Compositions and methods for alpha-1-antitrypsin deficiency

Abstract

Disclosed herein are compositions and methods useful for treating alpha-1-antitrypsin deficiency.

Description

[Background technology]

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

[0002] Serpins are a superfamily of structurally similar proteins that were first identified for their ability to inhibit proteases. The name serpin was originally coined because the first identified serpins were found to act against chymotrypsin-like serine proteases. As a result, ser ine p rotease in The acronym serpin was chosen to incorporate the combination of the first letters of the word hibitor (serine protease inhibitor).

[0003] Serpins are of interest due to their unusual mechanism of action: they irreversibly inhibit target proteases by undergoing large conformational changes that disrupt their active sites. Serpin inhibition of proteases regulates numerous biological processes, including coagulation and inflammation. While this mechanism of protease inhibition offers certain advantages, it also has drawbacks. One major drawback is that serpins are particularly vulnerable to mutations that can result in serpinopathies, such as protein misfolding and the formation of inactive long-chain polymers.

[0004] Alpha-1-antitrypsin or α1-antitrypsin ("A1AT," "A1A," or "AAT") is a protein belonging to the serpin superfamily. In humans, alpha-1-antitrypsin is encoded by the SERPINA1 gene, which has four coding exons and three noncoding exons and is located at q31-31.2 on chromosome 14. Alpha-1-antitrypsin is also known as alpha-1-proteinase inhibitor or alpha-1-antiproteinase because it inhibits various proteases, not just trypsin. Alpha-1-antitrypsin is a 52-kD glycoprotein that functions as an antiprotease. Alpha-1-antitrypsin has a single chain of 394 amino acids, followed by a 24-amino acid signal peptide. Alpha-1-antitrypsin, as a type of enzyme inhibitor, protects tissues from inflammatory cell enzymes, particularly neutrophil elastase and proteinase 3 ("PR3"), and has a normal reference range in blood of 1.5-3.5 g / L, which can increase many-fold during acute inflammation.

[0005] Neutrophil elastase is a serine protease that destroys elastase, a rubbery macromolecule that provides elastic recoil to the lungs. When blood does not contain sufficient alpha-1-antitrypsin or contains functionally defective alpha-1-antitrypsin, neutrophil elastase is free to destroy elastin excessively, resulting in reduced lung elasticity. This reduced elasticity can result in respiratory complications such as chronic obstructive pulmonary disease.

[0006] PR3 is a serine protease enzyme expressed primarily in neutrophil granulocytes. Its exact role in neutrophil function is unknown, but in human neutrophils, proteinase 3 contributes to the proteolysis of antimicrobial peptides.

[0007] Alpha-1-antitrypsin deficiency ("AATD") is a genetic disorder that results in insufficient production of alpha-1-antitrypsin. AATD is an autosomal recessive genetic disorder caused by reduced serum levels of AATD. This disease leads to decreased alpha-1-antitrypsin. This disease often causes lung and liver disease and is called hereditary emphysema. AATD is one of the most common fatal genetic disorders in Caucasians of European descent.

[0008] Alpha-1 antitrypsin deficiency comes in several forms and degrees, depending on whether the patient has one or two copies of the defective allele. Severe alpha-1 antitrypsin deficiency can cause panlobular emphysema or COPD-like symptoms in adulthood in many affected individuals, especially if they are exposed to tobacco smoke. The disease can also lead to various liver diseases in children and adults, and in some cases, more rare problems. Alpha-1 antitrypsin deficiency usually results in some degree of disability and a shortened life expectancy.

[0009] Alpha-1 antitrypsin deficiency is treated by intravenous infusion of alpha-1 antitrypsin or by liver or lung transplantation, avoiding harmful inhalants. Recombinant forms of alpha-1 antitrypsin are also known but are currently used in medical research rather than as treatments. Approved formulations for alpha-1 antitrypsin deficiency are purified from human plasma, require frequent weekly infusions at high doses (60–120 mg / kg), and provide limited efficacy. Alpha-1 antitrypsin-boosting therapy is not ideal due to poor pharmacokinetics, as evidenced by peak and trough circulating AAT levels, despite administering high concentrations of plasma-derived AAT.

[0010] Some recent research has focused on developing long-acting alpha-1-antitrypsin fusions via protein engineering. For example, AAT Fc has been expressed and developed by two groups: Soohyun Kim (Konkuk University, Seoul, Korea); licensed to OmniBio ("Kim / OmniBio molecule") (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 Fe-fused (AAT-Fc) protein on the inhibition of inflammatory cytokine production and streptozotocin-induced diabetes. Mol Med. 2013 May 20;19:65-71. doi:10.2119 / molmed.2012.00308. PubMed PMID: 23552726; PubMed Central See PMCID: PMC3667213, which is incorporated by reference in its entirety, including any drawings) and InhibRx ("InhibRx molecule") (see, e.g., U.S. Pat. No. 8,980,266 B2, Serpin Fusion Polypeptides and Methods and Use Thereof, which is incorporated by reference in its entirety, including any drawings). Both molecules are N-terminal AAT fusions with IgG1 Fc. The Kim / OmniBio molecule does not inhibit human neutrophil elastase as well as plasma-derived AAT, which may result in low pH lability upon elution from Protein A chromatography during purification. The InhibRx molecule has been reported to inhibit human neutrophil elastase as well as plasma-derived AAT.

[0011] Others have demonstrated that N-glycans are more easily cleaved 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 by reference in its entirety, including any drawings), or by adding 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 US A. 2011 May 3;108(18):7397-402. doi:10.1073 / pnas.1019266108. Epub 2011 Apr 18. PubMed PMID: 21502532; PubMed Central PMCID: PMC3088639, which is incorporated by reference in its entirety, including any drawings), modified the glycosylation pattern of AAT.Some have produced aglycosylated AAT (see, e.g., Cantin AM, Woods DE, Cloutier D, Heroux 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. 2002 Summer;15(2):141-8. PubMed PMID: 12184864, which is incorporated by reference in its entirety, including any drawings). Still others have developed aglycosylated AAT that has been PEGylated to extend its 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 by reference in its entirety, including any drawings).

[0012] Because AAT is prone to Met oxidation in the lung, some groups have constructed mutant AAT variants that are resistant to methionine oxidation but retain activity. In particular, 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 by reference in its entirety, including any drawings). Baek et al. introduced a disulfide bond (K168C-F189C) into AAT, substantially increasing its resistance to loop-sheet polymerization (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 by reference in its entirety, including any drawings).Single point mutations (F51L, G117F, K331F, K335A) engineered into AAT increased stability and resistance to loop / sheet polymerization (Dafforn TR, Mahadeva R, Elliott PR, Sivasothy P, Lomas DA. A kinetic mechanism for the polymerization of alpha1-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 alpha1-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. J Biol Chem. 1999 Apr 16; 274(16):11072-7. PubMed PMID: 10196190, each of which is incorporated by reference herein in its entirety, including any drawings. However, despite the above-mentioned advances, there is a need for additional therapeutic options. [Prior art documents]

Charter Documents

[0013] [Patent Document 1] U.S. Patent No. 8,980,266

Non-licensed literature

[0014]

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[0015] 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. Some embodiments include recombinant proteins comprising an alpha-1-antitrypsin serpin domain and a human serum albumin binding domain or a human serum albumin domain. In some embodiments, the alpha-1-antitrypsin serpin domain has a sequence comprising all or a portion 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 serpin domain is fused to the N-terminus of human serum albumin or the human serum albumin binding domain in the recombinant protein. Some embodiments include recombinant proteins comprising an alpha-1-antitrypsin serpin domain and a human serum albumin domain, wherein neither the alpha-1-antitrypsin serpin domain nor the human serum albumin domain is wild-type alpha-1-antitrypsin or wild-type human serum albumin.

[0016] In some embodiments, the alpha-1-antitrypsin serpin domain comprises one or more mutations. In some embodiments, the alpha-1-antitrypsin serpin domain retains activity compared to a wild-type alpha-1-antitrypsin serpin 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 at residues numbered according to SEQ ID NO: 1. In some embodiments, the one or more mutations include K168C and F189C at residues numbered according to SEQ ID NO: 1. In some embodiments, the one or more mutations confer substantially increased resistance to loop-sheet polymerization compared to wild-type alpha-1-antitrypsin. In some embodiments, the one or more mutations are one or more point mutations selected from F51L, G117F, K331F, or K335A at residues 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 the wild-type alpha-1-antitrypsin serpin domain.

[0017] In some embodiments, the one or more mutations comprise a mutation at 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. In some embodiments, the one or more mutations include one or more of the following mutations in SEQ ID NO: 1: F51L, L100F, L100C, T114F, G117F, K163T, G164V, T165S, K168C, K168I, K168A, K169V, L172V, L172A, L172C, V173C, K174T, A183V, F189C, F 189I, F189V, C232S, S283C, K300A, V302A, L303A, G304A, 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 in SEQ ID NO: 1. In some embodiments, the one or more mutations are both K168C and F189C in SEQ ID NO: 1. In some embodiments, the one or more mutations are both P361C and S283C in SEQ ID NO: 1.

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

[0019] In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including F51L, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including G117F, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S, K331F, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S, K335A, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including K168C, F189C, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S, S283C, M351V, M358L, and P361C. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including F51L, G117F, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including F51L, C232S, K331F, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including F51L, C232S, K335A, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including K51L, K168C, F189C, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including F51L, C232S, S283C, M351V, M358L, and P361C.In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including G117F, C232S, K331F, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including G117F, K335A, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including G117F, K168C, F189C, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including G117F, C232S, S283C, M351V, M358L, and P361C. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S, K331F, K335A, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including K168C, F189C, C232S, K331F, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S, S283C, K331F, M351V, M358L, and P361C. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including K168C, F189C, C232S, K335A, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1, including C232S, S283C, K335A, M351V, M358L, and P361C. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1, including K168C, F189C, C232S, S283C, M351V, M358L, and P361C.In some embodiments, the alpha-1-antitrypsin serpin domain comprises the point mutation K335A in SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serpin domain comprises the point mutation K331F in SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serpin domain comprises the point mutation G117F in SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serpin domain comprises the point mutation S283C in SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serpin domain comprises the point mutation P361C in 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 NOs: 22-36. In some embodiments, the human serum albumin binding domain comprises one or more CDRs selected from SEQ ID NOs: 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 serpin domain compared to wild-type alpha-1-antitrypsin. In some embodiments, the recombinant protein has a higher number of N-glycans or polysialylation in the alpha-1-antitrypsin serpin domain than the wild-type alpha-1-antitrypsin serpin 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, which are one or more residues numbered according to SEQ ID NO: 5. In some embodiments, the one or more mutations are one or more of residues L407A, L408V, V409A, R410A, L413Q, and L414Q, which are one or more residues 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 a human serum albumin protein having the sequence set forth 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 that encodes a recombinant protein provided herein. In some embodiments, the nucleic acid comprises a vector. In some embodiments, the vector comprises a host cell.

[0025] Some embodiments are directed to methods for producing a recombinant protein, comprising culturing a host cell with one of the nucleic acids provided herein and recovering 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 recovered using a pH-neutral alpha-1-antitrypsin-Fc capture selection medium.

[0026] Some embodiments are directed to methods of treating alpha-1-antitrypsin deficiency in a patient in need thereof, comprising administering to the patient a recombinant protein provided herein. In some embodiments, the recombinant protein comprises a pharmaceutical composition. In some embodiments, the recombinant protein is administered about or more than once a week, about or more than once every 10 days, about or more than once every 15 days, about or more than once every 20 days, about or more than once every 25 days, about or more than once every month, or about or more than once every two months.

[0027] Some embodiments are directed to methods of improving the pharmacokinetics of a protein comprising an alpha-1-antitrypsin serpin domain. In some embodiments, the protein is any of the recombinant proteins described herein. In some embodiments, the improved pharmacokinetics refers to an improvement relative to prolastin. In particular embodiments, for example, the following items are provided: (Item 1) (a) an alpha-1-antitrypsin serpin domain; (b) Human serum albumin binding domain and A recombinant protein comprising: (Item 2) 2. The recombinant protein of item 1, further comprising a linker consisting of SEQ ID NO: 63. (Item 3) 3. The recombinant protein of any one of items 1 to 2, wherein the C-terminus of the alpha-1-antitrypsin serpin domain is fused to the N-terminus of the human serum albumin binding domain. (Item 4) 4. The recombinant protein of any one of items 1 to 3, wherein the human serum albumin binding domain is at least a portion of human serum albumin having either SEQ ID NO: 3 or SEQ ID NO: 5. (Item 5) 5. The recombinant protein of any one of items 1 to 4, wherein the half-life of the alpha-1-antitrypsin serpin domain is increased compared to wild-type alpha-1-antitrypsin. (Item 6) 6. The recombinant protein of any one of items 1 to 5, wherein the number of N-glycans or polysialylation sites in the alpha-1-antitrypsin serpin domain of the recombinant protein is greater than that of a wild-type alpha-1-antitrypsin serpin domain. (Item 7) 7. The recombinant protein of any one of items 1 to 6, wherein the alpha-1-antitrypsin serpin domain has one or more mutations compared to the wild-type alpha-1-antitrypsin serpin domain. (Item 8) 8. The recombinant protein of item 7, wherein the one or more mutations cause resistance to methionine oxidation compared to the wild-type alpha-1-antitrypsin serpin domain. (Item 9) 9. The recombinant protein according to item 7 or 8, wherein the alpha-1-antitrypsin serpin domain retains activity compared to the wild-type alpha-1-antitrypsin serpin domain. (Item 10) 9. The recombinant protein according to item 7 or 8, wherein the one or more mutations comprise M351V and / or M358V at residues numbered according to SEQ ID NO: 1. (Item 11) 11. The recombinant protein of any one of items 7 to 10, wherein the one or more mutations comprise K168C and F189C at residues numbered according to SEQ ID NO: 1, and wherein the one or more mutations confer increased resistance to loop-sheet polymerization compared to wild-type alpha-1-antitrypsin. (Item 12) 12. The recombinant protein according to any one of items 7 to 11, wherein the one or more mutations are one or more point mutations selected from F51L, G117F, K331F, or K335A at residues numbered according to SEQ ID NO: 1. (Item 13) 13. The recombinant protein of any one of items 1 to 12, wherein the alpha-1-antitrypsin retains activity against human neutrophil elastase and / or PR3 compared to wild-type alpha-1-antitrypsin. (Item 14) 14. The recombinant protein according to any one of items 1 to 13, wherein the human serum albumin binding domain comprises a polypeptide having a sequence set forth in any one of SEQ ID NOs: 22 to 36. (Item 15) 15. An isolated nucleic acid encoding the recombinant protein of any one of items 1 to 14. (Item 16) A vector comprising the isolated nucleic acid of item 15. (Item 17) A host cell comprising the vector according to item 16. (Item 18) 18. A method for producing a recombinant protein, comprising culturing the host cell of item 17 and recovering the recombinant protein. (Item 19) 19. The method of claim 17 or 18, wherein the host cell is a mammalian host cell. (Item 20) 20. The method of item 19, wherein the mammalian host is a CHO cell. (Item 21) 21. The method of any one of items 18 to 20, wherein the recombinant protein is recovered using a pH-neutral alpha-1-antitrypsin-Fc capture selection medium. (Item 22) 15. A method for treating or prophylactically treating alpha-1-antitrypsin deficiency in a patient in need thereof, comprising administering to said patient a recombinant protein according to any one of items 1 to 14. (Item 23) 23. The method of item 22, wherein the recombinant protein is administered about or more than once a week, about or more than once every 10 days, about or more than once every 15 days, about or more than once every 20 days, about or more than once every 25 days, about or more than once every month, or about or more than once every two months. (Item 24) (a) an alpha-1-antitrypsin serpin domain; (b) Human serum albumin domain and A recombinant protein comprising: (Item 25) 25. The recombinant protein of item 24, wherein the human serum albumin domain is wild-type human serum albumin. (Item 26) 26. The recombinant protein of any one of items 24 to 25, comprising a linker having the sequence set forth in SEQ ID NO: 63. (Item 27) 27. The recombinant protein of any one of items 24 to 26, wherein the C-terminus of the alpha-1-antitrypsin serpin domain is fused to the N-terminus of the human serum albumin domain. (Item 28) 28. The recombinant protein of any one of items 24 to 27, wherein the human serum albumin domain increases the plasma half-life of the alpha-1-antitrypsin serpin domain compared to wild-type alpha-1-antitrypsin. (Item 29) 29. The recombinant protein of any one of items 24 to 28, wherein the number of N-glycans or polysialylation sites in the alpha-1-antitrypsin serpin domain of the recombinant protein is greater than that of a wild-type alpha-1-antitrypsin serpin domain. (Item 30) 30. The recombinant protein of any one of items 24 to 29, wherein the alpha-1-antitrypsin serpin domain has one or more mutations compared to wild-type alpha-1-antitrypsin. (Item 31) 31. The recombinant protein of item 30, wherein the one or more mutations cause resistance to methionine oxidation compared to wild-type alpha-1-antitrypsin. (Item 32) 32. The recombinant protein according to item 30 or 31, wherein the one or more mutations comprise C232S, M351V, M358L and / or M358V at residues numbered according to SEQ ID NO: 1. (Item 33) 33. The recombinant protein of any one of items 30 to 32, wherein the one or more mutations comprise K168C and F189C at residues numbered according to SEQ ID NO: 1, and wherein the one or more mutations confer substantially increased resistance of alpha-1-antitrypsin to loop-sheet polymerization compared to wild-type alpha-1-antitrypsin. (Item 34) 34. The recombinant protein of any one of items 30 to 33, wherein the one or more mutations are one or more point mutations selected from F51L, G117F, S283C, K331F, K335A, or P361C at residues numbered according to SEQ ID NO: 1. (Item 35) 35. The recombinant protein of any one of items 30 to 34, wherein the one or more mutations comprise a mutation selected from C232S in SEQ ID NO: 1, M351V and M358V in SEQ ID NO: 1, M351V and M358L in SEQ ID NO: 1, and C232S, M351V and M358V in SEQ ID NO: 1. (Item 36) The alpha-1 antitrypsin serpin domain may be one or more mutations in SEQ ID NO:1 including F51L, C232S, M351V and M358L, one or more mutations in SEQ ID NO:1 including G117F, C232S, M351V and M358L, one or more mutations in SEQ ID NO:1 including C232S, K331F, M351V and M358L, one or more mutations in SEQ ID NO:1 including C232S, K335A, M351V and M358L, one or more mutations in SEQ ID NO:1 including K168C, F189C, C232S ... one or more mutations including C232S, S283C, M351V, M358L and P361C in SEQ ID NO: 1, one or more mutations including F51L, G117F, C232S, M351V and M358L in SEQ ID NO: 1, one or more mutations including F51L, C232S, K331F, M351V and M358L in SEQ ID NO: 1, one or more mutations including F51L, C232S, K335A, M351V and M358L in SEQ ID NO: 1, one or more mutations including K51L, K168C, F189C, C232S, M351V and M358L in SEQ ID NO: 1, 1, one or more mutations including G117F, C232S, K331F, M351V and M358L in SEQ ID NO: 1, one or more mutations including G117F, K335A, C232S, M351V and M358L in SEQ ID NO: 1, one or more mutations including G117F, K168C, F189C, C232S, M351V and M358L in SEQ ID NO: 1, one or more mutations including G117F, C232S, S283C, M351V, M358L and P361C in SEQ ID NO: 1 a number of mutations, one or more mutations including C232S, K331F, K335A, M351V and M358L in SEQ ID NO: 1, one or more mutations including K168C, F189C, C232S, K331F, M351V and M358L in SEQ ID NO: 1, one or more mutations including C232S, S283C, K331F, M351V, M358L and P361C in SEQ ID NO: 1, one or more mutations including K168C, F189C, C232S, K335A, M351V and M358L in SEQ ID NO: 1, C232S, S283C, K335A, M351V,36. The recombinant protein according to any one of items 30 to 35, having one or more mutations including M358L and P361C, or one or more mutations in SEQ ID NO: 1 including K168C, F189C, C232S, S283C, M351V, M358L and P361C. (Item 37) 37. The recombinant protein of any one of items 24 to 36, wherein the alpha-1-antitrypsin retains activity compared to wild-type alpha-1-antitrypsin. (Item 38) 38. The recombinant protein of any one of items 24 to 37, wherein the alpha-1-antitrypsin retains activity against human neutrophil elastase and / or PR3 compared to wild-type alpha-1-antitrypsin. (Item 39) 39. An isolated nucleic acid encoding the recombinant protein of any one of items 24 to 38. (Item 40) A vector comprising the isolated nucleic acid of item 39. (Item 41) A host cell comprising the vector of Item 40. (Item 42) 42. A method for producing a recombinant protein, comprising culturing the host cell of item 41 and recovering the recombinant fusion protein. (Item 43) 43. The method of item 42, wherein the host cell is a eukaryotic cell. (Item 44) 44. The method of item 43, wherein the eukaryotic cell is a yeast cell. (Item 45) 45. The method of item 44, wherein the host cell is a mammalian host cell. (Item 46) 46. ​​The method of item 45, wherein the mammalian host is a CHO cell. (Item 47) 47. The method of any one of items 40 to 46, wherein the recombinant protein is recovered using a neutral pH alpha-1-antitrypsin-Fc selection medium. (Item 48) 39. A method for treating or prophylactically treating alpha-1-antitrypsin deficiency in a patient in need thereof, comprising administering to said patient a recombinant protein according to any one of items 1 to 14 or 24 to 38. (Item 49) 49. The method of item 48, wherein the recombinant protein is administered about or more than once a week, about or more than once every 10 days, about or more than once every 15 days, about or more than once every 20 days, about or more than once every 25 days, about or more than once every month, or about or more than once every two months. (Item 50) (a) an alpha-1-antitrypsin serpin domain; (b) Human serum albumin domain and A recombinant protein comprising: The alpha-1-antitrypsin serpin domain and the human serum albumin are recombinant proteins, wherein the alpha-1-antitrypsin serpin domain and the human serum albumin are not wild-type alpha-1-antitrypsin and wild-type human serum albumin. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 depicts the protein and gene structure of alpha-1-antitrypsin. The upper diagram shows the normal protein in its linear form, a 394 amino acid single-chain protein with three asparaginyl-linked complex carbohydrate side chains at residues 46, 83, and 247 according to SEQ ID NO: 1. The lower diagram shows the gene (SEQ ID NO: 69), which consists of three non-coding exons (IA, IB, and IC) and four coding exons (II to V). Alpha helices are indicated by boxes, and beta sheets are indicated by zigzag lines. The start codon is within exon II, followed by a 24-residue signal peptide. The carbohydrate attachment site is indicated in the diagram as CHO. [Figure 2] Figure 2 shows the location of the alpha-1-antitrypsin gene and associated mutations associated with alpha-1-antitrypsin deficiency. [Figure 3] FIG. 3 shows the alpha-1-antitrypsin secondary structure. [Figure 4A]Figure 4 shows purified CE-SDS for constructs of the invention. Figure 4A shows purified CE-SDS for PP11288. Figure 4B shows purified CE-SDS for PP11289. Figure 4C shows purified CE-SDS for PP11290. Figure 4D shows purified CE-SDS for PP11291. Figure 4E shows purified CE-SDS for PP11292. Figure 4F shows purified CE-SDS for purified samples (reducing overlay). Figure 4G shows purified CE-SDS for purified samples (non-reducing conditions). [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 4D] Same as above. [Figure 4E] Same as above. [Figure 4F] Same as above. [Figure 4G] Same as above. [Figure 5] FIG. 5 depicts the stability analysis of five AAT-albumin fusions. [Figure 6] FIG. 6 shows the results of an hNE inhibition assay with five AAT-albumin fusions. [Figure 7A]Figure 7 shows purified CE-SDS for constructs of the present invention. Figure 7A shows purified CE-SDS for PP13579. Figure 7B shows purified CE-SDS for PP13580. Figure 7C shows purified CE-SDS for PP13581. Figure 7D shows purified CE-SDS for PP13582. Figure 7E shows purified CE-SDS for PP13583. Figure 7F shows purified CE-SDS for PP13584. Figure 7G shows purified CE-SDS for PP13585. Figure 7H shows purified CE-SDS for PP13586. Figure 71 shows purified CE-SDS for PP13587. Figure 7J shows purified CE-SDS for PP13588. Figure 7K shows purified CE-SDS for PP13589. Figure 7L shows purified CE-SDS for PP13590. Figure 7M represents purified CE-SDS for PP13592. Figure 7N represents purified CE-SDS for PP13593. Figure 7O represents purified CE-SDS for PP13594. Figure 7P represents purified CE-SDS for PP13595. Figure 7Q represents purified CE-SDS for PP13596. Figure 7R represents purified CE-SDS for PP13597. Figure 7S represents purified CE-SDS for PP13598. Figure 7T represents purified CE-SDS for PP13599. Figure 7U represents purified CE-SDS for PP13600. [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 7D] Same as above. [Figure 7E] Same as above. [Figure 7F] Same as above. [Figure 7G] Same as above. [Figure 7H] Same as above. [Figure 7I] Same as above. [Figure 7J] Same as above. [Figure 7K] Same as above. [Figure 7L] Same as above. [Figure 7M] Same as above. [Figure 7N] Same as above. [Figure 7O] Same as above. [Figure 7P] Same as above. [Figure 7Q] Same as above. [Figure 7R] Same as above. [Figure 7S] Same as above. [Figure 7T] Same as above. [Figure 7U] Same as above. [Figure 8] FIG. 8 depicts the stability analysis of 21 AAT-albumin fusions. [Figure 9A] Figure 9 shows IC50 analysis of AAT mutants. Figure 9A shows IC50 analysis of AAT mutants. Figure 9B shows IC50 analysis of AAT mutants. Figure 9C shows IC50 analysis of AAT mutants. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 10] FIG. 10 presents the normalized EpiMatrix protein scores plotted on a standardized scale. [Figure 11] FIG. 11 shows the stability analysis of AAT variants (K335A, S283C / P361C) at the base (C232S, M351V, M351L). [Figure 12] FIG. 12 depicts cynomolgus monkey (cyno) PK data. [Figure 13] FIG. 13 shows that AAT-HSA and prolastin after PK in monkeys at 10 mg / kg were three compartments. DETAILED DESCRIPTION OF THE INVENTION

[0029] Terminology As used herein, the term "about" refers to approximate mathematical quantities and may be used for percentages as well as integer values. When an upper or lower limit includes an integer value, the term about refers to the approximate boundary, and the term also includes everything above that boundary (relative to the lower limit) or below that boundary (relative to the upper limit). For example, a portion of a 393-residue sequence that includes "about" 20 residues includes the boundary that is approximately 20 residues and all integer values ​​above that value.

[0030] As used herein, "albumin" refers to albumin protein, or a fragment, portion, or variant of albumin having one or more functional activities of albumin, or a nucleic acid sequence encoding an albumin protein, fragment, portion, or variant thereof having one or more functional activities of albumin. Albumin can be from any vertebrate, particularly any mammal. In some embodiments, albumin is from human, bovine, ovine, or porcine. Non-mammalian albumins include, but are not limited to, hen and salmon. In some embodiments, albumin from any vertebrate can be substituted for human albumin or human serum albumin.

[0031] As used herein, the term "antibody" refers to a type of immunoglobulin molecule and is used in its broadest sense. Antibodies specifically include intact antibodies (e.g., intact immunoglobulins) and antibody fragments. Antibodies contain at least one antigen-binding domain. An example of an antigen-binding domain is V H -V L The antigen-binding domain is formed by a dimer. Other examples of antibodies are described in the following sections.

[0032] V H and V L The regions can be further subdivided into regions of hypervariability ("hypervariable regions (HVRs)", also referred to as CDRs as defined herein), interspersed with more conserved regions. The more conserved regions are referred to as framework regions (FRs).H and V L Each contains three CDRs and four FRs, generally arranged in the following order (N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, which is incorporated by reference in its entirety.

[0033] Light chains from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of their constant domain.

[0034] 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 designated α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on sequence and functional differences. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0035] "Antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments.

[0036] An "Fv" fragment comprises a non-covalently associated dimer of one heavy- and one light-chain variable domain.

[0037] A "Fab" fragment contains the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ) Fab fragments can be generated, for example, by papain digestion of a full-length antibody.

[0038] An "F(ab')2" fragment contains two Fab' fragments linked near the hinge region by a disulfide bond. F(ab')2 fragments can be generated, for example, by pepsin digestion of an intact antibody. F(ab')2 fragments can be isolated, for example, by treatment with β-mercaptoethanol.

[0039] "Single-chain Fv" or "sFv" or "scFv" antibody fragments consist of VFs in a single polypeptide chain linked by a peptide linker. H Domain and V L See Pluckthun A. (1994). An "scFv-Fc" fragment comprises an scFv attached to an Fc domain. For example, the Fc domain can be attached to the C-terminus of the scFv. The Fc domain can be attached to the V H or V L This can be followed by the arrangement of the variable domains in the scFv (i.e., V H -V L or V L -V H Any suitable Fc domain known in the art or described herein can be used.

[0040] "Humanized" forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are generally human immunoglobulins (recipient antibody) in which residues from one or more CDRs are substituted by residues from one or more CDRs of a non-human antibody (donor antibody).

[0041] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or human antibody coding sequences (e.g., obtained from a human or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0042] As used herein, a "single domain antibody" refers to an antibody whose CDRs are 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 traditional 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, mouse, human, camel, llama, goat, rabbit, shark, and cow. By way of example, single domain antibodies can be raised in camelid species such as camel, dromedary, alpaca, and guanaco. In some embodiments, a single domain antibody comprises the sequence set forth in any one of SEQ ID NOS: 22-28 and 29-36.

[0043] As used herein, a "complementarity determining region" or "CDR" is a portion of the variable chain in an antibody. CDRs are important for a specific antibody. Those skilled in the art will recognize how to determine the location of CDRs in an antibody for a specific antibody, and several systems exist for determining the location of CDRs. For clarity, in this application, the CDRs of single domain antibodies will be determined according to U.S. Patent Application Publication No. 2010 / 011339, which is incorporated herein by reference in its entirety.

[0044] The term "epitope" refers to a portion of an antigen that is capable of specific binding to a binding protein, such as, but not limited to, an antibody. Epitopes often consist of surface-accessible amino acid residues and / or sugar side chains and may have specific charge characteristics as well as specific three-dimensional structural characteristics. Epitopes may include amino acid residues directly involved in the binding and other amino acid residues not directly involved in the binding. The epitope to which an antibody binds can be determined using known techniques for determining epitopes.

[0045] As used herein, "human albumin" refers to human albumin protein, or a fragment, variant, or portion of human albumin having one or more functional activities of human albumin, or a nucleic acid sequence encoding human albumin, a fragment, portion, or variant thereof, having one or more functional activities of human albumin. Human albumin is a 609 amino acid polypeptide sequence, the first 18 amino acids of which constitute a leader sequence not found in final blood-derived products. In some embodiments, human albumin is set forth as SEQ ID NO: 3.

[0046] As used herein, "human serum albumin" refers to human serum albumin present in human blood, which typically lacks the first 18 amino acid leader sequence. In some embodiments, the human serum albumin has the sequence set forth in SEQ ID NO: 5. A human serum albumin domain also refers to any human serum albumin protein, or a fragment or variant or portion of human serum albumin that has one or more functional activities of human serum albumin.

[0047] As used herein, "human serum albumin binding protein" or "human albumin binding domain" refers to any protein or protein fragment or variant that has one or more binding activities for human albumin or human serum albumin.

[0048] As used herein, "alpha-1-antitrypsin" or "α1-antitrypsin" (also "A1AT," "A1A," or "AAT") refers to an alpha-1-antitrypsin protein, or a fragment or variant of alpha-1-antitrypsin having one or more functional activities of alpha-1-antitrypsin, or a nucleic acid sequence encoding an alpha-1-antitrypsin protein, 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 set forth in SEQ ID NO:1.

[0049] As used herein, "alpha-1-antitrypsin serpin domain" or "α1-antitrypsin serpin domain" (also "A1AT domain," "A1A domain," or "AAT domain") is intended to refer to an alpha-1-antitrypsin serpin domain, or a fragment or variant of an alpha-1-antitrypsin serpin domain that has one or more functional activities of an alpha-1-antitrypsin protein or fragment, or a nucleic acid sequence that encodes an alpha-1-antitrypsin protein serpin domain or fragment or variant thereof that has one or more functional activities of an alpha-1-antitrypsin protein.

[0050] As used herein, "alpha-1-antitrypsin deficiency" or "AATD" refers to a genetic disorder that results in insufficient production of alpha-1-antitrypsin. This disorder, also known as hereditary emphysema, leads to reduced levels of alpha-1-antitrypsin and results in lung and liver disease. The deficiency exists in several forms and degrees, depending on whether the patient has one or two copies of the defective allele. Severe alpha-1-antitrypsin deficiency leads to panlobular emphysema or COPD in adulthood in many affected individuals, especially if they are exposed to tobacco smoke. The disorder can also lead to various liver diseases in a small number of children and adults, and in some cases, to rarer problems. Alpha-1-antitrypsin deficiency usually results in some degree of disability and a shortened life expectancy.

[0051] An "effective amount" of a recombinant protein or functional fragment or variant thereof as described herein refers to that amount of polypeptide or functional fragment or variant thereof that, when administered as a collection of multiple doses, or as part of any other type of defined treatment regimen, results in a measurable statistical improvement in prognosis or prevention as evidenced by at least one clinical parameter associated with the complication.

[0052] As used herein, "neutrophil elastase" refers to a serine protease that destroys elastase, a rubber-like macromolecule that provides elastic recoil to the lungs. When blood contains insufficient AAT, neutrophil elastase can destroy elastin, reducing lung elasticity and resulting in respiratory complications such as chronic obstructive pulmonary disease.

[0053] As used herein, the term "moiety" is intended to mean a part or fragment of something whole, such as a protein or polypeptide.

[0054] As used herein, "recombinant" with respect to a protein or polypeptide molecule refers to a protein or polypeptide molecule that is expressed using an isolated or recombinant nucleic acid molecule.

[0055] As used herein, the term "variant" refers to different sequences of the protein, including but not limited to, for example, insertions, deletions, and substitutions, either conservative or non-conservative, which do not substantially alter one or more of the colloidally useful ligand-binding and non-immunogenic properties of albumin or albumin-binding domains, or which do not alter the serpin domain.

[0056] 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. Calculations of "identity" or "sequence homology" between two sequences (the terms are used interchangeably herein) are performed by alignment for optimal comparison (e.g., gaps can be inserted in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). 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 that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent 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 either been chemically modified and / or contain one or more amino acid sequence alterations compared to the human wild-type polypeptide.

[0057] Variants of the polypeptides described herein can have amino acid modifications (e.g., deletions, additions, or substitutions, such as conservative substitutions) relative to the wild-type amino acid sequence of the polypeptide. In some embodiments, variants of AAT can 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 can vary from human serum albumin (SEQ ID NO: 5) by at least 1, 2, 3, 4, or 5 residues.

[0058] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having 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), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and 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.

[0059] The term "fusion" or "fusion molecule" can refer to a fusion of polypeptides or nucleic acids, depending on the context. It can include full-length sequences or proteins or nucleic acids or fragments thereof. It can also include recombinant proteins with or without linker molecules.

[0060] An "isolated" protein refers to a protein that is recovered from at least about 90% of at least one component of the natural sample from which it is obtained. A protein may be "at least approximately" pure if the species or population of species of interest is at least about 5, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on a weight-by-weight basis.

[0061] As used herein, the term "colloidal" refers to colloid osmotic pressure or oncotic colloid osmotic pressure. It is the form of colloid osmotic pressure exerted by proteins, particularly albumin, in vascular plasma that draws water into the circulatory system. It is the counter force to hydrostatic pressure. It has a major influence on the pressure across the glomerular filter.

[0062] A "patient," "subject," or "host" (these terms are used interchangeably) to be treated by the subject method can refer to either a human or non-human animal. Preferably, patient, subject, or host refers to a human patient.

[0063] The term "prophylactically treating" a disease in a subject refers to subjecting the subject to a drug treatment, e.g., administering an agent to prophylactically treat at least one symptom of the disease, i.e., administering the agent prior to the clinical appearance of the undesired symptom, such that the agent protects the host from developing the undesired symptom. "Prophylactically treating" a disease can also be referred to as "prevention." In some embodiments, prophylactic treatment prevents the disease.

[0064] As used herein, "treating" an alpha-1-antitrypsin deficiency-associated disorder in a subject in need thereof or "treating" a subject having an alpha-1-antitrypsin deficiency-associated disorder refers to subjecting the subject to drug therapy, e.g., administering a drug to cure, alleviate, or relieve at least one symptom of the disease.

[0065] Any of the treatments 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, where the use or action of the agents or therapies overlaps in time. The agents or therapies can be administered simultaneously (e.g., as a single dosage form administered to the patient or as two separate dosage forms administered simultaneously) or sequentially in any order.

[0066] Recombinant proteins Some embodiments provide a recombinant protein comprising an alpha-1-antitrypsin serpin 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.

[0067] Alpha-1-antitrypsin serpin domain In some embodiments, the alpha-1-antitrypsin serpin domain comprises full-length alpha-1-antitrypsin. In some embodiments, the alpha-1-antitrypsin is a protein comprising the sequence set forth in SEQ ID NO:1.

[0068] In some embodiments, the alpha-1-antitrypsin serpin domain comprises one or more mutations, hi some embodiments, the one or more mutations comprise a mutation at 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. In some embodiments, the one or more mutations are selected from the following mutations in SEQ ID NO: 1: 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 , G304A, 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 in SEQ ID NO: 1. In some embodiments, the one or more mutations are both K168C and F189C in SEQ ID NO: 1. In some embodiments, the one or more mutations are both P361C and S283C in SEQ ID NO: 1.

[0069] In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including M351V and M358V. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including M351V and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S, M351V, and M358V.

[0070] In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including F51L, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including G117F, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S, K331F, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S, K335A, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including K168C, F189C, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S, S283C, M351V, M358L, and P361C. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including F51L, G117F, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including F51L, C232S, K331F, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including F51L, C232S, K335A, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including K51L, K168C, F189C, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including F51L, C232S, S283C, M351V, M358L, and P361C.In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including G117F, C232S, K331F, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including G117F, K335A, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including G117F, K168C, F189C, C232S, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including G117F, C232S, S283C, M351V, M358L, and P361C. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S, K331F, K335A, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including K168C, F189C, C232S, K331F, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including C232S, S283C, K331F, M351V, M358L, and P361C. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1 including K168C, F189C, C232S, K335A, M351V, and M358L. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1, including C232S, S283C, K335A, M351V, M358L, and P361C. In some embodiments, the alpha-1-antitrypsin serpin domain has one or more mutations in SEQ ID NO: 1, including K168C, F189C, C232S, S283C, M351V, M358L, and P361C.In some embodiments, the alpha-1-antitrypsin serpin domain comprises the point mutation K335A in SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serpin domain comprises the point mutation K331F in SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serpin domain comprises the point mutation G117F in SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serpin domain comprises the point mutation S283C in SEQ ID NO: 1. In some embodiments, the alpha-1-antitrypsin serpin domain comprises the point mutation P361C in SEQ ID NO: 1.

[0071] In some embodiments, the glycan site within the alpha-1-antitrypsin serpin domain is truncated and deleted. In some embodiments, the first residue of SEQ ID NO: 1 is mutated to delete the glycan site. In some embodiments, residues 1-46 of SEQ ID NO: 1 are deleted.

[0072] In some embodiments, the one or more mutations provide improved stability, including improved stability against oxidation, retained potency against human neutrophil elastase, resistance to polymerization, and ease of production, where the improved stability is measured relative to the wild type.

[0073] In some embodiments, the one or more mutations are one or more mutations as set forth in Tables 1 and 2-5 at the residue positions set forth according to SEQ ID NO:1. AAT-albumin fusion Base WT (wild type) Base C232S Base M351V, M358V Base M351V, M358L Base C232S, M351V, M358L

[0074] [Table 1]

[0075] In some embodiments, the alpha-1-antitrypsin serpin domain comprises a portion of alpha-1-antitrypsin, which in some embodiments has equal or greater activity than full-length human alpha-1-antitrypsin, such as, but not limited to, an alpha-1-antitrypsin serpin domain having the sequence set forth in SEQ ID NO:1. In some embodiments, the moiety 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 110 residues of alpha-1-antitrypsin, at least about 120 residues of alpha-1-antitrypsin, at least about 130 residues of alpha-1-antitrypsin, at least about 140 residues of alpha-1-antitrypsin, at least about 160 residues of alpha-1-antitrypsin, at least about 180 residues of alpha-1-antitrypsin, at least about 190 residues of alpha-1-antitrypsin, at least about 200 residues of alpha-1-antitrypsin, 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 at least about 150 residues of alpha-1-antitrypsin, at least about 160 residues of alpha-1-antitrypsin, at least about 170 residues of alpha-1-antitrypsin, at least about 180 residues of alpha-1-antitrypsin, at least about 190 residues of alpha-1-antitrypsin, at least about 200 residues of alpha-1-antitrypsin, 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.

[0076] In some embodiments, the portion comprises at least about 20 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 30 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 40 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 50 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 60 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 70 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 80 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 90 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 100 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 110 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 120 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 130 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 140 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 150 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 160 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 170 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, 20 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 130 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 140 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 150 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 160 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 170 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1 at least about 180 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 190 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 200 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 210 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 220 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 230 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1,at least about 240 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 250 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 260 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 270 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 280 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 290 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 300 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 310 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, the sequence at least about 320 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 330 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 340 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 350 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 360 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 370 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, at least about 380 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1, or at least about 390 residues of alpha-1-antitrypsin having the sequence set forth in SEQ ID NO:1.

[0077] In some embodiments, the alpha-1-antitrypsin serpin domain comprises a variant of the conventional alpha-1-antitrypsin set forth in SEQ ID NO: 1. In some embodiments, the variant comprises an active portion of the alpha-1-antitrypsin serpin domain.

[0078] albumin In some embodiments, the human serum albumin domain comprises full-length human albumin. In some embodiments, the human serum albumin domain comprises the sequence set forth 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 ID NO: 5.

[0079] In some embodiments, the human serum albumin domain comprises one or more mutations. In some embodiments, the one or more mutations comprise a mutation at one or more of the following positions: 407, 408, 409, 410, 413, and 414, where the position numbering is relative to 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, where the position numbering is relative to SEQ ID NO: 5 (see, e.g., WO 95 / 23857, herein incorporated by reference in its entirety). In some embodiments, the one or more mutations comprise L407A, L408V, V409A, and R410A, where the position numbering is relative to SEQ ID NO: 5. In some embodiments, the one or more mutations include R410A, L413Q, and L414Q, where the position numbers are relative to SEQ ID NO: 5. In some embodiments, the one or more mutations improve stability and / or increase production of the recombinant protein.

[0080] In some embodiments, the human serum albumin domain comprises a portion of human albumin, in some embodiments, the portion comprises 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, or at least about 110 residues of human serum albumin. 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 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 550 residues of human serum albumin, at 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 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.

[0081] In some embodiments, the portion comprises 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, at least about 110 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5 at least about 120 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 130 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 140 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 150 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 160 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 170 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 180 residues of human serum albumin having the sequence set forth in SEQ ID 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 230 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 240 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 250 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 260 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 270 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 280 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 290 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 300 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 310 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 320 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5 at least about 330 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 340 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 350 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 360 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 370 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 380 residues of human serum albumin having the sequence set forth 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:3 or 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 set forth in SEQ ID NO:3 or SEQ ID NO:5,At least about 440 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 450 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 460 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 470 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 480 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 490 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5, at least about 500 residues of human serum albumin having the sequence set forth in SEQ ID 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, or at least about 600 residues of human serum albumin having the sequence set forth in SEQ ID NO:3 or SEQ ID NO:5.

[0082] In some embodiments, the portion of human serum albumin comprises or consists of at least one amino acid sequence or variant thereof 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. Each domain is itself composed of two homologous subdomains, namely, 1-105, 120-194, 195-291, 316-387, 388-491, and 512-585, with a flexible inter-subdomain linker region (see, e.g., European Patent Application Publication No. 2 090 589 A1, incorporated herein by reference, including any drawings). In some embodiments, the portion consists of or comprises at least one subdomain.

[0083] In some embodiments, the portion of the human serum albumin domain has an activity greater than or equal to full-length human albumin, etc. In some embodiments, the human serum albumin has a sequence comprising the sequence set forth in SEQ ID NO:5.

[0084] In some embodiments, the human serum albumin domain comprises a variant of wild-type human serum albumin.

[0085] Albumin-binding proteins In some embodiments, the human serum albumin binding domain comprises an amino acid sequence capable of binding to human serum albumin. In some embodiments, the human serum albumin has the sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5. In some embodiments, the human serum albumin comprises a portion of human serum albumin set forth in SEQ ID NO: 5. In some embodiments, the portion of human albumin comprises an epitope.

[0086] 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.

[0087] 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 essentially consisting 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 essentially consisting 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 essentially consisting of a sequence selected from SEQ ID NOs: 17-21. In some embodiments, the single domain antibody comprises any one or more of the framework regions having a sequence comprising SEQ ID NOs: 37-62.

[0088] In some embodiments, single domain antibodies comprise a combination of one or more binding domains. In some embodiments, single domain antibodies comprise a combination of one or more of CDR1, CDR2, and CDR3. In some embodiments, single domain antibodies comprise a CDR1 sequence comprising, consisting of, or essentially consisting of a sequence selected from SEQ ID NOs: 6-9, and a CDR2 sequence comprising, consisting of, or essentially consisting of a sequence selected from SEQ ID NOs: 10-16. In some embodiments, single domain antibodies comprise a CDR1 sequence comprising, consisting of, or essentially consisting of a sequence selected from SEQ ID NOs: 6-9, and a CDR3 sequence comprising, consisting of, or essentially consisting of a sequence selected from SEQ ID NOs: 17-21. In some embodiments, single domain antibodies comprise a CDR2 sequence comprising, consisting of, or essentially consisting of a sequence selected from SEQ ID NOs: 10-16, and a CDR3 sequence comprising, consisting of, or essentially consisting of a sequence selected from SEQ ID NOs: 17-21. In some embodiments, the single domain antibody comprises a CDR1 sequence comprising, consisting of, or essentially consisting of a sequence selected from SEQ ID NOs: 6 to 9; a CDR2 sequence comprising, consisting of, or essentially consisting of a sequence selected from SEQ ID NOs: 10 to 16; and a CDR3 sequence comprising, consisting of, or essentially consisting of a sequence selected from SEQ ID NOs: 17 to 21.

[0089] In some embodiments, the single domain antibody comprises a sequence selected from SEQ ID NOs: 22-28.

[0090] 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.

[0091] In some embodiments, the single domain antibody comprises a variant of a humanized sequence selected from SEQ ID NOs: 29-36. 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 of the sequences 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. nucleic acid

[0092] Some embodiments include an isolated nucleic acid molecule encoding a recombinant protein comprising a nucleic acid encoding an alpha-1-antitrypsin serpin domain optionally linked to human serum albumin or a nucleotide sequence encoding 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.

[0093] In some embodiments, the disclosure features a nucleic acid molecule including a fragment of an alpha-1-antitrypsin serpin domain encoding sequence and a human serum albumin binding domain or a fragment of a human serum albumin domain encoding 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 fragment of a human serum albumin domain encoding a sequence of SEQ ID NO: 3 or SEQ ID NO: 5 or any of SEQ ID NOs: 22-36.

[0094] In some embodiments, the nucleic acid comprises a vector, as described below.

[0095] Domains, Domain Order and Linkers In some embodiments, the recombinant proteins of the present invention have one human serum albumin domain or one human serum albumin-binding domain and one alpha-1-antitrypsin serum domain. However, in some embodiments, multiple domains from each protein may be used to produce the recombinant proteins of the present invention. Similarly, one or more alpha-1-antitrypsin serum domains can be used to produce the recombinant proteins of the present invention. For example, the alpha-1-antitrypsin serum domain can be fused to both the N-terminal and C-terminal ends of the human serum albumin domain or human serum albumin-binding domain.

[0096] In some embodiments, the recombinant protein does not contain a linker between the fused portions of the polypeptides (e.g., a portion of the alpha-1-antitrypsin serpin domain is fused to a portion of the human serum albumin domain and / or human serum albumin binding domain). In some embodiments, the recombinant protein includes a linker peptide between the fused portions of the polypeptides. The linker may be a peptide linker. The linker may improve expression yield of the recombinant protein. The linker may improve and / or enhance the biological activity of the alpha-1-antitrypsin serpin domain.

[0097] Those of skill in the art will recognize that there are numerous linkers that can be used in the present invention (see, e.g., Chen at al., Fusion Protein Linkers: Property, Design, and Functionality, Adv Drug Deliv Rev. 2013 October 15;65(10):1357-1369 (2012), which is incorporated by reference herein in its entirety). In some embodiments, the linker is a peptide having a sequence comprising, consisting of, or consisting essentially of the sequence set forth in SEQ ID NO:63.

[0098] composition The present disclosure provides pharmaceutical compositions comprising any of the recombinant proteins described herein. The pharmaceutical compositions may take the form of any acceptable pharmaceutical formulation. Pharmaceutical compositions can be formulated in a variety of different forms, including liquid, semi-solid, and solid dosage forms, such as solutions (e.g., injectable solutions and insoluble solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form may depend on the intended mode of administration and therapeutic application.

[0099] Exemplary pharmaceutical compositions are described below, including 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 suitable route can depend, for example, on the condition and disorder of the recipient.

[0100] The pharmaceutical compositions described herein can be administered systemically, for example, parenterally (e.g., intravenously, subcutaneously, intradermally, intramuscularly, and intraarticularly). The pharmaceutical compositions described herein can be administered locally, for example, to the area affected by the condition or disorder being treated by administering the pharmaceutical composition.

[0101] Compositions for parenteral administration include aqueous and nonaqueous sterile injectable solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the compositions isotonic with the blood of the intended recipient, as well as aqueous and nonaqueous sterile suspensions, which may contain suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example, physiological 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, a suitable nontoxic parenterally acceptable diluent or solvent, such as EDTA, mannitol, 1,3-butanediol, water, Ringer's solution, isotonic sodium chloride solution, or other suitable dispersing or wetting suspending agent. The compositions may contain pharmacologically acceptable substances or adjuvants, including, but not limited to, EDTA, e.g., 0.5 mM EDTA; pH adjusting and buffering agents and / or isotonicity adjusting agents, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents or preservatives.

[0102] treatment In some embodiments, the disclosure features a method of treating a disorder associated with alpha-1 antitrypsin deficiency, the method comprising administering to a patient or subject having a disorder associated with alpha-1 antitrypsin deficiency in need thereof an effective amount of a recombinant protein or pharmaceutical composition provided herein. In some embodiments, the disclosure features a method of prophylactically treating a disorder associated with alpha-1 antitrypsin deficiency, the method comprising administering to a patient or subject having a disorder associated with alpha-1 antitrypsin deficiency in need thereof an effective amount of a recombinant protein or pharmaceutical composition described herein. In some embodiments, the recombinant protein or pharmaceutical composition can be administered to a patient in need thereof alone or in combination with another compound.

[0103] In some embodiments, the recombinant protein or pharmaceutical composition is administered about or more than once a week, about or more than once every 10 days, about or more than once every 15 days, about or more than once every 20 days, about or more than once every 25 days, about or more than once every month, about or more than once every two months, or about or more than once every three months.

[0104] In some embodiments, the subject has been diagnosed with or is at risk of being diagnosed with alpha-1-antitrypsin deficiency. In some embodiments, the alpha-1-antitrypsin deficiency indicates evidence of liver disease or lung disease. In some embodiments, the alpha-1-antitrypsin deficiency occurs in a subject who has not previously demonstrated liver or lung disease. In some embodiments, the evidence of liver disease or lung disease is at least one selected from the group consisting of emphysema, liver failure, hepatitis, hepatomology, jaundice, cirrhosis, nephrotic syndrome, autosomal recessive inheritance, COPD-like symptoms, dyspnea, elevated liver transaminases, and hepatocellular carcinoma.

[0105] In some embodiments, alpha-1 antitrypsin deficiency is caused by a deletion mutation in the SERPINA1 gene. Over 120 deletion mutations in the SERPINA1 gene have been identified and may be useful in diagnostic methods according to the present invention. Some deletion mutations do not affect the production of alpha-1 antitrypsin, while others cause a shortage or deficiency of the protein. In some embodiments, a deletion mutation in the SERPINA1 gene causes a substitution of the amino acid glutamic acid with the amino acid lysine in the protein at position 342 according to SEQ ID NO: 1.

[0106] Abnormal alpha-1 antitrypsin proteins can bind together to form large molecules that cannot leave the liver. The accumulation of these macromolecules often results in liver damage. Furthermore, lung tissue is destroyed because insufficient alpha-1 antitrypsin is available for protection from neutrophil elastase or PR3. Large alpha-1 antitrypsin molecules may also contribute to excessive inflammation, which may explain some of the other features of alpha-1 antitrypsin deficiency, such as the skin condition called panniculitis. Other SERPINA1 deficiency mutations result in the production of an abnormally small form of alpha-1 antitrypsin that is quickly destroyed in the liver. As a result, little or no alpha-1 antitrypsin is available for the lungs. In individuals with these deficiency mutations, the liver remains healthy, but the lungs remain unprotected from neutrophil elastase or PR3.

[0107] In some embodiments, the deletion mutation is a loss-of-function mutation. In some embodiments, the deletion mutation results in a stop codon. In some embodiments, the deletion mutation results in an amino acid substitution. In some embodiments, the deletion mutation is a point mutation, insertion, deletion, and / or substitution. In some embodiments, the deletion mutation is a point mutation. In some embodiments, the deletion 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 deletion mutation is a deletion of the entire gene. In some embodiments, the deletion mutation is a partial gene deletion. In some embodiments, the deletion mutation results in an intronic change that affects splicing. In some embodiments, the deletion mutation is an alteration in the 3'-untranslated region of the gene. In some embodiments, the deletion mutation is a gross chromosomal rearrangement. In some embodiments, the deletion mutation results in truncation of the SERPINA1 gene product.

[0108] In some embodiments, the subject has abnormal expression of alpha-1-antitrypsin protein 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 have a disorder associated with alpha-1-antitrypsin deficiency. In some embodiments, the subject has reduced or substantially absent expression of alpha-1-antitrypsin protein compared to the reference standard. In some embodiments, the reference standard is the expression level of alpha-1-antitrypsin protein in a subject who does not have a disorder associated with alpha-1-antitrypsin deficiency.

[0109] In some embodiments, the alpha-1-antitrypsin serpin domain is an allele of a normal alpha-1-antitrypsin domain (see Crystal, RG The alpha-1-antitrypsin gene and its deficiency states. Trends Genet. 5:411-417, 1989. [PubMed:2696185], which is incorporated by reference herein in its entirety). In some embodiments, the allele is an allele containing a valine at position 213 (M1V; 107400.0002) or an allele containing an alanine at position 213 (M1A; 107400.0001) according to SEQ ID NO: 1 (see Nukiwa, T., Brantly, M. L., Ogushi, F., Fells, G. A., Crystal, R. G. 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 by reference herein in its entirety). In some embodiments, the allele is a deficient or null allele.

[0110] In some embodiments, the subject has been diagnosed with or is at risk for a disorder associated with alpha-1-antitrypsin deficiency. In some embodiments, the subject has a mutation in a gene encoding the alpha-1-antitrypsin domain. In some embodiments, the mutation is in one of the exons of alpha-1-antitrypsin. In some embodiments, the mutation is in exon 1 of alpha-1-antitrypsin, the 5 prime coding region of exon 2 of alpha-1-antitrypsin, or the 3 prime 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 by reference in its entirety herein).

[0111] 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.

[0112] Vectors and host cells, and expression of polypeptides In some embodiments, the nucleic acid is a vector. In some embodiments, the vector comprises a host cell. Some embodiments are directed to a method for producing a recombinant protein, comprising culturing a host cell with one of the nucleic acids provided herein and recovering the recombinant protein. In some embodiments, the recombinant protein is recovered using a pH-neutral alpha-1-antitrypsin-Fc capture selection medium.

[0113] The recombinant proteins described herein can be produced as recombinant molecules, for example, by secretion from any suitable host cell. Numerous expression systems are known and can be used, including bacteria (e.g., E. coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae, Kluyveromyces lactis, and Pichia pastoris), filamentous fungi (e.g., Aspergillus species), plant cells, animal cells, and insect cells.

[0114] 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.

[0115] In some embodiments, the host cell is a yeast cell. Exemplary genera of yeast that can be used as hosts described herein include, but are not limited to, Pichia, Saccharomyces, Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces, Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, and the like. Examples of yeast species (Saccharomyces spp) include S. cerevisiae, S. italicus, and S. rouxii. Yeast strains suitable for producing the fusion polypeptides described herein include, but are not limited to, D88, DXY1, and BXP10.

[0116] Also referred to herein is the transformed host cells themselves, as well as a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. If the recombinant protein is secreted, the medium will contain the recombinant protein, with the cells, or without the cells if the cells have been filtered or centrifuged away.

[0117] Recombinant proteins can be produced in conventional manner, for example, from coding sequences inserted into the host chromosome or on a free plasmid. Host cells are transformed with the coding sequence for the recombinant protein by any method known in the art, for example, electroporation. Successfully transformed cells, i.e., cells containing the DNA constructs described herein, can be identified by techniques known to those skilled in the art. For example, cells resulting from the introduction of an expression construct can be grown to produce recombinant protein. Cells can be harvested, lysed, and their DNA content examined for the presence of DNA using methods known in the art. The presence of recombinant protein in the supernatant can be detected using any method known in the art (e.g., antibody-based detection methods).

[0118] Also disclosed herein are plasmid vectors containing nucleic acids for use in expressing the recombinant proteins described herein. To express the recombinant proteins described herein, a nucleotide sequence encoding the appropriate recombinant protein, or a functional equivalent, can be inserted into a suitable vector. Suitable vectors will contain the necessary and appropriate transcriptional and translational control sequences for expression of the inserted nucleic acid sequence. Standard methods known to those skilled in the art may 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 will depend on the properties of the particular nucleotide fragment and may be determined by one of skill in the art.

[0119] Vectors suitable for use herein may contain an origin of replication and a restriction endonuclease sequence site. Those skilled in the art will be familiar with suitable origins of replication and restriction endonuclease sequences for use in host cells. Vectors suitable for use herein may contain sequence elements that support transcription, including, but not limited to, promoter elements and enhancer elements. Those skilled in the art will be familiar with various transcription control elements, including, but not limited to, promoters, inducible promoters, and enhancer elements, that will be suitable in host cells.

[0120] Suitable vectors for use herein may also contain a selectable marker gene that encodes a product necessary for a host cell to grow and survive under certain conditions, thereby facilitating the selection of host cells into which the vector has been introduced. Typical selectable 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.). One of skill in the art would be knowledgeable as to the coding sequences for suitable selectable marker and reporter genes for use in host cells.

[0121] The expression vectors described herein can be introduced into host cells via conventional transformation or transfection techniques. Transformation and transfection techniques include, but are not limited to, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofectamine, electroporation, microinjection, and viral-mediated transfection (see U.S. Pat. No. 6,632,637, which is incorporated herein by reference in its entirety). Those skilled in the art will be knowledgeable about suitable transformation and transfection methods based on the host cell / vector combination. For long-term, high-yield production of recombinant proteins, stable expression of the recombinant protein may be preferred. Host cells that stably express recombinant proteins can be engineered. Yeast plasmid vectors may include, but are not limited to, pRS403 to 406, pRS413 to 416, pRS403, pRS404, pRS405, pRS406, pRS413 to 416, pPPC0005, pScCHSA, pScNHSA, and pC4.

[0122] In another embodiment, the recombinant mammalian expression vector can direct expression of a polypeptide preferentially in a particular cell type (e.g., expressing a polypeptide using tissue-specific regulatory elements). 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 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., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreatic-specific promoters (Edlund et al. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), and the pancreatic-specific promoters (Edlund et al. (1989) Cell 33:741-748). al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., whey promoters; U.S. Pat. No. 4,873,316 and European Patent Application No. 264,166). Developmentally regulated promoters are also encompassed, such as the mouse hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the alpha-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev 3:537-546). Each of the above-identified references is incorporated by reference in its entirety, including any drawings.

[0123] 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, hydroxylapatite chromatography, hydrophobic charge interaction chromatography, and lectin chromatography. In some embodiments, high-performance liquid chromatography ("HPLC") is used for purification. Polypeptides produced and recovered by the recombinant and molecular biological 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, reversed-phase chromatography, or gel filtration. Depending on the host used in the recombinant production procedure, the polypeptides of the invention can be glycosylated or non-glycosylated. In some embodiments, recombinant proteins will be purified using a neutral pH alpha-1-antitrypsin-Fc capture selection medium.

[0124] In some embodiments, nucleic acids encoding the recombinant proteins described herein are optimized for expression in yeast. Exemplary genera of yeast that can be used as hosts for expressing the fusion polypeptides described herein include, but are not limited to, Pichia, Saccharomyces, Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces, Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis, and the like. Yeast strains suitable for producing the fusion polypeptides described herein include, but are not limited to, D88, DXY1, and BXP10. Yeast plasmid vectors can include, but are not limited to, pRS403-406, pRS413-416, pRS403, pRS404, pRS405, pRS406, pRS413-416, pPPC0005, pScCHSA, pScNHSA, and pC4. Vectors for generating albumin fusion proteins for expression in yeast include pPPC0005, pScCHSA, pScNHSA, and pC4:HSA.

[0125] In some embodiments, nucleic acids encoding the recombinant proteins described herein are expressed in the yeast Saccharomyces. Preferred exemplary species of Saccharomyces include S. cerevisiae, S. italicus, 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.

[0126] Other organisms may be used for expression. Further exemplary species of the genus Pichia include P. pastoris. Exemplary species of the genus Aspergillus include A. niger and A. nidulans. Preferred exemplary species of the genus Yarrowia include Y. lipolytica. Many preferred yeast species are available from ATCC® (American Type Culture Collection).

[0127] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Example]

[0128] Example 1: Construction of AAT-Albumin Wild-type alpha-1-antitrypsin (alpha-1-antitrypsin, SEQ ID NO: 1) and four variants (C232S, SEQ ID NO: 64; M351V, M358V, SEQ ID NO: 65; M351V, M358L, SEQ ID NO: 66; C232S, M351V, M358L, SEQ ID NO: 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 in the AAT gene are generated by overlap 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 alpha-1-antitrypsin gene is cloned in frame with a gene encoding human serum albumin and a linker into a mammalian expression vector containing a mammalian secretory signal sequence upstream of the alpha-1-antitrypsin gene insertion site.

[0129] Initial AAT variants were selected because they were resistant to either cysteine ​​oxidation and / or methionine oxidation. Methionine oxidation has been shown to reduce AAT inhibitory activity against human neutrophil elastase. Once expressed in Chinese hamster ovary (CHO) cells, each AAT human serum albumin (wild-type (WT) or variants, SEQ ID NOS: 1-5) was purified using anti-AAT resin (GE) and tested for inhibition of human neutrophil elastase (hNE). Each AAT human serum albumin (wild-type (WT) or variants, SEQ ID NOS: 1-5) was also tested for whole protein stability using dynamic light scattering (DLS) and dynamic scanning fluorimetry (DSF) instruments (UNcle, Unchained).

[0130] Overall AAT protein stability has been shown to correlate with AAT's resistance to self-polymerization. This data will be used to select AAT-human serum albumin for further mutagenesis with at least one additional mutation (see Table 1 above). These mutations have been shown to further increase AAT stability and prevent self-polymerization. Each AAT-human serum albumin variant will be expressed in CHO, purified, and tested for hNE inhibition and protein stability.

[0131] By combining multiple mutations together, an unexpected synergistic increase in stability was observed with little or no loss of hNE inhibition.

[0132] Each gene sequence was cloned into a proprietary high-expression mammalian vector. Each completed construct was sequence verified before proceeding to DNA scale-up. Each DNA expression construct was scaled up to an appropriate amount for transfection. Plasmid DNA was run on an agarose gel for quality assessment and sequence verified before proceeding to transfection.

[0133] Example 2: Construction of AAT albumin binding domain A preferred AAT domain is fused to an albumin-binding single domain antibody in place of human serum albumin as described in Example 1. For example, a single domain antibody having any of the respective sequences SEQ ID NOS: 22-36 is fused to AAT as provided in Example 1. The AAT albumin-binding domain variants are expressed in CHO, purified, and tested for hNE inhibition and protein stability as described in Example 1.

[0134] Each gene sequence was cloned into a proprietary high-expression mammalian vector. Each completed construct was sequence verified before proceeding to DNA scale-up. Each DNA expression construct was scaled up to an appropriate amount for transfection. Plasmid DNA was run on an agarose gel for quality assessment and sequence verified before proceeding to transfection.

[0135] Example 3: CHO transient transfection Suspension CHO cells were seeded into shake flasks and grown in serum-free, chemically defined medium. On the day of transfection, the grown cells were seeded into new flasks containing fresh medium. Each DNA construct was transiently transfected into CHO cells. Cells were maintained in fed-batch culture (C1603 and C1604, Medna) until the end of the production run.

[0136] [Table 3]

[0137] [Table 4-1] [Table 4-2]

[0138] Example 4: Affinity purification of CHO albumin Conditioned medium from transient production runs was collected and purified by centrifugation and filtration. The supernatant was loaded onto a column packed with alpha-1 antitrypsin-selective resin and pre-equilibrated with PBS, pH 7.4. The column was washed with PBS to remove impurities until the OD280 (measured by NanoDrop, Thermo Scientific) fell to around zero. The target protein was eluted with high-salt elution buffer (20 mM Tris, 2 M MgCl2, pH 7.4), and fractions were collected and recorded for OD280. Fractions containing the target protein were pooled and dialyzed from the final buffer into PBS, pH 7.4. The final product was filtered through a 0.2 μm membrane filter. After buffer exchange, the protein concentration and final yield were calculated from the OD280 value, and the extinction coefficient was calculated.

[0139] CE-SDS analysis of the target protein was performed using LabChip GXII (Perkins Elmer).

[0140] The results can be seen in FIGS.

[0141] Example 5: Stability analysis For analysis, the samples were run on an UNcle system (Unchained Labs). A temperature ramp of 1°C / min was performed from 25°C to 95°C for dynamic light scattering ("DSF") and static light scattering ("SLS") with observation. The UNcle measured SLS at 266 nm and 473 nm. DLS was measured at the start of the temperature ramp. UNcle Analysis software was used to calculate and analyze Tm, Tagg, and DLS measurements. The measured Tm is indicated by a solid drop line on the DSF graph.

[0142] The results can be seen in FIGS.

[0143] Example 6: hNA analysis A neutrophil elastase inhibition assay was performed using a screening kit from Abcam (catalog number ab118971), and the results are shown in Figure 6.

[0144] Example 7: IC50 IC50 analysis of the AAT mutants was also performed using a screening kit from Abcam (catalog number ab118971). The concentration range used for IC50 analysis was 400 nM to 98 pM, and data from two duplicates were analyzed using GraphPad Prism.

[0145] [Table 5]

[0146] Twenty-one AAT mutant proteins and one AAT serum control were also used to generate IC50 analysis curves. Table 5 shows the IC50s for the mutant AATs.

[0147] [Table 6]

[0148] Figure 9 shows the IC50 analysis of AAT mutants, where the x-axis represents the log concentration (nM) of AAT protein and the y-axis represents the percentage of inhibition. Curves with higher slopes indicate highly cooperative inhibitory properties. Mutants G11F / K335A, G117F / K331F, F51L / K331F, and K331F / K335A all have highly cooperative inhibitory properties.

[0149] Example 8: In silico immunogenicity Using a proprietary screen, proteins were compared to those already in clinical use (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 drawings). AAT albumin fusion ("AAT_MLA"), AAT mutant ("AAT_Mutant"), and albumin protein ("Albumin") were screened for the presence of putative T cell epitopes. The AAT mutant has the following mutations in SEQ ID NO: 1: C232S, M351V, M358L, K335A, S283C, and P361C. The immunogenicity of each T cell epitope or AAT-albumin fusion, AAT mutant, and albumin protein was compared with each other, normalized, and ranked.

[0150] The input sequence was broken down into overlapping 9-mer frames, and each frame was evaluated against a panel of eight common HLA type II alleles. These alleles are referred to as epitypes. Each epitype is functionally equivalent or nearly equivalent to the alleles of a number of additional family members. Collectively, the eight epitype alleles, along with their respective family members, provide good coverage of 95% of the human population. Each frame-to-allele evaluation provides a description of the predicted HLA binding affinity.

[0151] The evaluation scores range from approximately -3 to +3. Evaluation scores higher than 1.64 were defined as "hits" and considered to be potentially immunogenic and worthy of further consideration. Often, approximately 5% of all evaluations can be expected to score higher than 1.64. These peptides have a significant chance of binding HLA molecules with moderate to high affinity and therefore have a significant chance of being present on the surface of APCs, such as dendritic cells or macrophages, where they can be matched by passing T cells. The greater the amount of HLA ligands (i.e., hits) a given protein contains, the greater the likelihood that the protein will induce an immune response.

[0152] The EpiMatrix protein score is the difference between the number of putative T cell epitopes one can expect to find in a protein of a given size and the number of putative epitopes predicted by the EpiMatrix system. EpiMatrix protein scores can be "normalized" and plotted on a standardized scale (see, for example, Figure 10). The EpiMatrix protein score of an "average" protein is zero. An EpiMatrix protein score above zero suggests the presence of an excess of MHC ligands and indicates a higher likelihood of immunogenicity, while a score below zero suggests the presence of fewer potential MHC ligands than expected and a lower likelihood of immunogenicity.

[0153] In analyzing the complete AAT_MLA sequence, the algorithm performed a total of 7,880 frame-to-allele evaluations. In analyzing the AAT_MUTANT domain, the algorithm performed a total of 3,088 frame-to-allele evaluations. In analyzing the ALBUMIN domain, the algorithm performed a total of 4,616 frame-to-allele evaluations. The results are shown in Table 6. The mutant AAT exhibits a slightly higher immunogenicity score compared to wild-type AAT. Fusion of the high-scoring AAT_MUTANT domain with a low-scoring glycine-serine linker and a low-scoring ALBUMIN domain reduces the overall epitope density of the AAT_MLA fusion. AAT has a negative score when fused to albumin (AAT_MLA), suggesting lower immunogenicity compared to wild-type or the mutant AAT alone.

[0154] [Table 7-1] [Table 7-2]

[0155] For the complete fusion sequence AAT_MLA, 384 peptide-to-HLA hits were identified, resulting in an overall score of -10.72. For the complete fusion sequence AAT_MUTANT, 206 hits were identified, resulting in an overall score of 26.87. For the complete fusion sequence AAT_WT, 202 hits were identified, resulting in an overall score of 21.01. For the complete fusion sequence AAT_MLA, 178 hits were identified, resulting in an overall score of -32.04. The results are shown in Figure 10.

[0156] Example 9: Stability analysis of AAT variants (K335A, S283C / P361C) in the base (C232S, M351V, M351L) For analysis, samples were run on UNcle (Unchained Labs). A temperature range of 1°C / min was run from 25°C to 95°C for DSF and SLS. SLS was measured at 266 nm and 473 nm on UNcle. DLS was measured at the start of the temperature ramp. UNcle Analysis software was used to calculate and analyze Tm, Tagg, and DLS measurements. Tm was measured as represented by the solid drop line in Figure 11. As can be seen, the variant AAT albumin fusions are highly stable.

[0157] Example 10: Cyno PK The potential toxicity of recombinant alpha-1-antitrypsin (AAT) K335A / S283C / P361C fused to human serum albumin (AAT albumin fusion) was determined when given as a single intravenous bolus infusion to cynomolgus monkeys. Cynomolgus monkeys were selected as the animal model for this study because they are a non-rodent species permitted by regulatory authorities for preclinical toxicology studies and most accurately reflect the pharmacokinetics and pharmacology of test agents expected in human subjects. The pharmacokinetic properties of the AAT albumin fusion compared with human plasma-derived AAT (Prolastin C) were also determined.

[0158] Table 7 shows the test and control substances.

[0159] [Table 8]

[0160] The control substance was used for dilution of the test substance. A 2 mg / mL dose formulation of the test substance was prepared by diluting the test substance stock (7.8 mg / mL) in serial dilutions of up to four times with the control substance to meet the dose level requirements. The dose level was selected based on previously published studies (Journal of Chronic Obstructive Pulmonary Disease (2013) 10(6):687-95; Lancet (2015) 386(9991):360-8). A weekly 60 mg / kg intravenous (IV) infusion of human plasma-derived alpha-1 antitrypsin (AAT) has been approved for individuals with alpha-1 antitrypsin deficiency. The safety and pharmacokinetic profile of weekly injections of 120 mg / kg AAT was considered safe and well tolerated in adults with AATD.

[0161] Animals were randomized and assigned to groups by a computer-based procedure before entering the study. The experimental design used is shown in Table 8.

[0162] [Table 9]

[0163] Animals were administered the test article by intravenous (slow bolus) injection once on Day 1. The dose per animal was based on the most recent body weight measurement. Animals were briefly restrained but not sedated for dose administration.

[0164] Blood was collected by venipuncture. Samples were collected according to Table 9.

[0165] [Table 10]

[0166] Blood was collected at a target volume of 1 mL with potassium (K3) EDTA as an anticoagulant. Blood smears were prepared from each blood sample. The smears were examined to assess the health status of the animals as follows: No unhealthy animals were observed.

[0167] Clinical chemistry was performed without anticoagulant at a target volume of 2 mL.

[0168] Samples were taken according to Table 10.

[0169] [Table 11-1] [Table 11-2]

[0170] Plasma from the samples was analyzed for test article concentration, and bronchoalveolar lavage samples were taken from anesthetized animals and analyzed (data not shown).

[0171] The plasma results are shown in Figure 12 and were fitted to a two-phase exponential decay model in Graphpad Prism. As can be seen in Figure 12, the variants have significantly longer elimination half-lives. The half-life (beta phase, slow) of the AAT-HSA variant (Test Article 1) was over 30 hours compared to less than 10 hours for Prolastin (plasma AAT).

[0172] The plasma results were further analyzed using a three-compartment model (Tables 11-13) and are also shown in Figure 13, which shows the median (line), 5th, and 95th percentiles (areas) from 1000 simulated PK profiles, along with individual measurements (symbols). Measurements below the limit of quantitation (BLQ, crosses) were not included in the analysis.

[0173] As can be seen above, the PK in monkeys from 10 mg / kg onwards was three compartments.max is the C of test substance 1 max The median volume of distribution was 0.213 L for test article 2 and 8 L for test article 1. The total volume of distribution was 1.45 L for test article 2 and 42 L for test article 1. The volume of distribution of Aralast (plasma-derived AAT) in humans was 5.6 L [https: / / www.drugbank.ca / drugs / DB00058]. Allometric scaling for NHPs predicts a volume of distribution of 0.240 L, which is comparable to test article 2 from this study.

[0174] The elimination half-life of HSA in monkeys is approximately 5 days. The β and γ half-lives of test substance 1 were 0.75 and 5.8 days, respectively. The β and γ half-lives of test substance 2 were 0.36 and 1.49 days, respectively. The γ half-life of test substance 2 is approximately 3.9 times longer than that of test substance 1. The γ elimination half-life of HSA (based on literature) and that of test substance 1 are comparable in monkeys.

[0175] Table 11 shows the PK parameters for Test Article 2 and Test Article 1.

[0176] [Table 12-1] [Table 12-2]

[0177] Table 12 shows the population PK parameters for Test Article 2:

[0178] [Table 13]

[0179] Table 13 shows the population PK parameters for Test Article 1:

[0180]

Table 14-1

Table 14-2

Claims

1. (a) an alpha-1-antitrypsin serpin domain; (b) a human serum albumin domain; wherein the alpha-1-antitrypsin serpin domain comprises wild-type alpha-1-antitrypsin (SEQ ID NO: 1) only with the base mutations C232S, M351V, and M358L, and the additional mutations S283C, K335A, and P361C; A recombinant protein, wherein the human serum albumin domain has at least 90% identity with wild-type human serum albumin, and the human serum albumin domain increases the plasma half-life of the alpha-1-antitrypsin serpin domain compared to wild-type alpha-1-antitrypsin.

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

3. 3. A recombinant protein according to any one of claims 1 to 2, comprising a linker having the sequence set forth in SEQ ID NO:

63.

4. 4. The recombinant protein of claim 1, wherein the C-terminus of the alpha-1-antitrypsin serpin domain is fused to the N-terminus of the human serum albumin domain.

5. 5. The recombinant protein of claim 1, wherein the number of N-glycans or polysialyations in the alpha-1-antitrypsin serpin domain of the recombinant protein is greater than that of a wild-type alpha-1-antitrypsin serpin domain.

6. The recombinant protein of any one of claims 1 to 5, wherein the mutation causes resistance to methionine oxidation compared to wild-type alpha-1-antitrypsin.

7. 7. The recombinant protein of any one of claims 1 to 6, wherein the alpha-1-antitrypsin retains activity compared to wild-type alpha-1-antitrypsin.

8. The recombinant protein of any one of claims 1 to 7, wherein the alpha-1-antitrypsin retains activity against human neutrophil elastase and / or PR3 compared to wild-type alpha-1-antitrypsin.

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