., dialysis, ion exchange chromatography, affinity chromatography, SDS gel electrophoresis, etc.). The polypeptides and / or fusion polypeptides described herein can be purified to homogeneity by ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, or gel filtration. Depending on the host used in the recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In some embodiments, the recombinant protein will be purified at neutral pH by capturing the selection medium with α-1-antitrypsin-Fc.

[0149] In some embodiments, nucleic acids encoding recombinant proteins described herein are optimized for expression in yeast. Exemplary yeasts that can be used as hosts for expressing fusion polypeptides described herein include, but are not limited to, Pichia, Saccharomyces, Kluyveromyces, Aspergillus, Candida, Torulopsis, Spore Torulina, Schizosaccharomyces, Solid Saccharomyces, Pachysaccharomyces, Zygosaccharomyces, Debarysaccharomyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Meiqi yeast, Red winter spore yeast, White winter spore yeast, Botryoascus, Lock Saccharomyces, Pseudomonas, etc. Yeast strains suitable for producing fusion polypeptides described herein may include, but are not limited to, D88, DXY1, and BXP10. Yeast plasmid vectors may include, but are not limited to, pRS403-406, pRS413-416, pRS403, pRS404, pRS405, pRS406, pRS413-416, pPPC0005, pScCHSA, pScNHSA, and pC4. Vectors for preparing albumin fusion proteins for expression in yeast include pPPC0005, pScCHSA, pScNHSA, and pC4:HSA.

[0150] In some embodiments, nucleic acids encoding recombinant proteins described herein are expressed in yeast. Preferred exemplary species of the genus Saccharomyces include Saccharomyces cerevisiae, Saccharomyces italicum, S. diastaticus, and Zygosaccharomyces rouxii. Preferred exemplary species of the genus Kluyveromyces include K. fragilis and K. lactis. Preferred exemplary species of the genus Hansenula include H. polymorpha (now Pichia angusta), H. anomala (now Pichia anomala), and Pichia capsulata.

[0151] Other organisms can be used for expression. Additional exemplary species of Pichia include P. pastoris. Exemplary species of Aspergillus include A. niger and A. nidulans. Preferred exemplary species of Yarrowia include Y. lipolytica. Many preferred yeast species can be obtained from (American Type Culture Collection).

[0152] Table S: Sequences.

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] Examples

[0172] Example 1: Construction of AAT-albumin

[0173] Wild-type alpha-1-antitrypsin (alpha-1-antitrypsin, SEQ ID: 1) and four variants (C232S, SEQ ID: 64; M351V, M358V, SEQ ID: 65; M351V, M358L, SEQ ID: 66; C232S, M351V, M358L, SEQ ID: 67) were fused to the N-terminus of human serum albumin via a long glycine-serine linker (GGSGGSGGSGGSGG (SEQ ID NO: 63)). The gene encoding human AAT can be PCR amplified from human liver cDNA (Zyagen). Specific point mutations within the AAT gene were generated by overlapping PCR (see, e.g., Higuchi R, Krummel B, Saiki R (1988) "A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions". Nucleic Acids Res. 16(15):7351-67, which is incorporated herein by reference in its entirety, including any drawings). The α-1-antitrypsin gene was cloned in frame with the gene encoding human serum albumin and a linker upstream of the α-1-antitrypsin gene insertion site into a mammalian expression vector containing a mammalian secretion signal sequence.

[0174] The initial AAT variant is selected because it is resistant to cysteine ​​oxidation and / or methionine oxidation. It is known that methionine oxidation can reduce the inhibitory activity of AAT to human neutrophil elastase. Once expressed in Chinese hamster ovary (CHO) cells, each AAT-human serum albumin (WT or variant, SEQ ID: 1-5) is purified using anti-AAT resin (GE), and its inhibition to human neutrophil elastase (hNE) is tested. The overall protein stability of each AAT-human serum albumin (WT or variant, SEQ ID: 1-5) is also tested using dynamic light scattering (DLS) and dynamic scanning fluorescence determination (DSF) instruments (UNcle, Unchained).

[0175] It is known that overall AAT protein stability is correlated with resistance to self-aggregation of AAT. Using this data, AAT-human serum albumin was selected for further mutagenesis with at least one additional mutation (see Table 1 above). These mutations are known to further improve AAT stability and prevent self-aggregation. Each AAT-human serum albumin variant was expressed in CHO, purified and tested for hNE inhibition and protein stability.

[0176] By combining multiple mutations together, an unexpected synergistic increase in stability was observed with little loss of hNE inhibition.

[0177] Each gene sequence is cloned into a proprietary high expression mammalian vector. Before DNA amplification, the sequence of each completed construct is confirmed. Each DNA expression construct is scaled up to an amount suitable for transfection. Plasmid DNA is run on an agarose gel for quality assessment and the sequence is confirmed before transfection.

[0178] Example 2: Construction of AAT-albumin binding domain

[0179] The preferred AAT domain is fused to a single domain antibody that binds to albumin instead of human serum albumin as described in Example 1. For example, a single domain antibody having any of the corresponding sequences SEQ ID NOs 22-36 is fused to AAT provided in Example 1. As described in Example 1, the AAT-albumin binding domain variants are expressed in CHO, purified and tested for hNE inhibition and protein stability.

[0180] Each gene sequence is cloned into a proprietary high expression mammalian vector. Before DNA amplification, the sequence of each completed construct is confirmed. Each DNA expression construct is scaled up to an amount suitable for transfection. Plasmid DNA is run on an agarose gel for quality assessment and the sequence is confirmed before transfection.

[0181] Example 3: CHO transient transfection

[0182] Suspension CHO cells are seeded in shake flasks, and the culture medium determined using serum-free chemical composition is increased. On the transfection day, the cells of amplification are inoculated in new flasks with fresh culture medium. Every kind of DNA construct is transiently transfected into CHO cells. Cells are maintained as batch feed cultures (C1603 and C1604, Medna), until production runs end.

[0183] The results can be seen in Tables 2 and 4.

[0184]

[0185] Table 2

[0186]

[0187]

[0188] Table 4

[0189] Example 4: CHO albumin affinity purification

[0190] Harvest the conditioned medium from the instantaneous production run, and make the conditioned medium clarified by centrifugation and filtration.The supernatant is loaded onto a post equipped with α-1 antitrypsin selection resin and pre-equilibrated with the PBS of pH 7.4.The post is washed with PBS to remove impurities until OD280 (measured by NanoDrop (Thermo Scientific)) is reduced to about zero.The target protein is eluted with high salt elution buffer (20mM Tris, 2M MgCl2, pH 7.4), fractions are collected, and OD280 is measured and recorded.The fractions containing the target protein are collected and dialyzed to finally buffer the PBS of pH 7.4.The final product is filtered by a membrane filter of 0.2 μm.Protein concentration and final yield are calculated according to the OD280 value, and the extinction coefficient after the buffer exchange is calculated.

[0191] CE-SDS analysis of target proteins was performed using LabChip GXII (Perkin Elmer).

[0192] The results can be seen in Figures 4 and 7.

[0193] Example 5: Stability Analysis

[0194] The samples were sent to the UNcle system (Unchained Labs) for analysis. For dynamic light scattering ("DSF") and static light scattering ("SLS"), a temperature slope of 1°C / min was performed with monitoring from 25°C to 95°C. UNcle measured SLS at 266nm and 473nm. DLS was measured at the beginning of the thermal slope. Tm, Tagg and DLS measurements were calculated and analyzed using UNcle analysis software. The determined Tm is represented by a solid dropline in the DSF graph.

[0195] The results can be found in Figure 5 and Figure 8 Seen in.

[0196] Example 6: hNA Assay

[0197] Neutrophil elastase inhibition assay was performed using a screening kit from Abcam (Cat. No. ab118971). Figure 6 Shown in.

[0198] Example 7: IC50

[0199] IC50 analysis of AAT mutants was also performed using a screening kit from Abcam (Cat. No. ab118971). 400 nM-98 pM was the concentration range used for IC50 analysis, and data obtained from duplicate experiments were analyzed using GraphPad Prism.

[0200] Table 3 shows the IC50s for the basal albumin fusions.

[0201]

[0202] Table 3

[0203] IC50 analysis curves were also generated using 21 AAT mutant proteins and 1 AAT serum control. Table 5 shows the IC50 of mutant AAT.

[0204]

[0205]

[0206] Table 5

[0207] Fig. 9 shows the IC50 analysis of AAT mutants, wherein the x-axis shows the logarithmic concentration (nM) of AAT protein, and the y-axis shows the inhibition percentage. The curve with higher slope shows a very collaborative inhibition spectrum. Mutants G11F / K335A, G117F / K331F, F51L / K331F and K331F / K335A all have a very collaborative inhibition spectrum.

[0208] Example 8: In silico immunogenicity analysis

[0209] The proteins were compared to proteins already in clinical use using a proprietary screen (see Jawa et al., T-cell dependent immunogenicity of protein therapeutics: Preclinical assessment and mitigation, 149 Clinical Immunology, 534-555 (2013), which is incorporated herein by reference in its entirety, including any figures). AAT-albumin fusions ("AAT_MLA"), AAT mutants ("AAT_mutants"), and albumin ("albumin") were screened for the presence of putative T-cell epitopes. The AAT mutants had the following mutations according to SEQ ID NO: 1: C232S, M351V, M358L, K335A, S283C, and P361C. The immunogenic potential of each T-cell epitope or AAT-albumin fusion, AAT mutant, and albumin was compared, normalized, and ranked with the others.

[0210] The input sequence is parsed into overlapping 9-mer frames, and each frame is evaluated relative to eight common class II HLA allele groups. The alleles are called supertypes. Each supertype is functionally equivalent to or nearly equivalent to many other family member alleles. The eight supertype alleles and their corresponding family members together cover more than 95% of the human population. Each frame* allele assessment (frame-by-allele assessment) is a statement about the predicted HLA binding affinity.

[0211] The evaluation scores range from about -3 to +3. Evaluation scores above 1.64 are defined as "hits" and are considered to be potentially immunogenic and worthy of further consideration. It is generally expected that about 5% of all evaluations will obtain scores above 1.64. These peptides have a great chance of binding to HLA molecules with moderate to high affinity, and therefore, have a great chance of appearing on the surface of APCs such as dendritic cells or macrophages, where the peptides may be interrogated by passing T cells. The greater the load of HLA ligands (i.e., hits) contained in a given protein, the greater the likelihood that the protein will induce an immune response.

[0212] The EpiMatrix protein score is the difference between the number of predicted T cell epitopes expected to be found in a protein of a given size and the number of putative epitopes predicted by the EpiMatrix system. EpiMatrix protein scores are "normalized" and can be plotted on a standardized scale (see, e.g., Fig.10 ). The "average" protein has an EpiMatrix protein score of zero. EpiMatrix protein scores above zero indicate the presence of excess MHC ligands and represent a higher immunogenic potential, while scores below zero indicate the presence of fewer potential MHC ligands than expected and a lower immunogenic potential.

[0213] When analyzing the complete AAT_MLA sequence, the algorithm performed a total of 7,880 framework * allele evaluations. When analyzing the AAT_mutant domain, the algorithm performed a total of 3,088 framework * allele evaluations. When analyzing the albumin domain, the algorithm performed a total of 4,616 framework * allele evaluations. The results are shown in Table 6. Compared with wild-type AAT, mutant AAT showed a slightly increased immunogenicity score. The fusion of high-scoring AAT mutant domains with low-scoring glycine-serine linkers and low-scoring albumin domains reduced the overall epitope density of the AAT_MLA fusion. When fused to albumin (AAT_MLA), AAT scored negative, indicating that the immunogenic potential was reduced compared to wild-type or mutant AAT alone.

[0214] Protein labeling length Evaluate EpiMatrix hit count EpiMatrix Scoring AAT MLA 993 7,880 384 -10.72 AAT mutant 394 3,088 206 26.87 AATWT 394 3,088 202 21.01 albumin 585 4,616 178 -32.04

[0215] Table 6

[0216] 384 peptide to HLA hits were identified in the complete fusion sequence AAT_MLA, resulting in a total score of -10.72. 206 hits were identified in the complete fusion sequence AAT_Mutant, resulting in a total score of 26.87. 202 hits were identified in the complete fusion sequence AAT_WT, resulting in a total score of 21.01. 178 hits were identified in the complete fusion sequence AAT_MLA, resulting in a total score of -32.04. The results are in Fig.10 Shown in.

[0217] Example 9: Stability analysis of AAT variants (K335A, S283C / P361C) on the base (C232S, M351V, M351L)

[0218] The samples were sent to UNcle (Unchained Laboratory) for analysis. For DSF and SLS, a temperature ramp of 1°C / min was performed while monitoring from 25°C to 95°C. UNcle measured SLS at 266nm and 473nm. DLS was measured at the beginning of the thermal ramp. Tm, Tagg and DLS measurements were calculated and analyzed using UNcle analysis software. Fig.11 The Tm is determined as shown by the solid vertical line in. As can be seen, the variant AAT albumin fusion is very stable.

[0219] Example 10: The PK toxicity potential of recombinant alpha-1-antitrypsin (AAT) K335A / S283C / P361C fused to human serum albumin was determined in cynomolgus monkeys when administered once by intravenous bolus injection. The cynomolgus monkey was selected as an animal model for this study because it is a non-rodent species accepted by regulatory agencies for preclinical toxicity testing and most accurately reflects the pharmacokinetics and pharmacodynamics of the test agent expected in human subjects. The pharmacokinetic properties of the AAT albumin fusion compared to human plasma-derived AAT (Prolastin C) were also determined.

[0220] Table 7 provides the test and control articles.

[0221]

[0222] Table 7

[0223] The control product is used to dilute the test product. A 2 mg / mL test product dose formulation is prepared by diluting the test product stock solution (7.8 mg / mL) with the control product to less than or equal to 4 times the serial dilution to meet the dose level requirements. The dose level is selected based on previously published studies (Journal of Chronic Obstructive Pulmonary Disease (2013) 10 (6): 687-95; Lancet (2015) 386 (9991): 360-8). For individuals with alpha-1 antitrypsin deficiency, weekly 60 mg / kg intravenous (IV) infusions of human plasma-derived alpha-1 antitrypsin (AAT) have been approved. The safety and pharmacokinetic profile of weekly infusions of 120 mg / kg doses of AAT are considered safe and well tolerated in adults with AATD.

[0224] Prior to transfer to the study, animals were randomized and grouped using a computer-based program. Table 8 provides the experimental design used.

[0225]

[0226] Table 8

[0227] Test articles were administered to animals by intravenous (slow bolus) injection on day 1. The dose volume for each animal was based on the most recent body weight measurement. Animals were temporarily restrained from dosing and were not given sedatives.

[0228] Blood was collected by venipuncture. Samples were collected according to Table 9.

[0229] Group No. Time hematology Clinical Chemistry All animals Preprocessing X X 1 and 2 Day 29 X X

[0230] Table 9

[0231] Hematology analysis was performed with a target volume of 1 mL and potassium (K3) EDTA anticoagulant. Blood smears were prepared from each hematology sample. The smears were examined to assess the health of the animals. No animal was considered unhealthy.

[0232] Clinical chemistry analysis was performed with a target volume of 2 mL and without anticoagulant.

[0233] Samples were collected according to Table 10.

[0234]

[0235] Table 10

[0236] Plasma from samples was analyzed for test article concentrations, and bronchoalveolar lavage samples were collected from anesthetized animals and analyzed (data not shown).

[0237] The plasma results were shown in Fig.12 and fitted to a 2-phase exponential decay model in Graphpad Prism. Fig.12 As can be seen, the variants have a significantly longer terminal half-life. The AAT-HSA variant (Test Article 1) has a half-life (beta phase, slow) of more than 30 hours compared to Prolastin (plasma AAT) which has a half-life of less than 10 hours.

[0238] The plasma results were further analyzed by a three-compartment model (Tables 11-13) and also in Fig.13 The plasma results are shown in . Fig.13 The median (line), 5th and 95th percentiles (areas) from 1000 simulated PK profiles and individual observations (symbols) are shown. Observations below the limit of quantitation (BLQ, crosses) were not included in the analysis.

[0239] As can be seen from the above, monkey PK is three-compartmental after 10 mg / kg. max Compared with the C of test product 1 max =-40 times higher. The central volume of distribution of Test Article 2 was 0.213 L, and the central volume of distribution of Test Article 1 was 8 L. The total volume of distribution of Test Article 2 was 1.45 L, and the total volume of distribution of Test Article 1 was 42 L. The volume of distribution of Aralast (plasma-derived AAT) in humans is 5.6 L [https: / / www.drugbank.ca / drugs / DB00058]. Allometric scaling to NHP would predict a volume of distribution of 0.240 L, which is equivalent to Test Article 2 from this study.

[0240] The terminal half-life of HSA in monkeys is approximately 5 days. The beta and gamma half-lives of test article 1 are 0.75 days and 5.8 days, respectively. The beta and gamma half-lives of test article 2 are 0.36 days and 1.49 days, respectively. The gamma half-life of test article 2 is approximately 3.9 times longer than the gamma half-life of test article 1. The gamma terminal half-lives of HSA (from the literature) and test article 1 are equivalent in monkeys.

[0241] Table 11 shows the PK parameters of Test Article 2 and Test Article 1.

[0242]

[0243] Table 11

[0244] Table 12 shows the overall PK parameters for Test Article 2:

[0245]

[0246] Table 12

[0247] Table 13 shows the overall PK parameters for Test Article 1:

[0248]

[0249] Table 13 Sequence Listing <110> SPIN THERAPEUTICS LLC A. Sarto M. DeSouza <120> Compositions and methods for use in alpha-1-antitrypsin disorders <130> 118030.00008 <150> US 62 / 752,182 <151> 2018-10-29 <160> 71 <170> PatentIn Version 3.5 <210> 1 <211> 394 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: alpha-1-antitrypsin <400> 1 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350 Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys 385 390 <210> 2 <211> twenty four <212> PRT <213> Artificial Sequence <220> <223> Synthesis: alpha-1-antitrypsin leader sequence <400> 2 Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys 1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala 20 <210> 3 <211> 498 <212> PRT <213> Homo sapiens <220> <221> misc_feature <223> Human Albumin <400> 3 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 Ala Ser Ser Ala Lys Gln Gly Leu Lys Cys 210 215 220 Ala 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 Gly 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 Glu 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 Cys Leu Ser Val Phe 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 <210> 4 <211> 18 <212> PRT <213> Homo sapiens <220> <221> misc_feature <223> Human albumin leader sequence <400> 4 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser <210> 5 <211> 480 <212> PRT <213> Homo sapiens <220> <221> misc_feature <223> Human serum albumin <400> 5 Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala His Arg 1 5 10 15 Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala 20 25 30 Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu 35 40 45 Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser 50 55 60 Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu 65 70 75 80 Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys 85 90 95 Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys 100 105 110 Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val 115 120 125 Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Tyr 130 135 140 Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu 145 150 155 160 Leu Phe Phe Ala Lys Arg Tyr Lys Ala Phe Ala Thr Glu Cys Cys Gln 165 170 175 Ala Ala Asp Lys Ala Ala Cys Leu Pro Lys Leu Asp Glu Leu Arg 180 185 190 Asp Glu Gly Lys Ser Ala Ser Ala Lys Gln Gly Leu Lys Cys Ala Ser 195 200 205 Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg 210 215 220 Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu 225 230 235 240 Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu 245 250 255 Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu 260 265 270 Asn Gln Asp Ser Ile Ser Leu Lys Glu Cys Cys Glu Lys Pro 275 280 285 Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met 290,295,300 Pro Ala Asp Leu Pro Ser Ser Leu Ala Ala Asp Phe Val Gly Ser Lys Asp 305 310 315 320 Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe 325 330 335 Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu 340 345 350 Leu Arg Leu Glu Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Ala 355 360 365 Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys 370 375 380 Pro Leu Val Glu Glu Pro Gln Asn Liver Ile Lys Gln Asn Cys Liver Glu 385 390 395 400 Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Val Arg 405 410 415 Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val 420 425 430 Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu 435 440 445 Ala Lys Arg Met Pro Cys Ala Glu Asp Cys Leu Ser Val Phe Leu Asn 450 455 460 Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr 465 470 475 480 <210> 6 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR1 <400> 6 Leu Asn Leu Met Gly 1 5 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR1 <400> 7 I'm so tired, so tired 1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR1 <400> 8 Ser Phe Gly Met Ser 1 5 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR1 <400> 9 Asn Tyr Trp Met Tyr 1 5 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR2 <400> 10 Thr Cys Ile Thr Val Gly Asp Ser Thr Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 11 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR2 <400> 11 Thr Ile Thr Val Gly Asp Ser Thr Ser Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 12 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR2 <400> 12 Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR2 <400> 13 Ser Ile Asn Gly Arg Gly Asp Asp Thr Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR2 <400> 14 Ala Ile Ser Ala Asp Ser Ser Thr Lys Asn Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR2 <400> 15 Ala Ile Ser Ala Asp Ser Ser Asp Lys Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 16 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR2 <400> 16 Arg Asp Ile Ser Thr Gly Gly Gly Tyr Ser Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Gly <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR3 <400> 17 Arg Arg Thr Trp His Ser Glu Leu 1 5 <210> 18 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR3 <400> 18 Gly Gly Ser Leu Ser Arg 1 5 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR3 <400> 19 Gly Arg Ser Val Ser Arg Ser 1 5 <210> 20 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR3 <400> 20 Gly Arg Gly Ser Pro 1 5 <210> twenty one <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: CDR3 <400> twenty one Asp Arg Glu Ala Gln Val Asp Thr Leu Asp Phe Asp Tyr 1 5 10 <210> twenty two <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: SDA <400> twenty two Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Gly Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Glu Arg Ile Phe Asp Leu Asn 20 25 30 Leu Met Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Arg Glu Leu Val 35 40 45 Ala Thr Cys Ile Thr Val Gly Asp Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Met Asp Tyr Thr Lys Gln Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Leu Tyr Tyr Cys 85 90 95 Lys Ile Arg Arg Thr Trp His Ser Glu Leu Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 23 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: SDA <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Glu Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Glu Arg Ile Trp Asp Ile Asn 20 25 30 Leu Leu Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Arg Glu Leu Val 35 40 45 Ala Thr Ile Thr Val Gly Asp Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Tyr Asp Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Leu Tyr Tyr Cys Lys 85 90 95 Ile Arg Arg Thr Trp His Ser Glu Leu Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 24 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: SDA <400> 24 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 25 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: SDA <400> 25 Ala Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Gly Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Tyr Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Asn Gly Arg Gly Asp Asp Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Glu Tyr Tyr Cys 85 90 95 Thr Ile Gly Arg Ser Val Ser Arg Ser Arg Thr Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 26 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: SDA <400> 26 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Asp Gln Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asp Ser Ser Thr Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ser Ser Pro Gly Thr Gln Val Thr Val 100 105 110 Ser Ser <210> 27 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: SDA <400> 27 Gln Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Gly Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Ala Asp Ser Ser Asp Lys Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Tyr 65 70 75 80 Leu Glu Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Ile Gly Arg Gly Ser Pro Ala Ser Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser <210> 28 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: SDA <400> 28 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Tyr Trp Val Arg Val Ala Pro Gly Lys Gly Leu Glu Arg Ile 35 40 45 Ser Arg Asp Ile Ser Thr Gly Gly Gly Tyr Ser Tyr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr 85 90 95 Cys Ala Lys Asp Arg Glu Ala Gln Val Asp Thr Leu Asp Phe Asp Tyr 100 105 110 Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 29 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: hSDA <400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 30 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: hSDA <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 31 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: hSDA <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 32 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: hSDA <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 33 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: hSDA <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 34 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: hSDA <400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 35 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: hSDA <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 36 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: hSDA <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Ile Gly Gly Ser Leu Ser Arg Ser Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 37 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR1 <400> 37 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Gly Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Glu Arg Ile Phe Asp 20 25 30 <210> 38 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR1 <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Glu Gly Gly 1 5 10 15 Ser Leu Arg Leu Ala Cys Ala Ala Ser Glu Arg Ile Trp Asp 20 25 30 <210> 39 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR1 <400> 39 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg 20 25 30 <210> 40 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR1 <400> 40 Ala Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Gly 20 25 30 <210> 41 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR1 <400> 41 Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Arg 20 25 30 <210> 42 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR1 <400> 42 Gln Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Thr Cys Thr Ala Ser Gly Phe Thr Phe Gly 20 25 30 <210> 43 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR1 <400> 43 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 20 25 30 <210> 44 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: FR2 <400> 44 Trp Tyr Arg Gln Gly Pro Gly Asn Glu Arg Glu Leu Val Ala 1 5 10 <210> 45 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: FR2 <400> 45 Trp Val Arg Gln Ala Pro Gly Lys Glu Pro Glu Trp Val Ser 1 5 10 <210> 46 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: FR2 <400> 46 Trp Val Arg Gln Tyr Pro Gly Lys Glu Pro Glu Trp Val Ser 1 5 10 <210> 47 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: FR2 <400> 47 Trp Val Arg Gln Ala Pro Gly Lys Asp Gln Glu Trp Val Ser 1 5 10 <210> 48 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: FR2 <400> 48 Trp Val Arg Gln Ala Pro Gly Glu Gly Leu Glu Trp Val Ser 1 5 10 <210> 49 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic: FR2 <400> 49 Trp Val Arg Val Ala Pro Gly Lys Gly Leu Glu Arg Ile Ser 1 5 10 <210> 50 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR3 <400> 50 Arg Phe Thr Ile Ser Met Asp Tyr Thr Lys Gln Thr Val Tyr Leu His 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Leu Tyr Tyr Cys Lys Ile 20 25 30 <210> 51 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR3 <400> 51 Arg Phe Thr Ile Ser Arg Asp Tyr Asp Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Arg Pro Glu Asp Thr Gly Leu Tyr Tyr Cys Lys Ile 20 25 30 <210> 52 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR3 <400> 52 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile 20 25 30 <210> 53 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR3 <400> 53 Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Glu Tyr Tyr Cys Thr Ile 20 25 30 <210> 54 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR3 <400> 54 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Tyr Leu Glu 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val Ile 20 25 30 <210> 55 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR3 <400> 55 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Leu Tyr Leu Glu 1 5 10 15 Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Val Ile 20 25 30 <210> 56 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR3 <400> 56 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln 1 5 10 15 Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys 20 25 30 <210> 57 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR4 <400> 57 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 58 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR4 <400> 58 Ser Ser Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 59 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR4 <400> 59 Arg Thr Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 60 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR4 <400> 60 Ser Ser Pro Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 61 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR4 <400> 61 Ala Ser Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 62 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: FR4 <400> 62 Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser 1 5 10 <210> 63 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Linkers <400> 63 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5 10 <210> 64 <211> 1017 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: AAT-linker albumin fusion <400> 64 Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys 1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala 20 25 30 Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn 35 40 45 Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln 50 55 60 Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser 65 70 75 80 Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr 85 90 95 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro 100 105 110 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn 115 120 125 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu 130 135 140 Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 145 150 155 160 Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu 165 170 175 Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys 180 185 190 Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu 195 200 205 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val 210 215 220 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val 225 230 235 240 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys 245 250 255 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala 260 265 270 Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu 275 280 285 Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp 290 295 300 Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr 305 310 315 320 Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe 325 330 335 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys 340 345 350 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly 355 360 365 Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile 370 375 380 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 385 390 395 400 Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 405 410 415 Gln Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 420 425 430 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 435 440 445 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 450 455 460 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 465 470 475 480 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 485 490 495 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 500 505 510 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 515 520 525 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 530 535 540 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 545 550 555 560 Asp Asn Glu Glu Thr Phe Leu Lys Tyr Leu Tyr Glu Ile Ala Arg 565,570,575 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 580,585,590 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 595,600,605 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 610 615 620 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 625 630 635 640 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 645,650,655 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 660,665,670 Val His Thr Glu Cys His Gly Asp Leads To Glu Cys Ala Asp Asp 675,680,685 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 690,695,700 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 705 710 715 720 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 725 730 735 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 740,745,750 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 755,760,765 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 770,775,780 Tyr Glu Thr Thr Leu Glu Lys Cys Ala Ala Ala Asp Pro His Glu 785,790,795,800 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 805 810 815 Gln Asn With Lys Gln Asn Cys Glu To Phe Glu Gln To Gly Glu 820 825 830 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Val Pro 835 840 845 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 850 855 860 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 865 870 875 880 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 885 890 895 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 900 905 910 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 915 920 925 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 930 935 940 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 945 950 955 960 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 965 970 975 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 980 985 990 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 995 1000 1005 Ala Ala Ser Gln Ala Ala Leu Gly Leu 1010 1015 <210> 65 <211> 1017 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Mutant AAT-linker albumin fusion (C232S) <400> 65 Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys 1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala 20 25 30 Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn 35 40 45 Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln 50 55 60 Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser 65 70 75 80 Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr 85 90 95 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro 100 105 110 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn 115 120 125 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu 130 135 140 Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 145 150 155 160 Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu 165 170 175 Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys 180 185 190 Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu 195 200 205 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val 210 215 220 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val 225 230 235 240 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Ser 245 250 255 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala 260 265 270 Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu 275 280 285 Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp 290 295 300 Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr 305 310 315 320 Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe 325 330 335 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys 340 345 350 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly 355 360 365 Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile 370 375 380 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 385 390 395 400 Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 405 410 415 Gln Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 420 425 430 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 435 440 445 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 450 455 460 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 465 470 475 480 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 485 490 495 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 500 505 510 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 515 520 525 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 530 535 540 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 545 550 555 560 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 565 570 575 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 580,585,590 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 595,600,605 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 610 615 620 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 625 630 635 640 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 645,650,655 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 660,665,670 Val His Thr Glu Cys His Gly Asp Leads To Glu Cys Ala Asp Asp 675,680,685 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 690,695,700 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 705 710 715 720 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 725 730 735 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 740,745,750 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 755,760,765 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 770,775,780 Tyr Glu Thr Thr Leu Glu Lys Cys Ala Ala Ala Asp Pro His Glu 785,790,795,800 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 805 810 815 Gln Asn With Lys Gln Asn Cys Glu To Phe Glu Gln To Gly Glu 820 825 830 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Val Pro 835 840 845 Gln Will Be Thr Pro Thr Leu Val Glu Will Be Arg Asn Leu Gly Lys 850 855 860 Val Gly Ser Lys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 865 870 875 880 Only Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 885,890,895 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 900 905 910 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 915 920 925 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 930 935 940 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 945 950 955 960 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 965 970 975 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 980 985 990 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 995 1000 1005 Ala Ala Ser Gln Ala Ala Leu Gly Leu 1010 1015 <210> 66 <211> 1017 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Mutant AAT-linker albumin fusion (M351V, M358V) <400> 66 Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys 1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala 20 25 30 Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn 35 40 45 Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln 50 55 60 Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser 65 70 75 80 Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr 85 90 95 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro 100 105 110 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn 115 120 125 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu 130 135 140 Serum Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 145 150 155 160 Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu 165 170 175 Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys 180 185 190 Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu 195 200 205 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val 210 215 220 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val 225 230 235 240 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys 245 250 255 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala 260 265 270 Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu 275 280 285 Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp 290 295 300 Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr 305 310 315 320 Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe 325 330 335 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys 340 345 350 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly 355 360 365 Thr Glu Ala Ala Gly Ala Val Phe Leu Glu Ala Ile Pro Val Ser Ile 370 375 380 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 385 390 395 400 Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 405 410 415 Gln Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 420 425 430 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 435 440 445 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 450 455 460 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 465 470 475 480 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 485,490,495 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 500 505 510 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 515,520,525 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 530 535 540 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 545 550 555 560 Asp Asn Glu Glu Thr Phe Leu Lys Tyr Leu Tyr Glu Ile Ala Arg 565,570,575 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 580,585,590 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 595,600,605 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 610 615 620 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 625 630 635 640 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 645,650,655 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 660,665,670 Val His Thr Glu Cys His Gly Asp Leads To Glu Cys Ala Asp Asp 675,680,685 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 690,695,700 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 705 710 715 720 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 725 730 735 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 740,745,750 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 755,760,765 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 770,775,780 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 785 790 795 800 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 805 810 815 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 820 825 830 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 835 840 845 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 850 855 860 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 865 870 875 880 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 885 890 895 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 900 905 910 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 915 920 925 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 930 935 940 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 945 950 955 960 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 965 970 975 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 980 985 990 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 995 1000 1005 Ala Ala Ser Gln Ala Ala Leu Gly Leu 1010 1015 <210> 67 <211> 1017 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Mutant AAT-linker albumin fusion (M351V, M358L) <400> 67 Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys 1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala 20 25 30 Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn 35 40 45 Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln 50 55 60 Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser 65 70 75 80 Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr 85 90 95 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro 100 105 110 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn 115 120 125 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu 130 135 140 Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 145 150 155 160 Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu 165 170 175 Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys 180 185 190 Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu 195 200 205 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val 210 215 220 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val 225 230 235 240 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys 245 250 255 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala 260 265 270 Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu 275 280 285 Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp 290 295 300 Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr 305 310 315 320 Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe 325 330 335 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys 340 345 350 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly 355 360 365 Thr Glu Ala Ala Gly Ala Val Phe Leu Glu Ala Ile Pro Leu Ser Ile 370 375 380 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 385 390 395 400 Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 405 410 415 Gln Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 420 425 430 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 435 440 445 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 450 455 460 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 465 470 475 480 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 485 490 495 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 500 505 510 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 515,520,525 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 530 535 540 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 545 550 555 560 Asp Asn Glu Glu Thr Phe Leu Lys Tyr Leu Tyr Glu Ile Ala Arg 565,570,575 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 580,585,590 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 595,600,605 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 610 615 620 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 625 630 635 640 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 645,650,655 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 660,665,670 Val His Thr Glu Cys His Gly Asp Leads To Glu Cys Ala Asp Asp 675,680,685 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 690,695,700 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 705 710 715 720 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 725 730 735 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 740,745,750 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 755,760,765 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 770,775,780 Tyr Glu Thr Thr Leu Glu Lys Cys Ala Ala Ala Asp Pro His Glu 785,790,795,800 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 805 810 815 Gln Asn With Lys Gln Asn Cys Glu To Phe Glu Gln To Gly Glu 820 825 830 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Val Pro 835 840 845 Gln Will Be Thr Pro Thr Leu Val Glu Will Be Arg Asn Leu Gly Lys 850 855 860 Val Gly Ser Lys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 865 870 875 880 Only Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 885,890,895 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Thr Glu Ser 900 905 910 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 915,920,925 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 930,935,940 With Cys Thr Leu Ser Glu Lys Glu Arg Gln With Lys Gln Thr Ala 945 950 955 960 Leu Val Glu Leu Val Lys Lys Lys Pro Lys Ala Thr Lys Glu Gln Leu 965,970,975 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 980,985,990 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 995 1000 1005 Ala Ala Ser Gln Ala Ala Leu Gly Leu 1010 1015 <210> 68 <211> 1017 <212> PRT <213> Artificial Sequence <220> <223> Synthesis: Mutant AAT-linker albumin fusion (C232S, M351V, M358L) <400> 68 Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys 1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala 20 25 30 Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn 35 40 45 Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln 50 55 60 Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser 65 70 75 80 Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr 85 90 95 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro 100 105 110 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn 115 120 125 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu 130 135 140 Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 145 150 155 160 Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu 165 170 175 Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys 180 185 190 Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu 195 200 205 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val 210 215 220 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val 225 230 235 240 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Ser 245 250 255 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala 260 265 270 Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu 275 280 285 Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp 290 295 300 Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr 305 310 315 320 Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe 325 330 335 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys 340 345 350 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly 355 360 365 Thr Glu Ala Ala Gly Ala Val Phe Leu Glu Ala Ile Pro Leu Ser Ile 370 375 380 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 385 390 395 400 Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 405 410 415 Gln Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 420 425 430 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 435 440 445 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 450 455 460 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 465 470 475 480 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 485 490 495 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 500 505 510 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 515 520 525 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 530 535 540 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 545 550 555 560 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 565,570,575 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 580,585,590 Tyr Lys Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 595,600,605 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 610 615 620 The Ser Al of Lys Gln Arg Leu Lys Cys Al Ser Leu Gln Lys Phe Gly Glu 625 630 635 640 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 645,650,655 Lys Ala Glu Phe Ala Glu Val Ser Leu Val Thr Asp Leu Thr Lys 660,665,670 Val His Thr Glu Cys His Gly Asp Leads To Glu Cys Ala Asp Asp 675,680,685 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 690,695,700 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 705 710 715 720 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 725 730 735 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 740,745,750 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 755,760,765 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 770,775,780 Tyr Glu Thr Thr Leu Glu Lys Cys Ala Ala Ala Asp Pro His Glu 785,790,795,800 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 805 810 815 Gln Asn With Lys Gln Asn Cys Glu To Phe Glu Gln To Gly Glu 820 825 830 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Val Pro 835 840 845 Gln Will Be Thr Pro Thr Leu Val Glu Will Be Arg Asn Leu Gly Lys 850 855 860 Val Gly Ser Lys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 865 870 875 880 Only Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 885,890,895 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Thr Glu Ser 900 905 910 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 915,920,925 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 930,935,940 With Cys Thr Leu Ser Glu Lys Glu Arg Gln With Lys Gln Thr Ala 945 950 955 960 Leu Val Glu Leu Val Lys Lys Lys Pro Lys Ala Thr Lys Glu Gln Leu 965,970,975 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 980,985,990 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 995 1000 1005 Ala Ser Ser Gln Ala Leu Gly Leu 1010 1015 <210> 69 <211> 3178 <212> DNA <213> Humans (Homo sapiens) <220> <221> misc_feature <223> wild-type AAT nucleic acid <400> 69 tgggcaggaa ctgggcactg tgcccagggc atgcactgcc tccacgcagc aaccctcaga 60 gtcctgagct gaaccaagaa ggaggagggg gtcgggcctc cgaggaaggc ctagccgctg 120 ctgctgccag gaattccagg ttggaggggc ggcaacctcc tgccagcctt caggccactc 180 tcctgtgcct gccagaagag acagagcttg aggagagctt gaggagagca ggaaaggaca 240 atgccgtctt ctgtctcgtg gggcatcctc ctgctggcag gcctgtgctg cctggtccct 300 gtctccctgg ctgaggatcc ccagggagat gctgcccaga agacagatac atcccaccat 360 gatcaggatc acccaacctt caacaagatc acccccaacc tggctgagtt cgccttcagc 420 ctataccgcc agctggcaca ccagtccaac agcaccaata tcttcttctc cccagtgagc 480 atcgctacag cctttgcaat gctctccctg gggaccaagg ctgacactca cgatgaaatc 540 ctggagggcc tgaatttcaa cctcacggag attccggagg ctcagatcca tgaaggcttc 600 caggaactcc tccgtaccct caaccagcca gacagccagc tccagctgac caccggcaat 660 ggcctgttcc tcagcgaggg cctgaagcta gtggataagt ttttggagga tgttaaaaaag 720 780 atcaacgatt acgtggagaa gggtactcaa gggaaaattg tggatttggt caaggagctt 840 gagagagaca cagtttttgc tctggtgaat tacatcttct ttaaaggcaa atgggagaga 900 ccctttgaag tcaaggacac cgaggaagag gacttccacg tggaccaggt gaccaccgtg 960 aaggtgccta tgatgaagcg tttaggcatg tttaacatcc agcactgtaa gaagctgtcc 1020 agctgggtgc tgctgatgaa atacctgggc aatgccaccg ccatcttctt cctgcctgat 1080 1140 gaaaatgaag acagaaggtc tgccagctta catttaccca aactgtccat tactggaacc 1200 tatgatctga agagcgtcct gggtcaactg ggcatcacta aggtcttcag caatggggct 1260 gacctctccg gggtcacaga ggaggcaccc ctgaagctct ccaaggccgt gkataaggct 1320 gtgctgacca tcgacgagaa agggactgaa gctgctgggg ccatgttttt agaggccata 1380 cccatgcta tcccccccga ggtcaagttc aaaaccct ttgtcttctt aatgattgaa 1440 caaaatacca agtctcccct cttcatggga aaagtggtga atcccaccca aaaataactg 1500 cctcgctc ctcaacccct cccctccatc cctggccccc tcctggatg aattaaagaa 1560 gggttgagct ggtccctgcc tgcatgtgac tgtaaatccc tcccatgttt tctctgagtc 1620 tccctttgcc tgctgaggct gtatggggc tccaggtaac agtgctgtct tcgggccccc 1680 tgaactgtgt tcatggagca tctggctggg taggcacatg ctgggcttga atccaggggg 1740 gactgaatcc tcagcttacg gacctgggcc catctgtttc tggagggctc cagtcttcct 1800 tgtcctgtct tggagtcccc aagaaggaat cacaggggag gaaccagata ccagccatga 1860 ccccaggctc caccaagcat cttcatgtcc ccctgctcat cccccactcc cccccaccca 1920 gagtgctca tcctgccagg gctggctgtg cccaccccaa ggctgccctc ctgggggccc 1980 cagaactgcc tgatcgtgcc gtggcccagt tttgtggcat ctgcagcaac acaagagaga 2040 ggacaatgtc ctcctcttga ctcgggccct gcacctctca ggcacttctg gaaaatgact 2100 gaggcagatt cttcctgaag cccattctc atggggcaac aaggacacct attctgtcct 2160 tgtccttcca tcgctgcccc agaagccctc acatactcc gtttagaatc aggtccctc 2220 tccccagatg agaggagggg tctctgctt gtttctcta tctcctcctc agacttgacc 2280 agcccagca ggccccagaa gaccattacc ctatatccct tctcctccct agtcacatgg 2340 ccataggcct gctgatggct caggaaggcc attgcagga ctcctcagct atgggagg 2400 aagcacatca cccattgacc cccgcaaccc ctccctttcc tcctctgagt cccgactggg 2460 gccacatgca gcctgacttc ttgtgcctg tgctgtccc tgcagtctc agagggccac 2520 cgcagctcca gtgccacggc aggaggctgt tcctgaatag cccctgtggt aagggccagg 2580 agagtccttc catcctccaa gccctgcta aaggacacag cagccaggaa gtcccctggg 2640 cccctagctg aaggacagcc tgctccctcc gtctctacca ggaatggcct tgtcctatgg 2700 aaggcactgc cccatcccaa actaatctag gatcactgt ctaccactc actgtcatga 2760 atgtgtactt aaaggatgag gttgagtcat accaatagt gatttcgata gttcaaatg 2820 gtgaaattag caattctaca tgattcagtc taatcaatgg ataccgactg tttcccacac 2880 aagtctcctg ttctcttaag cttactcact gacagccttt cactctccac aaatacatta 2940 aagatatggc catcaccaag ccccctagga tgacaccaga cctgagagtc tgaagacctg 3000 gatccaagtt ctgacttttc cccctgacag ctgtgtgacc ttcgtgaagt cgccaaacct 3060 ctctgagccc cagtcattgc tagtaagacc tgcctttgag ttggtatgat gttcaagtta 3120 gataacaaaa tgtttatacc cattagaaca gagaataaat agaactacat ttcttgca 3178 <210> 70 <211> 19002 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Wild-type human serum albumin nucleic acid <400> 70 cctttcccag ggacttctac aaggaaaaag ctagagttgg ttactgactt ctaataaata 60 atgcctacaa tttctaggaa gttaaaagtt gacataattt atccaagaaa gaattatttt 120 cttaacttag aatagtttct tttttctttt cagatgtagg tttttctggc tttagaaaaa 180 atgcttgttt ttcttcaatg gaaaataggc acacttgttt tatgtctgtt catctgtagt 240 cagaaagaca agtctggtat ttcctttcag gactcccttg agtcattaaa aaaaatcttc 300 ctatctatct atgtatctat catccatcta gctttgattt tttcctcttc tgtgctttat 360 tagttaatta gtacccattt ctgaagaaga aataacataa gattatagaa aataatttct 420 ttcattgtaa gactgaatag aaaaaatttt ctttcattat aagactgagt agaaaaaata 480 atactttgtt agtctctgtg cctctatgtg ccatgaggaa atttgactac tggttttgac 540 tgactgagtt atttaattaa gtaaataac tggcttagta ctaattattg ttctgtagta 600 tcagagaaag ttgttcttcc tactggttga gctcagtagt tcttcatatt ctgagcaaaa 660 gggcagaggt aggatagctt ttctgaggta gagataagaa ccttgggtag ggaaggaaga 720 tttatgaaat atttaaaaaa ttatcttcc ttcgctttgt ttttagacat aatgttaaat 780 ttattttgaa atttaaagca acataaaaga acatgtgatt tttctactta ttgaaagaga 840 gaaaggaaaa aaatatgaaa cagggatgga aagaatccta tgcctggtga aggtcaaggg 900 ttctcataac ctacagagaa ttttcataac gcctgtccta ttgtatatta tggcaagat 960 aatcatcatc tcattgggt ccattttcct ctcatctct gcttactga agatcccatg 1020 agatatactc acactgaatc taaatagcct atctcagggc tgaatcaca tgtgggccac 1080 agcaggaatg ggaacatgga atttctaagt cctatcttac ttgttattgt tgctatgtct 1140 tttcttagt ttgcatctga ggcacatca gcttttcag acagaatggc ttggaatag 1200 taaaaaagac acagaagccc taaaatatgt atgtagtat atgtgtgtgt gcatgcgtga 1260 gtacttgtgt gtaaattttt cattactat aggtaaagc acacttgga tgcaatag 1320 atgcaatttg ggacttact ctttcagtat gtcttatttc taagcaaagt atttagttg 1380 gttagtatt actaacact gagaactaa ttgcaacac acactaaatgttcaag 1440 tgggaaatta cagttaaata ccatggtaat gataaaagg tacaaatcgt ttaactct 1500 atgtaaaatt tgataagatg ttttacacaa ctttaataca ttgacaaggt ctgtggaga 1560 aaacagttcc agatggtaaa tatacacaag ggatttagtc aaacatttt ttggcaagaa 1620 tattatgaat tttgtaatcg gttggcagcc aatgaaatac aaagatgagt ctagttaata 1680 atctacaatt attggttaaa gaagtatatt agtgctaatt tccctccgtt tgtcctagct 1740 tttctcttct gcaacccca cacgcctttg gcacaatgaa gtgggtaacc tttatttccc 1800 ttctttttct ctttagctcg gcttattcca ggggtgtgtt tcgtcgagat gcacgtaaga 1860 aatccatttt tctattgttc aacttttatt ctattttccc agtaaaataa agttttagta 1920 aactctgcat ctttaaagaa ttattttggc attatttct aaaatggcat agtattttgt 1980 atttgtgaag tcttacaagg ttatcttatt aataaaattc aaacatccta ggtaaaaaaa 2040 aaaaaaggtc agaattgttt agtgactgta atttcttttt gcgcactaag gaaagtgcaa 2100 agtaacttag agtgactgaa acttcacaga atagggttga agattgaatt cataactatc 2160 ccaaagacct atccattgca ctatgcttta tttaaaaacc acaaaacctg tgctgttgat 2220 ctcataaata gaacttgtat ttatattta tttcatttta gtctgtcttc ttggttgctg 2280 ttgatagaca ctaaaagagt attagatatt atctaagtttt gaatataagg ctataaatat 2340 ttaataattt ttaaaatagt attcttggta attgaattat tcttctgttt aaaggcagaa 2400 gaaataattg aacatcatcc tgagtttttc tgtaggaatc agagcccaat attttgaaac 2460 aaatgcataa tctaagtcaa atggaaagaa atataaaaag taacattat acttcttgtt 2520 ttcttcagta tttaacaatc cttttttttc ttcccttgcc cagacaagag tgaggttgct 2580 catcggttta aagaatttggg agaagaaaat ttcaaagcct tgtaagttaa aatattgatg 2640 aatcaaattt aatgtttcta atagtgttgt ttattattct aaagtgctta tatttccttg 2700 tcatcagggt tcagattcta aaacagtgct gcctcgtaga gttttctgcg ttgaggaaga 2760 tattctgtat ctgggctatc caataaggta gtcactggtc acatggctat tgagtacttc 2820 aaatatgaca agtgcaactg agaaacaaaa acttaaattg tatttaattg tagttaattt 2880 gaatgtatat agtcacatgt ggctaatggc tactgtattg gacagtacag ctctggaact 2940 tgcttggtgg aaaggacttt aatatagggtt tcctttggtg gcttacccac taaatcttct 3000 ttacatagca agcattcctg tgcttagttg ggaatattta atttttttt ttttttaaga 3060 caggctctcg ctctgtcgcc caggctggag tgcagtggcg caatctcggc tcactgcaaa 3120 ctccgctccc gggttcacgc cattctcctg cctcagccctc ccgagtagct gggactacag 3180 gcgcccgcca tcacgcccgg ctaatctttt gtatttttag tagagatggg gttcaccgt 3240 gtgccaggat gtctcaatc tcctgacatc gtgatctgcc cacctcggcc tcccaagtg 3300 ctgggattac aggagtgagt caccgcgccc ggccttatta aatgtttttt aatctagtaa 3360 aaaatgagaa attgtttt ttaaaagtct acctaatcct acagctaat tAAagacgtg 3420 tgtgggatc aggtgcggtg gttcacacct gtaatcccag cacttggaa ggctgatgca 3480 ggaggattgc ttgagcccag gagtacaaga ccagcctggg caagctctt taaaaaaaac 3540 aaaaaaaaaaaaaaaa taggcatgg tgcacatgc ctgtagtcct agctacttag 3600 gaggctgacg taggagatc gtttggacct gagaggtca ggctacagtg agccatgatt 3660 gtgccactgc actccagcct gggtgacaga gtgagactct gtctcaaaaa agaaaaagga 3720 aatctgtggg gtttgtttta gttttaagta attctaagga ctttaaaaat gcctagtctt 3780 gacaattaga tctatttggc atacaatttg cttgcttaat ctatgtgtgt gcatagatct 3840 actgacacac gcatacatat aaacattagg gaactaccat tctctttgcg taggaagcca 3900 catatgccta tctaggcctc agatcatacc tgatatgaat aggctttctg gataatggtg 3960 aagaagatgt ataaaagata gaacctatac ccatacatga tttgttctct agcgtagcaa 4020 cctgttacat attaaagttt tattatacta catttttcta catcctttgt ttcagggtgt 4080 tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta aaattagtga 4140 atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtcagctgaa aattgtgaca 4200 aatcacttgt aagtacattc taattgtgga gattctttct tctgtttgaa gtaatcccaa 4260 gcatttcaaa ggaattttt ttaagttttc tcaattatta ttaagtgtcc tgatttgtaa 4320 gaaacactaa aaagttgctc atagactgat aagccattgt ttctttgtg atagagatgc 4380 tttagctatg tccacagttt taaaatcatt tctttattga gaccaaacac aacagtcatg 4440 gtgtatttaa atggcaattt gtcatttata aacacctcttt tttaaaattt gaggtttggt 4500 ttctttttgt agaggctaat agggatatga tagcatgtat ttatttattt attatctta 4560 ttttattata gtaagaaccc ttaacatgag atctaccctg ttatattttt aagtgtacaa 4620 tccattattg ttaactacgg gtacactgtt gtatagctta ctcatcttgc tgtattaaaa 4680 cttgtgccc attgatgt aacccctcgt ttcgtcctcc cccagccact ggcaaccagc 4740 attatactct ttgattctat gagtttgact actttagcta ccttatataa gtggtattat 4800 gtactgttta tctttttatg actgacttat ttcccttagc atagtgcatt caaagtccaa 4860 ccatgttgtt gcctattgca gaatttcctt cttttcaagg ctgaataata ttccagtgca 4920 tgtgtgtacc acattttctt tatccattaa tttgttgatt gatagacatt taggttggtt 4980 ttctacatct tgactatcat gaatagtgtt gcaatgaaca caggagagct actatctctt 5040 agagatgata tcatggtttt tatcatcaga aaacacccac tgatttctat gctaattttg 5100 ttacctgggt ggaataatag tacagctata tattcctcat tttagatatc tttgtatttc 5160 tacatacaat aaaaaagcag agtacttagt catgttgaag aactttaaac ttttagtatt 5220 tccagatcaa tcttcaaaac aaggacaggt ttatctttct ctcaccactc aatctatata 5280 tacctcttgt gggcaaggcc agtttttatc actggagcct ttcccctttt tattatgtac 5340 ctctccctca cagcagagtc aggactttaa ctttacacaa tactatggct ctacatatga 5400 aatcttaaaa atacataaaa attaataaat tctgtctaga gtagtatatt ttccctgggg 5460 ttacgattac tttcataata aaaattagag ataaggaaag gactcattta ttggaaagtg 5520 attttaggta acatttctgg aagaaaaatg tctatatctt aatagtcact tatatatga 5580 tggattgtgt tactcctcag ttttcaatgg catatactaa aacatggccc tctaaaaagg 5640 gggcaaatga aatgagaaac tctctgaatg ttttctccc ctaggtgaat tcacctgctg 5700 cttagaagct tattttctct tgatttctgt tataatgatt gctcttaccc tttagtttta 5760 agtttcaaaa taggagtcat ataactttcc ttaaagctat tgactgtctt tttgtcctgt 5820 tttattcacc atgagtata gtgtgacagt taattcttat gaaaattata tagagatggt 5880 taaatcatca gaaactgtaa acctcgattg ggaggggaag cggattttta aatgatttcc 5940 tgaccaagct taaccagtat attaaatcct ttgtactgtt ctttggctat aaagaaaaaa 6000 ggtactgtcc agcaactgaa acctgctttc ttccatttag catacccttt ttggagacaa 6060 attatgcaca gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa 6120 acaagaacct gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc 6180 ccgattggtg agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac 6240 atttttgaaa aagtaagtaa tcagatgttt atagttcaaa attaaaaagc atggagtaac 6300 tccataggcc aacactctat aaaaattacc ataacaaaaa tattttcaac attaagactt 6360 ggaagttttg ttatgatgat tttttaaaga agtagtattt gataccacaa aattctacac 6420 agcaaaaaat atgatcaaag atattttgaa gtttattgaa acaggataca atctttctga 6480 aaaatttaag atagacaaat tatttaatgt attacgaaga tatgtatata tggttgttat 6540 aattgatttc gttttagtca gcaacattat attgccaaaa tttaaccatt tatgcacaca 6600 cacacacaca cacacacact taacctttt ttccacatac ttaaagaatg acagagacaa 6660 gaccatcatg tgcaaattga gcttaattgg ttaattagat atctttggaa tttggaggtt 6720 ctggggagaa tgtcgattac aattatttct gtaatattgt ctgctataga aaagtgactg 6780 ttttctttt tcaaaattta gatacttata tgaaattgcc agaagacatc cttactttta 6840 tgccccggaa ctccttttct ttgctaaaag gtataaagct gcttttacag aatgttgcca 6900 agctgctgat aaagctgcct gcctgttgcc aaaggtatta tgcaaaagaa tagaaaaaaa 6960 gagttcatta tccaacctga ttttgtccat tttgtggcta gatttaggga acctgagtgt 7020 ctgatacaaa cttccgaca tggtcaaaaa agccttcctt ttatctgtct tgaaaatctt 7080 tcatctttga aggcctacac tctcgtttct tctttaaga tttgccaatg atgatctgtc 7140 agaggtaatc actgtgcatg tgtttaaaga tttcaccact tttatggtg gtgatcacta 7200 tagtgaaata ctgaaacttg tttgtcaaat tgcacagcaa ggggacacag ttcttgttta 7260 tcttttcatg ataattttta gtagggaggg aattcaaagt agagaattttt actgcatcta 7320 gatgcctgag ttcatgcatt cattccataa atatatatta tggaatgctt tatttcttt 7380 tctgaggagt ttactgatgt tggtggagga gagactgaaa tgaattatac acaaaattta 7440 aaaattagca aaattgcagc ccctgggata ttagcgtact ctttctctga cttttctccc 7500 acttttaagg ctctttttcc tggcaatgtt tggttggt tctaagg tgtctc number 7560 ccgctgtgac cagaatgatc gatgatctt tccttttctt aggagcaa atcattattc 7620 gctaaaggga gtacttggga atttaggcat aaattatgcc ttcaaattt aatttggcac 7680 agtctcatct gagcttatgg aggggtgttt catgtagaat ttttctcta attttcatca 7740 aattattcct ttttgtagct cgatgaactt cgggatgaag ggaggctc gtctgccaaa 7800 cagagactca agtgtgccag tctccaaaaa tttggagaaa gagctttcaa agcatggtaa 7860 atacttttaa acatagttgg catctttata acgatgtaaa tgataatgct tcagtgacaa 7920 attgtacatt tttgtatt ttgcaaagtg ctgtcaaata cattctttg gttgtctaac 7980 aggtagaact ctaatagagg taaaaatcag atatcaatg acatttgac attattttta 8040 atctttcttt ttctaaatag ttgaatatt tagaggacgc tgtccttttt gtcctaaaaa 8100 aagggacaga tatttagtt ctatttattt aaaatctt ggacttat tctaatggtt 8160 cattatttt atagagctgt aggcatggtt ctttatta ttttaaag ttatttttaa 8220 tttttgtgga tacagtag gtatacatat ttcggggta tatgagat ttgatata 8280 gtatacaaca tatataatcc ctttatta tttatctc cccccaatga tctaaaacta 8340 tttgcttgtc cttttatgtc ttatagttaa attcagtcac caactaagtt gaagttactt 8400 cttattttg catagctcca gctctgatct tcatctcatg ttttgctg agccctgtt 8460 ttcatattac ttagttggtt ctgggagcat acttatag ccgagtcaag aaaatacta 8520 gctgccccgt cctcacctgc tagtcacag CAATCACA CACAGAA 8580 taaaatgaaa ataatagaca ttatgcatgc tcttagaaa ctgtcaattg aactgtattt 8640 gctcatcatt cctaccattct acaccaccaa atcaccaa atttatgaaa aaaaaacagc 8700 cccacataa aattatacac agataacag gctatgatg gtttgggaa agagtcacc 8760 tttacctgat ttaggcaact gtgaaatgac tagagaatga agaaattag acgtttacat 8820 cttgtcatag agtttgaaga tagtgctgga tctttctttt father atcaataaaa actccctcat tctgtagaag ttatgatttc ttttctaaga gacctttag agtcagaaaa aatgtgtttc aattgagaaa aaagataact ggagtttgtg tagtacttcc cagattaa aatgcttttg tatgtattat ctaatttaat cctcaaaact tcttcaattt agcatgttgt catgacactg cagaggctga agctcagaga cgctgagccc tctgctaaca agtcctactg ctaacaagtg ataaagccag agctggaagt cacatctgga ctccaaacct gatgcttctc agcctgttgc cccttttaga gttcctttttt aatttctgct tttatgactt gctagatttc 9240 tacctaccac acacactctt aaatggataa ttctgcccta aggataagtg attackattt ggttcagaac tags gaattttaa aattattct gtatgtccat tttgaatttt cttatgagaa atagtatttg cctagtgttt tcatataaaa tatcgcatga taataccatt ttgattggcg attttctttt tagggcagta gctcgcctga gccagagatt tcccaaagct 9480. 9540. gagtttgcag aagtttccaa gttagtgaca gatcttacca aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac agggtaaga gtcgtcgata tgctttttgg tagcttgcat gctcaagttg gtagaatgga tgcgtttggt atcattggtg atagctgaca gtgggttgag attgtcttct gtgctttcgt ctgtcctatc ttcaatcttt ccctgcctat 9720 ggtggtggta cctttctgtt tttaacctgc fathercagataacc cattcactga tttgtaactc ctttcagtca tgctctaact gtaaatgaag gcttaaactg aagtagaaca gttacaaggt tttacttggc aagaacatctt gcaaggtaga tgtctaagaa gatttttttt tcttttttta agacagagtt tcgctcttgt ttcccaggct ggggtgcaat ggtgtgatct 9960. tggctcagcg caacctctgc ctcctgggtt caagtgattt tcatgcctca gcctcccaag 10020. tagctgggat tacaggcatg cgccaccaca cctggctaat tttgtatttt tagtaggc ggggtttcac catattgtcc agactggtct cgaactcctg acctcaggtg atccacccgc 10140 cttggcctcc caaagtgctg ggattacagg catgagccac cttgcccagc ctaagaagat tttttgaggg aggtaggtgg acttggagaa ggtcactact tgaagagatt tttggaaatg atgtattttt cttctctata ttccttccct taattaactc tgtttgttag atgtgcaaat 10320 atttggaatg atactcttt tctcaaaact tataatattt tctttctccc tttcttcaag 10380 attaaactta tgggcaaata ctagaatcct aatctctcat ggcactttct ggaaaattta 10440 aggcggttat tttatatatg taagcagggc ctatgactat gatcttgact cattttcaa 10500 aaatcttcta tattttattt agttatttgg tttcaaaagg cctgcactta atttggggg 10560 attatttgga aaaacagcat tgagttttaa tgaaaaaaac ttaaatgccc taacagtaga 10620 aacataaaat tataaataa ctgagctgag cacctgctac tgattagtct atttaatta 10680 agtgggaatg ttttgtagt cctatctaca tctccaggtt taggagcaaa cagagtatgt 10740 tcatagaagg aatatgtgta tggtcttaga atacaatgaa catgttctgc caacttaata 10800 aaggtctgag gagaaagtgt agcaatgtca attcgtgttg aacaatttcc accaacttac 10860 ttataggcgg accttgccaa gtatatctgt gaaaatcaag attcgatctc cagtaaactg 10920 aaggaatgct gtgaaaacc tctgttggaa aaatcccact gcattgccga agtggaaaat 10980 gatgagatgc ctgctgactt gccttcatta gctgctgatt ttgttgaaag taaggatgtt 11040 tgcaaaaact atgctgaggc aaaggatgtc ttcctgggca tgtaagtaga tagaaatta 11100 ttcttttata gctttggcat gacctcacaa cttaggagga tagcctaggc ttttctgtgg 11160 agttgctaca atttccctgc tgcccagaat gtttcttcat ccttcccttt cccaggcttt 11220 11280 gcaaagctca acattcctta ctccttaggg gtatttctga aaatacgtct agaaacattt 11340 tgtgtatata taaattatgt atacttcagt cattcattcc aagtgtattt cttgaacatc 11400 tataatat gtgtgtgact atgtattgcc tgtctatcta actaatctaa tctaatctag 11460 11520 cacaggtgct ttatatttgg tgaaaagacc agaagttcag tataatggca atatggtagg 11580 11640 tttgttgttg gattatgata atgcactaaa taatattcc taaaattatg taccctacaa 11700 gatttcactc atacagagaa gaagagaat attttagaa catatctctg cccatctatt 11760 tatcagaatc cttttgagat gtagtttaaa tcaacaaa tgttataaaacaagt 11820 atcattcatc aaagacttca tatgtgccaa gcagtgtgtg ctttgtgtag attatgtcat 11880 atagttctca taatccacct tccgagacag attackattta tttttgaga cagagtttta 11940 ctcttgttgc ccaggctgga gtgcaatggt gccatctcgg ctcaccacaa ccttcgcctc 12000 ccaggttcaa gcgattctcc tgctcagcc tcctggtt acaggcatgc accaccatgc 12060 ctggctaatt ttgtatttt agtagagatg gggtttcacc atgttggtca gactggtctc 12120 aaactcctga cctctggtga tatgcctgcc tcagcctcct aaagtgctgg gattacaggc 12180 atgagccact gtgcccagcc bakagatact attattatt ccattctacc gagaaggaga 12240 ctaaggctct gatcatttaa atagttgcc taaggtgatg cagtgatata agtagcag 12300 ctaggaattg agccttgta acttactc tggacccca gtccttagct actaagcttt 12360 actgcatggg gtttagtcaa attaagactt ttggaatatg agttacttttt gagattagct 12420 ttgtgatatt ttttgtgctc atttgtccaa caaagtctat tttattttca tcttaattag 12480 gttttgtat gaatatgcaa gaaggcatcc tgattactct gtcgtgctgc tgctgagact 12540 tgccaagaca tatgaaacca ctctagagaa gtgctgtgcc gctgcagatc ctcatgaatg 12600 ctatgccaaa gtggtaggtt tattgttgga aaaaaatgta gttctttgac tgatgattcc 12660 aataatgaga aagaaaata atgcaagaat gtaaaatgat atacagtgca atttagatct 12720 tttcttgaga tggtttcaat tctggaatct taaacatgaa agaaaaagta gccttagaat 12780 gattaacaaa atttagacta gttagaatag aaagatctga atagagcaat ctctaaaaaa 12840 ttttgatcttt ttttctcttt tttcacaatc ctgagaacaa aaaaaaatta aatttaaatg 12900 ttaattagaa gatatttaac ttagatgtaa agtgagttaa cctgattcca ggattaatca 12960 agtactagaa ttagtatctt atggcaaatt atagaaccta tccctttaga atattttcaa 13020 atctttttga ggatgtttag gaatagtttt acaagaaatt aagttaggag aggaaatctg 13080 ttctggagga tttttagggt tcccactagc atatgtaatg gtttctgaac tattcagaat 13140 cagagaaaac tcatttttcc tgctttcaag aagctactgt atgccaggca ccatgcacaa 13200 acaatgacca acgtaaaatc tctcattttg gagagcctgg aatctaactg gaaaaggtgaa 13260 ctaataataa taatatgtac aatcatagcc atcatttatt aaacttttat tatatgcaag 13320 gcactgttta atttcattag cttacctggt ttacagagca gctctatgag atgagtgcca 13380 tctttgcccc tattttaggg ataaggattc cgaaatgtgg agatggtaag taaaattgca 13440 caactgaaga atgagttaca tgacttggct caaatactgg tcattgaact ccagagcctg 13500 aatattctta accacttaca tgatgcaagc tcaccaaata aatagttcga atgtattgtg 13560 acagagcggc attgatattc atctattcat gtggctttga gtaggaagaa gaaaaggatat 13620 cattctgacc agaggggtga aaaacaacct gcatctgatc ctgaggcata atactattaa 13680 cacaattctt ttatgtttca gttcgatgaa tttaaacctc ttgtggaaga gcctcagaat 13740 ttaatcaaac aaattgtga gcttttgag cagcttggag agtacaatt ccagaatgcg 13800 tagtaattt ttattgactg attttttta tcaatttgta atttattag acttaatata 13860 tgagccacct agcatagaac ttttagaat gaaaatacat tgcatatttc taatcactct 13920 ttgtcaagaa agataggaga ggagagataa atagttgat ggggtggaga ggtctatatt 13980 tgaatgtagt ctaaaaattg tctcttag attggagta tgtaggctgg gaggtaaat 14040 accaaatctt ggtatatcag aactgagcat gtcccttga ggttaagaaa tagttaatgg 14100 gcaaatagag catggcaata tttgtagag cagcaagtag taggccttga atagatgtcg 14160 ctcaaaagt atatgtag ctgaacaca aaatgtaca atgaattta gatacatatt 14220 tgaatattaa attcaggttg ttgggagat gcacctagtc ttgatggtt aaacctttcc 14280 ctccatagaa gagacagaga cagaatggct tgctggacta atgtcccaat tcaatagagt 14340 cttatctacg aaggttaaa acagagag acatattata cagtagatat ttattgtgtg 14400 14460 atagtttctt tcctcgagta gattaaagtc atacattgac tttaatggt gactggcatt 14520 cttaatacat gattattata tattaggtac catgtcagat taattataat actttactat 14580 ttttaattta acccttgaac tatccctatt gagtcagata tatttccttc cattttctac 14640 ttgtatcttt caagtttagc atatgctgat acatatgaag ctctctccag gttttattga 14700 aagaagaaat taataaattt attaatgtca ctgaattagg caactcactt tcccaagatt 14760 atgcaagtgg tacaaggtgga actcaaagcc aagtttaact agttgttcag gagaatgttt 14820 tctaccctcc actaacccac tactctgcag atggagataa tatgatgaat ggaacatagc 14880 aacatcttag ttgattccgg ccaagtgttc tctgttttat ctactatgtt agacagtttc 14940 ttgccttgct gaaaacacat gactttttt tttcaggcta ttagttcgtt acaccaagaa 15000 agtaccccaa gtgtcaactc caactcttgt agaggtctca agaaacctag gaaaagtggg 15060 cagcaaatgt tgtaaacatc ctgaagcaaa aagaatgccc tgtgcagaag actatgtgag 15120 tctttaaaaaattaata attaataatg aaaaaattttt acctttagat attgataatg 15180 ctagctttca taagcagaag gaagtaatgt gtgtgtgtgc atgtttgtgt gcatgtgtgt 15240 gtgcatgcac gtgtgtgtat gtgtgatatt ggcagtcaag gccccgagga tgataatttt 15300 ttttttttttgagacgga gtctcgcttt gttgtccagg ctggagtgca gtggtgccat 15360 ctcggctcac tgcacctcc gcctcccaag ttcagccat tctctgcct cagcctccca 15420 agtagctggg actacaggtg catgccacca tgcctggcta attttgta ttttagtag 15480 aaaatttca gctcacctc tttgaattt ctgctctcct gcctgttctt tagctatccg 15540 tggtcctgaa ccagttatgt gtgttgcatg agaaaacgcc agtaagtgac agagtcacca 15600 aatgctgcac agaatccttg gtgaacaggc gaccatgctt ttcagctg gaagtcgatg 15660 aaacatacgt tcccaagg tttaatgctg aaacattcac cttccatgca gatatatgca 15720 cactttctga gaggagaga caatcaga aaaacgtg aggagtattt cattactgca 15780 tgtgtttgta gtcttgatag caagaactgt caattcaagc tagcaacttt ttcctgaagt 15840 agtgattata tttcttagag gaaagtattg gagtgttgcc cttattatgc tgataagagt 15900 acccagaata aaatgaataa ctttttaaag acaaaatcct ctgttataat attgctaaaa 15960 ttattcagag taatattgtg gattaaagcc acaatagaat aacatgttag accatattca 16020 gtagaaaaag atgaacaatt aactgataaa tttgtgcaca tggcaaatta gttaatggga 16080 accataggag aatttatttc tagatgtaaa taattatttt aagtttgccc tatggtggcc 16140 ccacacatga gacaaacccc caagatgtga cttttgagaa tgagacttgg ataaaaaaca 16200 tgtagaaatg caagccctga agctcaactc cctattgcta tcacaggggt tataattgca 16260 taaaatttag ctatagaaag ttgctgtcat ctcttgtggg ctgtaatcat cgtctaggct 16320 taagagtaat attgcaaaac ctgtcatgcc cacacaaatc tctccctggc attgttgtct 16380 ttgcagatgt cagtgaaaga gaaccagcag ctcccatgag tttggatagc cttattttct 16440 atagcctccc cactgaaggg agcaaagttt aagaaccaaa tataaagttt ctcatcttta 16500 tagatgaga aaatttaaa taagtccaa gatatttaa tttttagg tcattttag 16560 ctctttaata gcataaaac tcaatgac attatatggc acttccaaaa tctgaataat 16620 ataatattgc atgacatac ttctttcag agatttactg aaagaaatt tgttgacact 16680 acataacgtg atgagtggtt tatactgat gtttcagttg gtctcccac caactccatg 16740 aaagtggatt ttattatcct catcatgcag atgagaat tgagacttat agcggtatgc 16800 ctggcccaag tactcagagt tgcctggctc cagatttat aatctttaat gatgggacta 16860 ccatccttac tctctccatt ttctatacg tgagtaatgt ttttctgtt tttttttt 16920 ctttttccat tcaactcag tgcactgtt gagctcgtga aacacaagcc caagcaca 16980 aaagagcaac tgaagctgt tattgat ttcgcagctt tgtagagaa gtgctgcaag 17040 gctgacgata aggagacctg ctttgccgag gaggtactac agttctctc attaatat 17100 gtccagtatt cattttgca tgttttgtta ggctagggct taggttta tatatcaaag 17160 gaggctttgt acatgtggga cagggatctt atttacaaa caattgtctt acaaaatgaa 17220 taaaacagca ctttgttttt atctcctgct ctattgtgcc atactgttga atgtttataa 17280 tgcatgttct gtttccaaat ttgtgatgct tatgaatatt aataggaata tttgtaaggc 17340 ctgaaatatt ttgatcatga aatcaaaaca ttaatttatt taaacattta cttgaaatgt 17400 ggtggttgt gatttagttg attttatagg ctagtgggag aatttacatt caaatgtcta 17460 aatcacttaa aatttccctt tatggcctga cagtaacttt ttttattca tttggggaca 17520 actatgtccg tgagcttcca tccagagatt atagtagtaa attgtaatta aaggatatga 17580 tgcacgtgaa atcactttgc aatcatcaat agcttcataa atgttaattt tgtatcctaa 17640 tagtaatgct aatattttcc taacatctgt catgtctttg tgttcagggt aaaaaacttg 17700 ttgctgcaag tcaagctgcc ttaggcttat aacatcacat ttaaaagcat ctcaggtaac 17760 tatattttga atttttaaa aaagtaacta tatagttat tattaaaata gcaaagattg 17820 accatttcca agagccatat agaccagcac cgaccactat tctaaactat ttatgtatgt 17880 aaatattagc tttaaaatt ctcaaaatag ttgctgagtt gggaaccact attatttcta 17940 ttttgtagat gagaaaatga agataaaacat caaagcatag attaagtaat tttccaaagg 18000 gtcaaaattc aaaattgaaa ccaaggtttc agtgttgccc attgtcctgt tctgacttat 18060 atgatgcggt acacagagcc atccaagtaa gtgatggctc agcagtggaa tactctggga 18120 attaggctga accacatgaa agagtgcttt atagggcaaa aacagttgaa tatcagtgat 18180 ttcacatggt tcaacctaat agttcaactc atcctttcca ttggagaata tgatggatct 18240 accttctgtg aactttatag tgaagaatct gctattacat ttccaatttg tcaacatgct 18300 gagctttaat aggacttatc ttcttatgac aacatttatt ggtgtgtccc cttgcctagc 18360 ccaacagaag aattcagcag ccgtaagtct aggacaggct taaattgttt tcactggtgt 18420 aaattgcaga aagatgatct aagtaatttg gcatttattt taataggttt gaaaacaca 18480 tgccatttta caaataagac ttatatttgt ccttttgttt ttcagcctac catgagaata 18540 agagaaagaa aatgaagatc aaaagcttat tcatctgttt ttctttttcg ttggtgtaaa 18600 gccaacaccc tgtctaaaaa acataaattt cttaatcat tttgcctctt ttctctgtgc 18660 ttcaattaat aaaaaatgga aagaatctaa tagagtggta cagcactgtt atttttcaaa 18720 gatgtgttgc tatcctgaaa attctgtagg ttctgtggaa gttccagtgt tctctcttat 18780 tccacttcgg tagaggattt ctagtttctt gtgggctaat taaataaatc attaatactc 18840 ttctaagtta tggattataa acattcaaaa tatattttg acattatgat aattctgaat 18900 aaaagaacaa aaaccatggt ataggtaagg atataaaac atggctttta ccttagaaaa 18960 aacaattcta aaattcatat ggaatcaaaa aagagcctgc ag 19002 <210> 71 <211> 993 <212> PRT <213> Artificial Sequence <220> <223> S283C, K335A, P361C) <400> 71 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Ser Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Cys Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Ala Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Val Phe 340 345 350 Leu Glu Ala Ile Pro Leu Ser Ile Cys Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Glu Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys Gly Gly Ser Gly Gly Ser 385 390 395 400 Gly Gly Ser Gly Gly Ser Gly Gly Asp Ala His Lys Ser Glu Val Ala 405 410 415 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 420 425 430 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 435 440 445 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 450 455 460 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 465 470 475 480 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 485,490,495 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 500 505 510 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 515,520,525 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 530 535 540 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 545 550 555 560 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Phe Thr Glu Cys 565,570,575 Cys Gln Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 580,585,590 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 595,600,605 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 610 615 620 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 625 630 635 640 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 645,650,655 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 660,665,670 Cys Glu Asn Gln Asp Ser Ile Ser Ser Leu Lys Glu Cys Cys Glu 675,680,685 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 690,695,700 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 705 710 715 720 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 725 730 735 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 740,745,750 Thr Thr Thr Thr Thr Glu Lys Cys 755,760,765 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 770,775,780 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 785,790,795,800 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 805 810 815 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 820 825 830 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 835 840 845 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 850 855 860 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 865 870 875 880 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 885 890 895 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 900 905 910 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 915 920 925 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 930 935 940 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 945 950 955 960 Ala Phe Val Glu Lys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 965,970,975 Glu Glu Gly Lys Lys Leu Val Val Ser Ser Gln Ala Leu Gly 980,985,990 Leu

Claims

1. A recombinant protein consisting of: (a) an α-1-antitrypsin serine protease inhibitor domain variant, wherein the α-1-antitrypsin serine protease inhibitor domain variant is generated by generating the following mutations based on the α-1-antitrypsin serine protease inhibitor domain shown in SEQ ID NO: 1: C232S, S283C, K335A, M351V, M358L and P361C; (b) a linker consisting of the sequence shown in SEQ ID NO: 63; and (c) Human serum albumin domain.

2. The recombinant protein according to claim 1, wherein the human serum albumin domain is wild-type human serum albumin.

3. The recombinant protein according to claim 1 or 2, wherein the recombinant protein consists of the sequence shown in SEQ ID NO:71.

Images