Engineering DNASE enzymes for manufacturing and therapeutic purposes

Abstract

To provide a DNASE useful for treating conditions characterized by neutrophil extracellular trap (NET) accumulation and / or release.SOLUTION: The present disclosure provides engineered human extracellular DNASE proteins (e.g., variants of DNASE1 (D1), DNASE1-LIKE 1 (D1L1), DNASE1-LIKE 2 (D1L2), DNASE1-LIKE 3 Isoform 1 (D1L3), DNASE1-LIKE 3 Isoform 2 (D1L3-2), DNASE2A (D2A), and DNASE2B (D2B)).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of and priority to U.S. Provisional Application Nos. 62 / 742,682, filed October 8, 2018, 62 / 775,563, filed December 5, 2018, 62 / 779,104, filed December 13, 2018, 62,808,601, filed February 21, 2019, and 62 / 846,904, filed May 13, 2019, the contents of which are incorporated herein by reference in their entireties.

[0002] The present invention relates to the field of engineered DNASE enzymes. [Background technology]

[0003] Inflammation is an essential host response for controlling invading microorganisms and healing damaged tissues. Uncontrolled and persistent inflammation causes tissue damage in many inflammatory disorders. Neutrophils are the primary leukocytes involved in acute inflammation. During infection, neutrophils produce neutrophil extracellular traps (NETs), lattices of DNA filaments decorated with toxic histones and enzymes that immobilize and neutralize bacteria. However, inappropriately released NETs can harm host cells due to their cytotoxic, pro-inflammatory, and prothrombotic activities.

[0004] DNASE1 (D1), along with DNASE1-like 1 (D1L1), DNASE1-like 2 (D1L2), and DNASE1-like 3 (D1L3), form the DNASE1 protein family, a group of homologous secreted DNase 1 protein enzymes. DNASE2A and DNASE2B form an additional group of homologous extracellular DNase enzymes. DNASE1 and DNASE2 protein family members are evolutionarily conserved and expressed in various species, including humans. Recombinant human DNASE1 and DNASE2 protein family members provide drug candidates for NET-associated diseases. While D1 has been developed for several therapeutic applications in patients, conditions for large-scale production of other members of the DNASE1 protein family have not been described. Furthermore, the physical, enzymatic, and pharmacokinetic properties of these enzymes are not ideal for clinical applications. Therefore, there is a need to define the manufacturing processes for D1L1, D1L2, and D1L3 enzymes and engineer DNases for therapeutic use, including degrading NETs. Summary of the Invention

[0005] The present invention provides engineered human extracellular DNASE proteins (e.g., variants of DNASE1 (D1), DNASE1-like 1 (D1L1), DNASE1-like 2 (D1L2), DNASE1-like 3 isoform 1 (D1L3), DNASE1-like 3 isoform 2 (D1L3-2), DNASE2A (D2A), and DNASE2B (D2B)) that are useful for treating conditions characterized by the accumulation and / or release of extracellular DNA, extracellular chromatin, and neutrophil extracellular traps (NETs). In accordance with aspects of the present invention, the DNase variants described herein are more suitable for treatment and / or are more amenable to large-scale manufacturing. In some embodiments, the DNase variants described herein have advantages for medical therapy, including systemic therapy. Such advantages include slower drug elimination, e.g., increased circulating half-life (e.g., serum half-life), extended duration of pharmacodynamic activity, enhanced chromatin degradation activity, and protease resistance.

[0006] In some aspects, the present invention provides a D1L3 variant, wherein the D1L3 variant is The present invention provides D1L3 variants that have one or more of increased protein stability, slower drug elimination, increased duration of pharmacodynamic activity, resistance to proteolysis, higher production levels in in vitro expression systems, better suitability for purification, and substantially less, the same, or better chromatin and / or NET degradation activity compared to the wild-type D1L3 isoform 1 enzyme of SEQ ID NO: 4 or the wild-type D1L3 isoform 2 enzyme of SEQ ID NO: 5.

[0007] In some embodiments, the D1L3 variant is a fusion protein comprising an amino acid sequence at least 80% identical to the mature enzyme defined by SEQ ID NO:4 or SEQ ID NO:5, an albumin amino acid sequence N-terminal to the mature enzyme, and optionally a linking amino acid sequence between the albumin amino acid sequence (albumin domain) and the D1L3 amino acid sequence (D1L3 domain). In these embodiments, D1L3 exhibits slower elimination (e.g., improved circulating or serum half-life) and extended duration of pharmacodynamic activity, including systemic therapy. In some embodiments, fusion of albumin with a linking sequence to the D1L3 domain does not substantially affect the chromatin degradation activity of the enzyme (e.g., as measured using an in vitro assay) compared to the enzyme without the albumin fusion.

[0008] In these embodiments, the D1L3 domain of the fusion protein lacks all or part of the C-terminal basic domain present in the wild-type D1L3 enzyme. Deletion or inactivation of the C-terminal basic domain substantially improves chromatin degradation activity. That is, removal of the C-terminal basic domain (BD) activates the wild-type D1L3 enzyme to degrade chromatin.

[0009] In some embodiments, the D1L3 variant has one or more structural element substitutions from D1. For example, the D1L3 variant can have a structural element substitution of Q282_S305delinsK, which includes a deletion of the C-terminal basic domain, which is absent in D1. In some embodiments, the D1L3 variant has an amino acid substitution at position 101 of SEQ ID NO: 4. The substitution can be Arg based on the corresponding structural element from D1, or in some embodiments, Lys. The substitution at this position can enhance the chromatin degradation activity of the D1L3 variant.

[0010] The linker present can be a flexible linker, a rigid linker, or a physiologically cleavable linker, such as a protease-cleavable linker. For example, the linker can be a hydrophilic amino acid sequence and can be constructed primarily from amino acids selected from Gly, Ala, Ser, Thr, and Pro. In some embodiments, variants include flexible linkers (e.g., (G y S) n linker, y is 1-5, and n is 1-20). In some embodiments, the linker is an α-helical linker. In some embodiments, the linker has at least 15 amino acids, or at least 25 amino acids. In various embodiments, longer linkers of at least 15 amino acids can provide improved yields upon expression in mammalian and non-mammalian expression systems, such as CHO cells or Pichia pastoris. Furthermore, surprisingly, longer linker sequences exhibited improved chromatin degradation activity in in vitro chromatin degradation assays compared to shorter linker sequences.

[0011] In various embodiments, the D1L3 variant comprises the amino acid sequence of any one of SEQ ID NOs: 17-30, optionally with 1-20 amino acid modifications, in each case independently selected from insertions, deletions, or substitutions. These sequences provide exemplary fusion proteins between D1L3 (or D1L3 variants) and albumin sequences, including various linker designs. In some embodiments, the amino acid modifications are located within the D1L3 domain, albumin. In some embodiments, the D1L3 variant has the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. In these embodiments, the D1L3 variant comprises, from N- to C-terminus, an albumin amino acid sequence, a medium or long flexible linker, and a D1L3 amino acid sequence (i.e., comprising the D1L3 variant). SEQ ID NO: 28 further comprises an albumin fusion at the C-terminus via a long flexible peptide linker.

[0012] In other embodiments, the linker is cleavable by a protease, e.g., a coagulation pathway protease, e.g., activated factor XII. In certain embodiments, the linker comprises the amino acid sequence of factor XI and / or prekallikrein. In other embodiments, the linker comprises a peptide sequence targeted for cleavage by a neutrophil-specific protease, such as neutrophil elastase, cathepsin G, or proteinase 3.

[0013] In some aspects, the present invention provides variants of extracellular DNASE enzymes that have been engineered to have manufacturing advantages and provide for the production of recombinant enzymes suitable for therapeutic use. In various embodiments, the present invention provides recombinant D1, D1L1, D1L2, and D1L3 variants that contain one or more amino acid substitutions at cysteine ​​residues (or PEGylation of Cys residues), resulting in reduced intra- and intermolecular cross-linking via disulfide bridges during protein expression.

[0014] In another aspect, the present invention provides variants of extracellular DNASE enzymes engineered to have advantages in protease resistance to improve in vivo exposure, e.g., slow clearance, extend half-life (e.g., serum half-life), and prolong duration of pharmacodynamic activity, as well as reduce proteolysis during recombinant enzyme production. The present disclosure identifies D1L3 residues that are susceptible to proteolysis by, e.g., plasmin, thrombin, and / or trypsin, as well as residues (e.g., paired basic amino acids) that are susceptible to proteases produced by mammalian and non-mammalian cell lines. Engineered mutations of these residues can confer these advantages in protease resistance.

[0015] In another aspect, the present invention provides methods for the recombinant production of extracellular DNASE proteins, including variants thereof described herein. In some embodiments, the methods utilize a non-mammalian expression system, e.g., a eukaryotic non-mammalian expression system such as Pichia pastoris. In some embodiments, the Pichia pastoris encodes a DNase enzyme with its native signal peptide that allows for secretion from the host cell. In some embodiments, the expression system is a mammalian cell expression system, such as Chinese hamster ovary (CHO) cells.

[0016] In some embodiments, the recombinant expression system has deletion or inactivation of one or more proteases that cleave at paired basic amino acids. Exemplary enzymes include furin (expressed by CHO cells) and aspartic proteinase 3 (Ysp1) and kexin (Kex2) expressed by Pichia pastoris. In some embodiments, these enzymes are not genetically deleted or inactivated, but their activity is inhibited with protease inhibitors during recombinant protein production.

[0017] In some embodiments, growth media for non-mammalian or mammalian expression systems are supplemented with polyanions such as dextran sulfate, heparin, ferric citrate, EDTA, etc. In further embodiments, growth media for Pichia pastoris or other expression systems are supplemented with dextran sulfate having an average molecular weight between 5 kDa and 100 kDa. For example, polyanions can be added to the culture in an amount sufficient to complex with the recombinant protein produced. In some embodiments, recombinant extracellular DNASE proteins and variants thereof from the culture medium of non-mammalian or mammalian expression systems are purified through a method that includes dissociation of the recombinant extracellular DNASE proteins and variants from polyanions such as dextran sulfate, heparin, and EDTA.

[0018] In other aspects, the present invention provides isolated polynucleotides encoding D1, D1L1, D1L2, or D1L3 variants, as well as vectors and host cells. The polynucleotides can be encoding mRNA or DNA. The host cells can be cells of recombinant expression systems, including bacteria or eukaryotes, whether non-mammalian, such as Pichia pastoris, or mammalian, such as CHO cells. In other embodiments, the host cells can be delivered for DNASE therapy. For example, in some embodiments, the present invention provides host cells, e.g., human cells, e.g., leukocytes, modified to secrete one or more of the extracellular DNASE proteins described herein and intended for administration as a therapeutic agent.

[0019] The present invention further provides a pharmaceutical composition comprising an extracellular DNASE protein or variant thereof described herein, or optionally a polynucleotide or vector described, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated for any route of administration.

[0020] In another aspect, the present invention provides methods for treating a subject in need of extracellular DNA degradation, extracellular chromatin degradation, extracellular trap (ET) degradation, and / or neutrophil extracellular trap (NET) degradation by administering a therapeutically effective amount of an extracellular DNASE or variant thereof or composition described herein.

[0021] Other aspects and embodiments of the present invention will become apparent from the following detailed description. Item 1 A DNase 1-like 3 (D1L3) variant, wherein the D1L3 protein variant has one or more of increased resistance to proteolysis, increased circulating half-life, higher production levels in an in vitro expression system, and substantially less, the same, or better chromatin and / or NET degradation activity in vitro compared to the wild-type D1L3 protein of SEQ ID NO: 4 and / or SEQ ID NO: 5, in each case. Section 2 Item 1. The D1L3 variant of item 1, wherein the D1L3 variant is a fusion protein comprising an amino acid sequence that is at least 80% identical to the mature enzyme defined by SEQ ID NO: 4 or SEQ ID NO: 5, an albumin amino acid sequence on the N-terminal side of the mature enzyme, and a linking amino acid sequence between the albumin amino acid sequence and the amino acid sequence of the mature enzyme. Section 3 Item 3. The D1L3 variant of item 2, wherein the D1L3 variant comprises an amino acid sequence that is at least 90% identical to the mature enzyme defined by SEQ ID NO:4 or SEQ ID NO:5. Section 4 Item 3. The D1L3 variant of item 2, wherein the D1L3 variant has a deletion of at least 5 amino acids in the C-terminal basic domain, and the C-terminal basic domain is defined by the 23 C-terminal amino acids of SEQ ID NO: 4 or SEQ ID NO: 5. Section 5 Item 5. The D1L3 variant of item 4, wherein the D1L3 has a deletion of at least 10 amino acids in the C-terminal basic domain. Section 6 Item 5. The D1L3 variant of item 4, wherein the D1L3 has a deletion of at least 15 amino acids in the C-terminal basic domain. Section 7 Item 7. The D1L3 variant of item 6, wherein the D1L3 has a deletion of the entire C-terminal basic domain. Section 8 Item 8. The D1L3 variant according to any one of Items 1 to 7, wherein the D1L3 has an amino acid substitution at a position corresponding to position 101 of SEQ ID NO:4. Section 9 The D1L3 variant of paragraph 8, wherein the D1L3 has an amino acid substitution of Q101R. Section 10 The D1L3 variant of paragraph 2, wherein the linker is flexible, rigid, or comprises a protease cleavage site. Section 11 The D1L3 variant of paragraph 10, wherein the linker is a flexible linker. Section 12 12. The D1L3 variant of paragraph 10 or 11, wherein the linker has at least 10 amino acids, or at least 15 amino acids. Section 13 Item 13. The D1L3 variant according to Item 12, wherein the linker has 15 to 35 amino acids. Section 14 Item 1. The D1L3 variant of item 1, wherein the variant comprises the amino acid sequence of any one of SEQ ID NOs: 8 to 30, and in each case, optionally has 1 to 20 amino acid modifications independently selected from an insertion, deletion, or substitution. Section 15 The D1L3 variant of paragraph 14, wherein the variant has the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. Section 16 15. The D1L3 variant of any one of items 1 to 14, comprising one or more component substitutions from D1 and / or one or more deletions or substitutions of cysteine ​​residues compared to the enzyme of SEQ ID NO: 4. Section 17 17. The D1L3 variant of paragraph 16, wherein the amino acid corresponding to C68 in SEQ ID NO: 4 is substituted. Section 18 Item 18. The D1L3 variant of item 17, wherein the amino acid corresponding to C68 in SEQ ID NO: 4 is substituted with an amino acid selected from Ala, Ser, and Gly. Section 19 17. The D1L3 variant of clause 16, wherein the variant comprises the substitution N64_I70delinsHLTAVGK, optionally modified by one, two, or three amino acid substitutions, deletions, or insertions, with the proviso that the component substitutions are not modified to include a cysteine ​​residue. Section 20 Item 1, wherein the D1L3 variant comprises PEG conjugated to the amino acid corresponding to C68 and / or C194. Section 21 Item 3. A D1L3 variant according to item 1 or 2, comprising one or more substitutions of arginine and lysine residues present in SEQ ID NO: 4 and / or SEQ ID NO: 5, wherein the substitutions render the D1L3 variant less susceptible to proteolysis by plasmin, thrombin, and / or trypsin compared to the protein of SEQ ID NO: 4 or the protein of SEQ ID NO: 5. Section 22 22. The D1L3 variant of paragraph 21, wherein one or more of the arginine and lysine substitutions correspond to position(s) K180, K200, K259, and R285 of SEQ ID NO: 4. Section 23 23. The D1L3 variant of paragraph 22, wherein the arginine or lysine substitution is selected from one or more of K180A, K200A, K259A, and R285A with respect to SEQ ID NO:4. Section 24 23. The D1L3 variant of clause 22, wherein the variant comprises a substitution selected from K180_A181delinsGL, P198_A201delinsRPSQ, and K259A with respect to SEQ ID NO: 4. Section 25 22. The D1L3 variant of claim 21, wherein the variant comprises one or more mutations of paired basic residues, and the paired basic residues correspond to positions selected from K50 / R51, R80 / R81, K114 / R115, K199 / K200, K226 / K227, K291 / K292, R297 / K298 / K299, and K303 / R304 of SEQ ID NO: 4. Section 26 26. The D1L3 variant of clause 25, wherein the one or more mutations in the paired basic residues comprise an amino acid substitution selected from R114T, R114A, R114D, R114Q, K227S, and K227E with respect to SEQ ID NO:4. Section 27 26. The D1L3 variant of clause 25, wherein the mutation of one or more of the paired basic residues comprises an amino acid substitution selected from R51K, R81K, R115K, and R304K with respect to SEQ ID NO:4. Section 28 The D1L3 variant of paragraph 10, wherein the linker is cleavable by a coagulation pathway protease. Section 29 29. The D1L3 variant of paragraph 28, wherein the linker is cleavable by factor XII or a neutrophil protease. Section 30 A DNase1 (D1) variant, comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 1, with one or more deletions or substitutions of cysteine ​​residues. Section 31 31. The D1 variant of paragraph 30, wherein one or more amino acids corresponding to C123 and C126 of SEQ ID NO: 1 are deleted or substituted. Section 32 32. The D1 variant of paragraph 31, wherein the cysteine(s) are substituted with an amino acid independently selected from Ala, Ser, and Gly. Item 33 The D1 variant of clause 30, wherein the variant comprises the substitution G122_N128delinsYQGDA. Section 34 Item 34. The D1 variant according to any one of Items 30 to 33, wherein the D1 variant is a fusion protein comprising an albumin amino acid sequence and, optionally, a peptide linker. Section 35 35. The D1 variant of claim 34, wherein the albumin domain of the fusion protein is located N-terminal to the D1 domain. Section 36 36. The D1 variant protein of claim 34 or 35, wherein the peptide linker is inserted between the albumin domain and the D1 domain. Section 37 37. The D1 variant of paragraph 36, wherein the peptide linker is a flexible linker, a rigid linker, or a protease-cleavable linker. Section 38 38. The D1 variant of paragraph 37, wherein the linker has at least 10 amino acids, or at least 15 amino acids. Section 39 Item 38. The D1 variant according to Item 37, wherein the linker has 15 to 35 amino acids. Section 40 40. The D1 variant of any one of items 34 to 39, wherein the D1 domain of the fusion protein comprises an amino acid sequence that is at least 80% identical to the mature enzyme defined by SEQ ID NO: 4 or SEQ ID NO: 5. Section 41 41. The D1 variant of claim 40, wherein the D1 variant has at least 80% of the DNA degradation activity compared to the wild-type D1 protein of SEQ ID NO:1. Section 42 42. The D1 variant of paragraph 40 or 41, wherein the D1 variant comprises an amino acid sequence that is at least 85%, or at least 90%, or at least 95%, or at least 98% identical to the enzyme of SEQ ID NO:1. Section 43 A D1 variant comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 1, and having PEG conjugated to the amino acids corresponding to C123 and / or C126. Section 44 A DNase1-like 1 (D1L1) variant, comprising an amino acid sequence at least 80% identical to the enzyme defined by SEQ ID NO: 2, with one or more deleted or substituted cysteine ​​residues. Section 45 45. The D1L1 variant of paragraph 44, wherein at least one substituted or deleted cysteine ​​residue is at a position corresponding to C22 or C50 of SEQ ID NO:2. Section 46 46. ​​The D1L1 variant of paragraph 45, wherein one or more of the substituted cysteine ​​residues is substituted with an amino acid that is Gly, Arg, or Ser. Section 47 47. The D1L1 variant of any one of paragraphs 44 to 46, wherein the D1L1 variant comprises an amino acid sequence that is at least 85%, or at least 90%, or at least 95%, or at least 98% identical to the enzyme of SEQ ID NO:2. Section 48 Item 48. The D1L1 variant of any one of items 44 to 47, wherein the variant is a fusion protein comprising an albumin amino acid sequence and, optionally, a peptide linker between the D1L1 domain and the albumin domain. Section 49 49. The D1L1 variant of paragraph 48, wherein the albumin domain is located N-terminal to the D1L1 domain. Item 50 50. The D1L1 variant of paragraph 48 or 49, wherein the fusion protein comprises a peptide linker, and the peptide linker is a flexible linker, a rigid linker, or a protease-cleavable linker. Section 51 A D1L1 variant comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 2, and having PEG conjugation to the amino acids corresponding to C22 and / or C50. Section 52 A DNase 1-like 2 (D1L2) variant, comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 3, with one or more substituted or deleted cysteine ​​residues. Section 53 53. The D1L2 variant of paragraph 52, wherein the cysteine ​​residue corresponding to C43 of SEQ ID NO: 3 is substituted or deleted. Section 54 54. The D1L2 variant of paragraph 52 or 53, wherein one or more of the substituted cysteine ​​residues is substituted with an amino acid that is Gly, Arg, or Ser. Section 55 55. The D1L2 variant of any one of paragraphs 52 to 54, wherein the D1L2 variant comprises an amino acid sequence that is at least 85%, or at least 90%, or at least 95%, or at least 98% identical to the enzyme of SEQ ID NO:2. Section 56 56. The D1L2 variant of any one of items 52 to 55, wherein the variant is a fusion protein comprising an albumin amino acid sequence and, optionally, a peptide linker between the D1L2 domain and the albumin domain. Section 57 57. The D1L2 variant of paragraph 56, wherein the albumin domain is located N-terminal to the D1L2 domain. Section 58 58. The D1L2 variant of paragraph 56 or 57, wherein a peptide linker is inserted between the albumin domain and the D1L2 domain. Section 59 59. The D1L2 variant of paragraph 58, wherein the peptide linker is a flexible linker, a rigid linker, or a protease-cleavable linker. Item 60 A D1L2 variant comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO:3, and having PEG conjugation to the amino acid corresponding to C43. Section 61 61. An isolated polynucleotide encoding the D1, D1L1, D1L2, or D1L3 variant of any one of paragraphs 1 to 60. Section 62 62. The isolated polynucleotide of Paragraph 61, wherein the polynucleotide is mRNA. Section 63 62. The isolated polynucleotide of Paragraph 61, wherein the polynucleotide is DNA. Section 64 A vector comprising the polynucleotide according to any one of Items 61 to 63. Section 65 A host cell comprising the vector of paragraph 64. Section 66 61. A host cell modified to express the D1, D1L1, D1L2, or D1L3 variant of any one of paragraphs 1 to 60. Section 67 A pharmaceutical composition comprising a D1, D1L1, D1L2, or D1L3 variant described in any one of Items 1 to 60, a polynucleotide described in any one of Items 61 to 63, a vector described in Item 64, or a host cell described in Item 65 or 66, and a pharmaceutically acceptable carrier. Section 68 68. The pharmaceutical composition of paragraph 67, formulated for topical, parenteral, or pulmonary administration. Section 69 69. The pharmaceutical composition of paragraph 68, formulated for intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial, oral, sublingual, pulmonary, or transdermal administration. Section 70 70. A method for treating a subject in need of extracellular DNA degradation, extracellular chromatin degradation, extracellular trap (ET) degradation, and / or neutrophil extracellular trap (NET) degradation, the method comprising administering a therapeutically effective amount of the composition of any one of paragraphs 67 to 69. Section 71 71. The method of paragraph 70, wherein the subject has a loss-of-function mutation in the D1L3 gene. Section 72 72. The method of paragraph 70 or 71, wherein the subject has a condition selected from chronic neutrophilia, neutrophil aggregation or leukostasis, thrombosis or vascular occlusion, ischemia-reperfusion injury, surgical or traumatic tissue injury, acute or chronic inflammatory response or disease, autoimmune disease, cardiovascular disease, metabolic disease, systemic inflammation, respiratory inflammatory disease, renal inflammatory disease, inflammatory disease associated with transplanted tissue, and cancer. Section 73 72. The method of clause 70 or 71, wherein the subject has or is at risk of having a NET that obstructs the ductal system, and the condition is optionally selected from pancreatitis, cholangitis, conjunctivitis, mastitis, dry eye disease, vascular obstruction, and renal disease. Section 74 72. The method of paragraph 70 or 71, wherein the subject has or is at risk of having an accumulation of NETs on the endothelial surface. Section 75 Item 75. The method of any one of items 70 to 74, wherein the D1L3 variant is a fusion protein comprising an albumin amino acid sequence and is administered parenterally to the subject, and the administration is approximately once a week. Section 76 61. A method for producing a recombinant DNase variant according to any one of items 1 to 60, comprising culturing a cell expressing a polynucleotide encoding the DNase variant, and recovering the recombinant protein. Section 77 77. The method of paragraph 76, wherein the cell is Pichia pastoris. Section 78 78. The method of paragraph 77, wherein the Pichia pastoris cells have a genetic deletion or inactivation, or pharmacological inhibition, of aspartic proteinase 3 and / or kexin. Section 79 77. The method of paragraph 76, wherein the cell is a CHO cell. Section 80 80. The method of paragraph 79, wherein the CHO cells have a genetic deletion or inactivation, or pharmacological inhibition, of the furin protease. Section 81 Item 81. The method according to any one of Items 76 to 80, wherein the DNase variant is a D1L3 variant comprising one or more mutations in paired basic residues, and the paired basic residues correspond to positions selected from K50 / R51, R80 / R81, K114 / R115, K199 / K200, K226 / K227, K291 / K292, R297 / K298 / K299, and K303 / R304 in SEQ ID NO: 4. Section 82 82. The method of claim 81, wherein the mutation of one or more of the paired basic residues comprises an amino acid substitution selected from R114T, R114A, R114D, R114Q, K227S, and K227E. Section 83 82. The method of claim 81, wherein the mutation of one or more of the paired basic residues comprises an amino acid substitution selected from R51K, R81K, R115K, and R304K. Section 84 1. A method for producing a recombinant D1 protein family member or a variant thereof, comprising culturing cells expressing a polynucleotide encoding the DNase variant in the presence of a polyanionic compound, and recovering the recombinant protein. Section 85 85. The method of paragraph 84, wherein the recombinant D1 protein family member is a variant of D1, D1L3, D1L1, or D1L2. Section 86 86. The method of claim 84 or 85, wherein the polyanion is selected from one or more of dextran sulfate, heparin, ferric citrate, and EDTA. Section 87 87. The method of claim 86, wherein the polyanion is dextran sulfate. Section 88 88. The method of any one of paragraphs 84 to 87, wherein the cell is prokaryotic or eukaryotic. Section 89 89. The method of paragraph 88, wherein the DNase is expressed using a non-mammalian expression system, which is optionally Pichia pastoris, Saccharomyces species, or Escherichia coli. Section 90 90. The method of claim 89, wherein the expression system is Pichia pastoris. Section 91 91. The method of paragraph 90, wherein the Pichia pastoris cells have a genetic deletion or inactivation, or pharmacological inhibition, of aspartic proteinase 3 and / or kexin. Section 92 89. The method of paragraph 88, wherein the cell is a CHO cell. Section 93 93. The method of paragraph 92, wherein the CHO cells have a genetic deletion or inactivation, or pharmacological inhibition, of the furin protease. Section 94 94. The method of any one of paragraphs 84 to 93, wherein the recombinant D1 family member comprises a fusion of albumin at the N-terminus via a peptide linker. Section 95 95. The method of claim 94, wherein the linker is a flexible linker. Section 96 96. The method of paragraph 95, wherein the linker is a cleavable linker, optionally cleavable by a coagulation pathway protease or a neutrophil protease. Section 97 97. The method of paragraph 96, wherein the coagulation factor protease is Factor XII. Section 98 98. The method of claim 97, wherein the peptide linker is selected from all or part of the FXII cleavage site in Factor XI or prekallikrein. Section 99 99. The method of any one of paragraphs 84 to 98, wherein the recombinant D1 family member or variant is expressed in a construct encoding a signal peptide that directs secretion of the recombinant protein from the cell. Item 100 Item 99. The method of any one of Items 84 to 99, wherein the recombinant D1 family member or variant is purified from the polyanionic compound using an anion exchange resin. Section 101 101. The method of paragraph 100, wherein the recombinant D1 family member or variant is further purified using a cation exchange resin. [Brief explanation of the drawings]

[0022] [Figure 1] This shows that the mutations Q101R and Q282_S305delinsK in SEQ ID NO: 4 increase the activity of DNASE1L3 to degrade high-molecular-weight chromatin. CHO cells were transiently transfected with wild-type DNASE1L3 or DNASE1L3 with component substitutions. Supernatants from transfected cells were incubated with purified nuclei (high-molecular-weight chromatin) or buffer. DNA was isolated and analyzed by agarose gel electrophoresis. The figure shows an agarose gel stained with a DNA dye. [Figure 2]The characteristics of two DNASE1L3 variants are shown. Different concentrations of supernatants from CHO cells transfected with wild-type DNASE1L3 or DNASE1L3 carrying the Q101R or Q282_S305delinsK mutations were analyzed by Western blot (WB) using an anti-DNASE1L3 antibody. In samples with wild-type DNASE1L3 and the Q101R mutation, larger (Variant 1) and smaller (Variant 2) bands were detected. Only the smaller band (Variant 2) was present in samples with the Q282_S305delinsK mutation. In parallel, chromatin degradation activity in supernatants at different concentrations was analyzed. The figure shows DNA analyzed by agarose gel electrophoresis. Both the Q101R and Q282_S305delinsK mutations increased chromatin degradation activity compared to wild-type DNASE1L3. [Figure 3] Figure 1 shows the presence of DNASE1L3 variants 1 and 2 in the supernatant of CHO cells stably transfected with wild-type DNASE1L3. Samples were analyzed by Western blot (WB) using an anti-DNASE1L3 antibody. A larger (variant 1) and smaller (variant 2) band were detected in five clones. [Figure 4] To identify frequent cleavage sites, the C-terminal amino acid sequence of wild-type D1L3 recombinantly expressed in Pichia pastoris is shown. Amino acid sequencing of purified wild-type D1L3 identified three C-terminal deletion mutants: K291_S305del, K292_S305del, and S293_S305del. The C-terminus of wild-type D1L3 was not detected. In parallel, the chromatin degradation activity of purified proteins at different concentrations was analyzed and compared with that of purified DNASE1 (D1) and basic domain-deleted DNASE1L3 (BDD-D1L3) carrying the F275Y / F279_K280delinsVM / Q282_S305delinsK mutations. The figure shows DNA analyzed by agarose gel electrophoresis. [Figure 5]This figure shows that adding dextran sulfate to CHO medium improves protein yield. Stable pools of CHO cells expressing wild-type D1L3 were incubated in standard CHO medium or CHO medium supplemented with dextran sulfate. Supernatants were analyzed by Western blot (WB) using an anti-DNASE1L3 antibody. The figure shows that D1L3 is poorly expressed in CHO cells, resulting in low yields. Adding dextran sulfate increases yield but does not prevent product fragmentation. [Figure 6] Figure 1 shows the use of anion-exchange and cation-exchange surfaces for affinity purification of dextran sulfate-conjugated D1L3. A shows that a polyanion, such as dextran sulfate (DS), forms a complex with D1L3. During the production process, the D1L3-DS complex is prevented from interacting with and being removed by the negatively charged surface. B and C show a two-step process for purifying D1L3 from the DS-D1L3 complex. [Figure 7] Trypsin cleavage site mutation strategies to limit D1L3 degradation are listed. [Figure 8] Alignment of human D1 (SEQ ID NO: 1) and human D1L3 (SEQ ID NO: 4) amino acid sequences with plasmin-sensitive KR residues indicated. [Figure 9] 1 shows a plasmin cleavage site mutation strategy to limit D1L3 degradation. [Figure 10] We demonstrate that D1L3 with mutated plasmin cleavage sites retains enzymatic activity. Supernatants from cells transiently transfected with DNASE1L3 containing mutations at four putative plasmid cleavage sites (K180_A181delinsGL, P198_A201delinsRPSQ, K259A, R285A) were incubated with purified nuclei (high molecular weight chromatin) or buffer. DNA was isolated and analyzed by agarose gel electrophoresis. The figure shows an agarose gel stained with a DNA dye. [Figure 11] Plasmin cleavage sites based on plasmin digestion are listed, and mutation strategies to limit D1L3 degradation are presented. [Figure 12](A) shows that D1L3 has a tendency to misfold when expressed in CHO cells. (B) shows a simple expression vector for D1L3 expression using the native secretory signal peptide. (C) The supernatant of the stable pool was analyzed by Western blot using an anti-DNASE1L3 antibody, showing the presence of high molecular weight aggregates under non-reducing conditions, which are degraded under reducing conditions. [Figure 13] Alignment of human D1 (SEQ ID NO: 1) and human D1L3 (SEQ ID NO: 4) amino acid sequences with conserved and non-conserved cysteine ​​residues indicated. [Figure 14] Cysteine ​​residues in D1L3 are listed and mutation strategies to limit high molecular weight aggregates during protein expression are presented. [Figure 15] This shows that the C68A and C194A mutations in D1L3 do not affect chromatin degradation activity. The mutations C24A and C52A abolished chromatin degradation activity. Supernatants from cells transiently transfected with mutant DNASE1L3 variants were incubated with purified nuclei or buffer. DNA was isolated and analyzed by agarose gel electrophoresis. The figure shows an agarose gel stained with a DNA dye. [Figure 16] Figure 1 shows expression of D1L3 in Pichia pastoris using either the native secretion signal or the α-mating factor (αMF) from Saccharomyces cerevisiae. (A) shows that the N-terminus of D1L3 is driven by the pre-pro secretory leader of the α-mating factor (αMF) from Saccharomyces cerevisiae. (B) shows that the secretion signal from αMF results in glycosylation and non-processing of the signal peptide. [Figure 17]A fusion construct of αME, human serum albumin (HSA), a linker sequence, and D1L3 is not glycosylated in the P. pastoris expression system and maintains chromatinolytic activity. (A) shows a fusion construct of αMF, human serum albumin (HSA), a linker sequence, and D1L3. (B) This fusion construct is not glycosylated in the P. pastoris expression system and maintains chromatinolytic activity. (C) [Figure 18] Figure 1 shows the expression levels of human serum albumin (HSA) fusion constructs of basic domain-deleted DNASE1L3 (BDD-D1L3) or wild-type DNASE1L3 (D1L3) in Pichia pastoris. HSA was fused to either the N- or C-terminus of BDD-D1L3 or D1L3. Two linker sequences, L1 and L2, were placed between HSA and BDD-D1L3 or D1L3. [Figure 19] Figure 1 shows the expression levels of wild-type DNASE1L3 (D1L3) fusion constructs with human serum albumin (HSA) in Pichia pastoris. HSA was fused to the N-terminus of D1L3. Three different linker sequences (L2, L3, and L4) were placed between HSA and D1L3. [Figure 20] Figure 1 shows the expression level and chromatin degradation activity of human serum albumin (HSA) fusion constructs of basic domain-deleted DNASE1L3 (BDD-D1L3) produced in Pichia pastoris. HSA was fused to the N-terminus of BDD-D1L3. Three different linker sequences (L5, L6, and L7) were placed between HSA and D1L3. [Figure 21] 1 shows the serum chromatin degrading activity and circulating half-life of albumin D1L3 fusion proteins. (A) Dnase1- / -Dnase1l3- / - mice injected with SEQ ID NO: 14 and SEQ ID NO: 19 exhibit similar chromatin degrading activity in serum. (B) SEQ ID NO: 19 has a circulating half-life of 3.3 days in mice expressing the human FcRn receptor. [Figure 22]Figure 1 shows the expression level and chromatin degradation activity of human serum albumin (HSA) fusion constructs of basic domain-deleted DNASE1L3 (BDD-D1L3) produced in Pichia pastoris. HSA was fused to the N- and C-termini of BDD-D1L3. Two different linker sequences (L7 and L8) were placed between HSA and BDD-D1L3. [Figure 23] Figure 1 shows the design of cleavable linkers. A shows a fusion construct of HSA and a linker. B shows a fusion construct of a linker cleavable by factor XIIa. A linker containing the human factor XI sequence (SEQ ID NO: 42) and a linker containing human prekallikrein (SEQ ID NO: 44) are shown. [Figure 24] 1 shows other constructs utilizing a Factor XIIa cleavable linker in half-life extending fusion proteins including human extracellular DNase, human coagulation factors, and human complement factors. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides candidate engineered human extracellular DNASE proteins (e.g., variants of DNASE1 (D1), DNASE1-like 1 (D1L1), DNASE1-like 2 (D1L2), DNASE1-like 3 isoform 1 (D1L3), DNASE1-like 3 isoform 2 (D1L3-2), DNASE2A (D2A), and DNASE2B (D2B)) that are useful for treating conditions characterized by the accumulation and / or release of extracellular DNA, extracellular chromatin, and neutrophil extracellular traps (NETs). In accordance with aspects of the present invention, the DNase variants described herein are more suitable and / or effective for therapy and / or more amenable to large-scale manufacturing. In some embodiments, the DNase variants described herein have advantages over systemic therapy. Such advantages include longer exposure (e.g., slower elimination, longer circulating half-life), extended duration of pharmacodynamic action, improved chromatin degradation activity, and protease resistance.

[0024] definition As used in the specification and claims, the singular forms "a," "an," and "the" include singular and plural references unless the context clearly dictates otherwise. Thus, for example, reference to an "agent" includes a single agent as well as a plurality of such agents.

[0025] The term "chromatin enzyme" refers to a class of deoxyribonuclease enzymes that have more than minor ability to cut, cleave, or digest chromatin, i.e., DNA associated with one or more histone proteins. Human DNASE1L3 is a chromatin enzyme. Generally, the various DNASE1L3 variants disclosed herein are chromatin enzymes. Not all DNASEs are chromatin enzymes. For example, human DNASE1 essentially does not have the ability to cut, cleave, or digest chromatin, and is not a chromatin enzyme.

[0026] As used herein with respect to drugs, "half-life" refers to the elimination half-life of the drug's concentration in an animal as measured in a matrix of interest, e.g., serum or plasma. Those skilled in the art will understand that not all drugs exhibit first-order kinetics or do so at all stages of elimination. In such cases, those skilled in the art will understand that the term "half-life extension" or "extended half-life" refers to a slower rate of elimination.

[0027] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but rather the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state. An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0028] As used herein, "neutrophil extracellular traps" and the acronym "NETs" refer to networks of extracellular fibers containing nuclear contents, e.g., DNA associated with histone proteins, that are released in a programmed manner from immune cells, typically neutrophils.

[0029] Unless otherwise specified, a "nucleotide sequence or nucleic acid encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that a nucleotide sequence encoding a protein may, in some forms, contain intron(s).

[0030] The terms "about" and "approximately" include amounts that are plus or minus 10% of the associated numerical value.

[0031] The term "extracellular DNASE" refers to extracellular DNASE proteins of the DNASE1 and DNASE2 families (e.g., DNASE1 (D1), DNASE1-like 1 (D1L1), DNASE1-like 2 (D1L2), DNASE1-like 3 isoform 1 (D1L3), DNASE1-like 3 isoform 2 (D1L3-2), DNASE2A (D2A), and DNASE2B (D2B)).

[0032] In some aspects and embodiments, the extracellular DNASE or variant thereof is optionally fused by an intercalated linker to a half-life extending moiety, such as albumin, transferrin, Fc or elastin-like protein, or variants thereof. See, e.g., US9,458,218, incorporated herein by reference in its entirety. In some embodiments, the extracellular DNASE or variant thereof is dimerized by an immunoglobulin hinge region. For example, the engineered enzymes described herein can be fused to an Fc or elastin-like protein, or variants thereof. Fusion domains (e.g., immunoglobulin hinge and CH2 domains and CH3 domains) may also be included. In some embodiments, the DNASE (e.g., a D1L3 variant) is fused to an albumin amino acid sequence or domain, e.g., human albumin, or a fragment or variant thereof. See, e.g., WO 2015 / 066550 and US 9,221,896, which are incorporated by reference in their entireties. Albumin can be linked to the DNASE at the N-terminus and / or C-terminus of the engineered extracellular DNASE or variant thereof, optionally using an inserted linker. An exemplary albumin amino acid sequence is provided by SEQ ID NO: 39. In some embodiments, the D1L3 and D1, or variants described herein, are dimerized together via the Fc hinge region to create a dimeric molecule with synergistic functional properties for degrading NETs. In some embodiments, the extracellular DNASE or variant thereof is fused to the albumin amino acid sequence at the N-terminus via a peptide linker. The peptide linker can be a flexible linker, a rigid linker, or in some embodiments, a physiologically cleavable linker (e.g., a protease-cleavable linker). In some embodiments, the linker is 5-100 amino acids in length, or 5-50 amino acids in length. In yet other embodiments, the linker is an organic molecule, group, polymer (e.g., PEG), or chemical moiety covalently attached to the extracellular DNASE and the half-life extending moiety (e.g., albumin).

[0033] In some aspects, the present invention provides D1L3 variants, wherein the D1L3 variants have one or more of increased protein stability, increased pharmacokinetic exposure and duration of pharmacodynamic activity, resistance to proteolysis, higher production levels in in vitro expression systems, better suitability for purification, and substantially the same, if not lower, or better chromatin and / or NET degradation activity compared to the wild-type D1L3 isoform 1 enzyme of SEQ ID NO: 4 or the wild-type D1L3 isoform 2 enzyme of SEQ ID NO: 5. As used herein, unless stated to the contrary, the term "D1L3" includes either isoform 1 or isoform 2.

[0034] The DNA and / or chromatin and / or NET-degrading activity of an enzyme, e.g., a D1L3 variant, can be measured in vitro, e.g., by incubating the enzyme with purified nuclei, DNA, or DNA, chromatin, or NETs obtained from ex vivo blood or neutrophils induced to form NETs. Alternatively, the DNA and / or chromatin and / or NET-degrading activity of an enzyme, e.g., a D1L3 variant, can be measured in vivo, e.g., by administering the enzyme to a subject, who produces or is induced to produce extracellular DNA, chromatin, or NETs, ​​and measuring the effect of the enzyme on the concentration of DNA, chromatin, or NET levels in the matrix, e.g., using serum, preferably a parallel negative control, or by comparing concentrations before and after enzyme administration over time.

[0035] In some embodiments, the D1L3 variant has about the same chromatin and / or NET degradation activity as the wild-type D1L3 isoform 1 enzyme of SEQ ID NO: 4 or the wild-type D1L3 isoform 2 enzyme of SEQ ID NO: 5. In some embodiments, the D1L3 variant has greater chromatin and / or NET degradation activity as the wild-type D1L3 isoform 1 enzyme of SEQ ID NO: 4 or the wild-type D1L3 isoform 2 enzyme of SEQ ID NO: 5.

[0036] In some embodiments, the D1L3 variant is a fusion protein comprising an albumin domain, an optional linker, and a D1L3 domain. In some embodiments, the albumin domain and optional linker are located N-terminal to the D1L3 domain. In some embodiments, the albumin domain and optional linker are located C-terminal to the D1L3 domain. In all such embodiments, an optional linker is inserted between the albumin domain and the D1L3 domain.

[0037] In some embodiments, the albumin amino acid sequence or domain of the fusion protein is at least about 75%, or at least about 80%, or at least about 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the reference albumin sequence defined by SEQ ID NO: 39. In some embodiments, the albumin amino acid sequence or domain comprises or consists of the reference albumin sequence defined by SEQ ID NO: 39. In various embodiments, the albumin amino acid sequence binds to a neonatal Fc receptor (FcRn), e.g., human FcRn. The albumin amino acid sequence can be a variant of wild-type HSA (e.g., represented by SEQ ID NO: 39). In various embodiments, the albumin variant can have 1 to 20, or 1 to 10, amino acid modifications independently selected from deletions, substitutions, and insertions with respect to SEQ ID NO: 39. In some embodiments, the albumin amino acid sequence is any mammalian albumin amino acid sequence.

[0038] In some embodiments, the albumin amino acid sequence or domain is a fragment of full-length albumin represented by SEQ ID NO: 39. The term "fragment," when used in the context of albumin, refers to any fragment of full-length albumin or a variant thereof that extends the half-life of a DNASE enzyme to which it is fused or conjugated compared to the corresponding unfused DNASE. In some embodiments, a fragment of albumin can refer to an amino acid sequence comprising a fusion of multiple domains of albumin, such as domains I and III and domains II and III (see, e.g., WO2011 / 124718). Generally, a fragment of albumin has at least about 100 amino acids, or at least about 200 or at least about 300 amino acids of the full-length sequence. In various embodiments, the albumin fragment maintains the ability to bind to human FcRn.

[0039] In some embodiments, the D1L3-like domain of the fusion protein is at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identical to the mature D1L3 enzyme reference sequence defined by SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the D1L3 domain comprises or consists of the reference sequence defined by SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the reference sequence does not include the C-terminal basic domain of SEQ ID NO: 4 or 5, which is defined by the C-terminal 23 amino acids.

[0040] In some embodiments, the fusion protein comprises a D1L3 domain, wherein the amino acid sequence of the D1L3 domain is at least about 80% identical to the mature enzyme defined by SEQ ID NO:4 or SEQ ID NO:5. The fusion protein may further comprise an albumin amino acid sequence or domain at the N-terminus of the mature enzyme, as well as a linking amino acid sequence between the albumin amino acid sequence and the amino acid sequence of the mature enzyme. In some embodiments, the D1L3 domain comprises an amino acid sequence that is at least about 90% identical to the mature enzyme reference sequence defined by SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the reference sequence does not include the C-terminal basic domain of SEQ ID NO:4 or SEQ ID NO:5, defined by the C-terminal 23 amino acids. Fusion proteins comprising the D1L3 domain exhibit improved circulating half-life and duration of pharmacodynamic effect, including systemic therapy. Additionally, the fusion of albumin to the linking sequence does not substantially affect (or, in some embodiments, does not negatively affect) chromatin degradation activity, as determined using an in vitro chromatin degradation assay, compared to a variant without the albumin fusion.

[0041] When referring to sequence identity with wild-type DNase enzymes, the sequences are shown in the signature unless otherwise stated. " refers to the mature enzyme lacking the signal peptide. Furthermore, unless otherwise specified, for clarity, amino acid positions are numbered with respect to the full translated DNase sequence, including the signal peptide. Thus, for example, reference to sequence identity to the enzyme of SEQ ID NO: 4 (human D1L3, isoform 1) refers to percent identity with the mature enzyme having M21 at the N-terminus. Similarly, reference to sequence identity to the enzyme of SEQ ID NO: 1 (human D1) refers to percent identity with the mature enzyme having L23 at the N-terminus.

[0042] In some embodiments, D1L3 has a deletion of all or part of its C-terminal basic domain. The C-terminal basic domain is defined as the C-terminal 23 amino acids of SEQ ID NO:4 or SEQ ID NO:5. Deletion or inactivation of the C-terminal basic domain of D1L3 substantially improves chromatin degradation activity. See Figures 1, 2, and 4. In some embodiments, the D1L3 variant has a deletion of C-terminal basic domain amino acids, such as at least 5 amino acids, or in some embodiments, at least 10 amino acids, or in some embodiments, at least 15 amino acids, or in some embodiments, at least 20 amino acids. In some embodiments, the D1L3 variant has a deletion of the entire C-terminal basic domain defined by the C-terminal 23 amino acids of SEQ ID NO:4 or SEQ ID NO:5. Exemplary BD deletions include Q282_S305delinsK (see SEQ ID NO: 9), S305delinsK (see SEQ ID NO: 10), K292_S305del (see SEQ ID NO: 11), and S293_S305del (see SEQ ID NO: 12). In some embodiments, the C-terminus of the D1L3 domain (with a BD deletion) has 1-10 or 1-5 amino acids at the C-terminus that do not align with the C-terminal BD and that do not adversely affect chromatin degradation activity in in vitro assays.

[0043] In some embodiments, the D1L3 variant is an engineered fusion protein comprising a DNASE1L3 domain of a sequence selected from SEQ ID NO:8-16, a linker of a sequence selected from SEQ ID NO:31-38, and an albumin domain having the sequence of SEQ ID NO:39 or a described variant or fragment. In some embodiments, the D1L3 variant has one or more component substitutions from D1, as described in PCT / US2018 / 04708, incorporated herein by reference.

[0044] In some embodiments, the D1L3 sequence or domain comprises component substitutions from D1, which are M21_R22delinsLK, C24_S25delinsAA, V28_S30delinsIQT, S34T, Q36_V44delinsMSNATLVSY, K47_K50delinsQILS, C52Y, I55_M58delinsIALVQE, I60_K61delinsVR, N64_I70 delinsHLTAVGK, M72_K74delinsLDN, R77_I83delinsQDAPD, N86H, I89V, S91_R92delinsEP, T97S, Q101R, A103L, L1 05V, K107_L110delinsRPDQ, V113_R115delinsAVD, H118Y, H120D, Y122_A127delinsGCEPCGN, V129T, S131N, F135_ V136delinsAI, W138R, Q140_H143delinFSRF, A145_D148delinsEVRE, V150A, I152V, T156_T157delinsAA, E159_S1 61delinsGDA, K163A, E167A, V169_E170delinsYD, T173L, K176_R178delinsQEK, K180_A181delinsGL, N183_F186d elinsDVML, P198_A201delinsRPSQ, K203_N204delinsSS, R208W, D210S, R212T, V214Q, G218P, Q220_E221delinsSA , V225_S228delinsATP, N230H, L238_R239delinsVA, Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256 N, K259A, K262G, T264_E267delinsSDQL, L269_V271delinsQAI, F275Y, F279_K280delinsVM, Q282_S205delinsK, each of the foregoing substitutions being numbered with respect to SEQ ID NO:4.

[0045] For example, the D1L3 variant may have a D1 component substitution of Q282_S305delinsK, which includes a deletion of the C-terminal basic domain and is absent in D1. In some embodiments, the D1L3 enzyme has an amino acid substitution at position 101 of SEQ ID NO: 4. The substitution may be Arg based on the corresponding component from D1, or in some embodiments, Lys. Substitutions at this position can enhance the chromatin degradation activity of D1L3. Other substitutions at this position are likely to exhibit similar properties.

[0046] The linker, when present, can be selected from flexible, rigid, and cleavable peptide linkers. Flexible linkers are composed primarily or entirely of small, non-polar, or polar residues, such as Gly, Ser, and Thr. Exemplary flexible linkers include (Gly y Ser) n Linkers include y=1-10 (e.g., 1-5) and n=1-10, and in some embodiments, 3-6. In exemplary embodiments, y=2-4 and n=3-8. Due to their flexibility, these linkers are unstructured. More rigid linkers include polyproline or polyPro-Ala motifs and α-helical linkers. An exemplary α-helical linker is A(EAAAK). n A, where n is as defined above (e.g., 1-10, or 2-6). Generally, the linker may be composed primarily of amino acids selected from Gly, Ser, Thr, Ala, and Pro. Exemplary linker sequences include at least 10 amino acids and can range from 15-35 amino acids. Exemplary linker designs are provided as SEQ ID NOs: 31-38.

[0047] In some embodiments, the variants include a linker, and the amino acid sequence of the linker is predominantly or essentially composed of glycine and serine residues. In some embodiments, the ratio of Ser to Gly in the linker is about 1:1 to about 1:10, about 1:2 to about 1:6, or about 1:4, respectively. Exemplary linker sequences include S(GGS)4GSS (SEQ ID NO: 36), S(GGS)9GS (SEQ ID NO: 37), and (GGS)9GS (SEQ ID NO: 39). In some embodiments, the linker has at least 10 amino acids, or at least 15 amino acids, or at least 20 amino acids, or at least 25 amino acids. For example, the linker can be 15-30 amino acids in length. In various embodiments, a longer linker of at least 15 amino acids can provide improved yields upon expression in Pichia pastoris. See Figure 20. Furthermore, surprisingly, longer linker sequences exhibited improved chromatin degradation activity compared to shorter linker sequences. See Figure 20.

[0048] In various embodiments, the D1L3 variant is a fusion protein comprising the amino acid sequence of any one of SEQ ID NOs: 17-30. In other embodiments, the D1L3 variant is a fusion protein comprising the amino acid sequence of any one of SEQ ID NOs: 17-30, and having 1-20, 1-10, or 1-5 amino acid modifications independently selected from amino acid insertions, deletions, or substitutions relative to a reference sequence selected from SEQ ID NOs: 17-30. In some embodiments, the amino acid modifications are in the D1L3 domain, the albumin domain, or both domains of the fusion protein. In some embodiments, the variant has the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. In these embodiments, the albumin domain is modified. The amino acid sequence is fused to the N-terminus or N-terminal side of D1L3 (or a variant) via a medium or long flexible linker.

[0049] In other embodiments, the linker is a physiologically cleavable linker, e.g., a protease-cleavable linker. For example, the protease can be a coagulation pathway protease, such as activated factor XII. In certain embodiments, the linker comprises the amino acid sequence of factor XI (SEQ ID NO: 42) and / or prekallikrein (SEQ ID NO: 44 or 45), or a physiologically cleavable fragment thereof. In selected embodiments, the linker amino acid sequence from factor XI comprises all or a portion of SEQ ID NO: 42 (e.g., a portion of SEQ ID NO: 42 containing a modification of SEQ ID NO: 42 that allows for cleavage by factor XIIa). In some embodiments, the linker amino acid sequence from prekallikrein comprises all or a portion of SEQ ID NO: 44 (e.g., a portion of SEQ ID NO: 44 containing a modification of SEQ ID NO: 44 that allows for cleavage by factor XIIa). In other embodiments, the linker comprises a peptide sequence targeted for cleavage by neutrophil-specific proteases, such as neutrophil elastase, cathepsin G, and proteinase 3.

[0050] Some exemplary embodiments of D1L3 fusion proteins include a combination of three amino acid sequences, which may be independently selected from the sequences disclosed herein, arranged in N-terminal to C-terminal order as follows:

[0051] Fusion 1: SEQ ID NO:4, SEQ ID NO:31, SEQ ID NO:39; Fusion 2: SEQ ID NO:5, SEQ ID NO:31, SEQ ID NO:39; Fusion 3: SEQ ID NO:8, SEQ ID NO:31, SEQ ID NO:39; Fusion 4: SEQ ID NO:9, SEQ ID NO:31, SEQ ID NO:39; Fusion 5: SEQ ID NO:10, SEQ ID NO:31, SEQ ID NO:39; Fusion 6: SEQ ID NO:11, SEQ ID NO:31, SEQ ID NO:39; Fusion 7: SEQ ID NO:12, SEQ ID NO:31, SEQ ID NO:39; Fusion 8: SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:39; Fusion 9: SEQ ID NO: 14, SEQ ID NO: 31, SEQ ID NO: 39; Fusion 10: SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 39; Fusion 11: SEQ ID NO: 16, SEQ ID NO: 31, SEQ ID NO: 39; Fusion 12: SEQ ID NO:4, SEQ ID NO:32, SEQ ID NO:39; Fusion 13: SEQ ID NO:5, SEQ ID NO:32, SEQ ID NO:39; Fusion 14: SEQ ID NO:8, SEQ ID NO:32, SEQ ID NO:39; Fusion 15: SEQ ID NO:9, SEQ ID NO:32, SEQ ID NO:39; Fusion 16: SEQ ID NO:10, SEQ ID NO:32, SEQ ID NO:39; Fusion 17: SEQ ID NO:11, SEQ ID NO:32, SEQ ID NO:39; Fusion 18: SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 39; Fusion 19: SEQ ID NO: 13, SEQ ID NO: 32, SEQ ID NO: 39; Fusion 20: SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 39; Fusion 21: SEQ ID NO: 15, SEQ ID NO: 32, SEQ ID NO: 39; Fusion 22: SEQ ID NO: 16, SEQ ID NO: 32, SEQ ID NO: 39; Fusion 23: SEQ ID NO:4, SEQ ID NO:33, SEQ ID NO:39; Fusion 24: SEQ ID NO:5, SEQ ID NO:33, SEQ ID NO:39; Fusion 25: SEQ ID NO:8, SEQ ID NO:33, SEQ ID NO:39; Fusion 26: SEQ ID NO:9, SEQ ID NO:33, SEQ ID NO:39; Fusion 27: SEQ ID NO: 10, SEQ ID NO: 33, SEQ ID NO: 39; Fusion 28: SEQ ID NO:11, SEQ ID NO:33, SEQ ID NO:39; Fusion 29: SEQ ID NO: 12, SEQ ID NO: 33, SEQ ID NO: 39; Fusion 30: SEQ ID NO: 13, SEQ ID NO: 33, SEQ ID NO: 39; Fusion 31: SEQ ID NO: 14, SEQ ID NO: 33, SEQ ID NO: 39; Fusion 32: SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 39; Fusion 33: SEQ ID NO: 16, SEQ ID NO: 33, SEQ ID NO: 39; Fusion 34: SEQ ID NO:4, SEQ ID NO:34, SEQ ID NO:39; Fusion 35: SEQ ID NO:5, SEQ ID NO:34, SEQ ID NO:39; Fusion 36: SEQ ID NO:8, SEQ ID NO:34, SEQ ID NO:39; Fusion 37: SEQ ID NO:9, SEQ ID NO:34, SEQ ID NO:39; Fusion 38: SEQ ID NO: 10, SEQ ID NO: 34, SEQ ID NO: 39; Fusion 39: SEQ ID NO:11, SEQ ID NO:34, SEQ ID NO:39; Fusion 40: SEQ ID NO: 12, SEQ ID NO: 34, SEQ ID NO: 39; Fusion 41: SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 39; Fusion 42: SEQ ID NO: 14, SEQ ID NO: 34, SEQ ID NO: 39; Fusion 43: SEQ ID NO: 15, SEQ ID NO: 34, SEQ ID NO: 39; Fusion 44: SEQ ID NO: 16, SEQ ID NO: 34, SEQ ID NO: 39; Fusion 45: SEQ ID NO:4, SEQ ID NO:35, SEQ ID NO:39; Fusion 46: SEQ ID NO:5, SEQ ID NO:35, SEQ ID NO:39; Fusion 47: SEQ ID NO:8, SEQ ID NO:35, SEQ ID NO:39; Fusion 48: SEQ ID NO:9, SEQ ID NO:35, SEQ ID NO:39; Fusion 49: SEQ ID NO:10, SEQ ID NO:35, SEQ ID NO:39; Fusion 50: SEQ ID NO:11, SEQ ID NO:35, SEQ ID NO:39; Fusion 51: SEQ ID NO: 12, SEQ ID NO: 35, SEQ ID NO: 39; Fusion 52: SEQ ID NO: 13, SEQ ID NO: 35, SEQ ID NO: 39; Fusion 53: SEQ ID NO: 14, SEQ ID NO: 35, SEQ ID NO: 39; Fusion 54: SEQ ID NO: 15, SEQ ID NO: 35, SEQ ID NO: 39; Fusion 55: SEQ ID NO: 16, SEQ ID NO: 35, SEQ ID NO: 39; Fusion 56: SEQ ID NO:4, SEQ ID NO:36, SEQ ID NO:39; Fusion 57: SEQ ID NO:5, SEQ ID NO:36, SEQ ID NO:39; Fusion 58: SEQ ID NO:8, SEQ ID NO:36, SEQ ID NO:39; Fusion 59: SEQ ID NO:9, SEQ ID NO:36, SEQ ID NO:39; Fusion 60: SEQ ID NO:10, SEQ ID NO:36, SEQ ID NO:39; Fusion 61: SEQ ID NO:11, SEQ ID NO:36, SEQ ID NO:39; Fusion 62: SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 39; Fusion 63: SEQ ID NO: 13, SEQ ID NO: 36, SEQ ID NO: 39; Fusion 64: SEQ ID NO: 14, SEQ ID NO: 36, SEQ ID NO: 39; Fusion 65: SEQ ID NO: 15, SEQ ID NO: 36, SEQ ID NO: 39; Fusion 66: SEQ ID NO: 16, SEQ ID NO: 36, SEQ ID NO: 39; Fusion 67: SEQ ID NO:4, SEQ ID NO:37, SEQ ID NO:39; Fusion 68: SEQ ID NO:5, SEQ ID NO:37, SEQ ID NO:39; Fusion 69: SEQ ID NO:8, SEQ ID NO:37, SEQ ID NO:39; Fusion 70: SEQ ID NO:9, SEQ ID NO:37, SEQ ID NO:39; Fusion 71: SEQ ID NO:10, SEQ ID NO:37, SEQ ID NO:39; Fusion 72: SEQ ID NO:11, SEQ ID NO:37, SEQ ID NO:39; Fusion 73: SEQ ID NO: 12, SEQ ID NO: 37, SEQ ID NO: 39; Fusion 74: SEQ ID NO: 13, SEQ ID NO: 37, SEQ ID NO: 39; Fusion 75: SEQ ID NO: 14, SEQ ID NO: 37, SEQ ID NO: 39; Fusion 76: SEQ ID NO: 15, SEQ ID NO: 37, SEQ ID NO: 39; Fusion 77: SEQ ID NO: 16, SEQ ID NO: 37, SEQ ID NO: 39; Fusion 78: SEQ ID NO:4, SEQ ID NO:38, SEQ ID NO:39; Fusion 79: SEQ ID NO:5, SEQ ID NO:38, SEQ ID NO:39; Fusion 80: SEQ ID NO:8, SEQ ID NO:38, SEQ ID NO:39; Fusion 81: SEQ ID NO:9, SEQ ID NO:38, SEQ ID NO:39; Fusion 82: SEQ ID NO:10, SEQ ID NO:38, SEQ ID NO:39; Fusion 83: SEQ ID NO:11, SEQ ID NO:38, SEQ ID NO:39; Fusion 84: SEQ ID NO: 12, SEQ ID NO: 38, SEQ ID NO: 39; Fusion 85: SEQ ID NO: 13, SEQ ID NO: 38, SEQ ID NO: 39; Fusion 86: SEQ ID NO: 14, SEQ ID NO: 38, SEQ ID NO: 39; Fusion 87: SEQ ID NO: 15, SEQ ID NO: 38, SEQ ID NO: 39; Fusion 88: SEQ ID NO: 16, SEQ ID NO: 38, SEQ ID NO: 39; Fusion 89: SEQ ID NO:4, SEQ ID NO:42, SEQ ID NO:39; Fusion 90: SEQ ID NO:5, SEQ ID NO:42, SEQ ID NO:39; Fusion 91: SEQ ID NO:8, SEQ ID NO:42, SEQ ID NO:39; Fusion 92: SEQ ID NO:9, SEQ ID NO:42, SEQ ID NO:39; Fusion 93: SEQ ID NO:10, SEQ ID NO:42, SEQ ID NO:39; Fusion 94: SEQ ID NO:11, SEQ ID NO:42, SEQ ID NO:39; Fusion 95: SEQ ID NO: 12, SEQ ID NO: 42, SEQ ID NO: 39; Fusion 96: SEQ ID NO: 13, SEQ ID NO: 42, SEQ ID NO: 39; Fusion 97: SEQ ID NO: 14, SEQ ID NO: 42, SEQ ID NO: 39; Fusion 98: SEQ ID NO: 15, SEQ ID NO: 42, SEQ ID NO: 39; Fusion 99: SEQ ID NO: 16, SEQ ID NO: 42, SEQ ID NO: 39 Fusion 100: SEQ ID NO:4, SEQ ID NO:43, SEQ ID NO:39; Fusion 101: SEQ ID NO:5, SEQ ID NO:43, SEQ ID NO:39; Fusion 102: SEQ ID NO:8, SEQ ID NO:43, SEQ ID NO:39; Fusion 103: SEQ ID NO:9, SEQ ID NO:43, SEQ ID NO:39; Fusion 104: SEQ ID NO:10, SEQ ID NO:43, SEQ ID NO:39; Fusion 105: SEQ ID NO:11, SEQ ID NO:43, SEQ ID NO:39; Fusion 106: SEQ ID NO: 12, SEQ ID NO: 43, SEQ ID NO: 39; Fusion 107: SEQ ID NO: 13, SEQ ID NO: 43, SEQ ID NO: 39; Fusion 108: SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 39; Fusion 109: SEQ ID NO: 15, SEQ ID NO: 43, SEQ ID NO: 39; Fusion 110: SEQ ID NO: 16, SEQ ID NO: 43, SEQ ID NO: 39; Fusion 111: SEQ ID NO:4, SEQ ID NO:44, SEQ ID NO:39; Fusion 112: SEQ ID NO:5, SEQ ID NO:44, SEQ ID NO:39; Fusion 113: SEQ ID NO:8, SEQ ID NO:44, SEQ ID NO:39; Fusion 114: SEQ ID NO:9, SEQ ID NO:44, SEQ ID NO:39; Fusion 115: SEQ ID NO: 10, SEQ ID NO: 44, SEQ ID NO: 39; Fusion 116: SEQ ID NO:11, SEQ ID NO:44, SEQ ID NO:39; Fusion 117: SEQ ID NO: 12, SEQ ID NO: 44, SEQ ID NO: 39; Fusion 118: SEQ ID NO: 13, SEQ ID NO: 44, SEQ ID NO: 39; Fusion 119: SEQ ID NO: 14, SEQ ID NO: 44, SEQ ID NO: 39; Fusion 120: SEQ ID NO: 15, SEQ ID NO: 44, SEQ ID NO: 39; Fusion 121: SEQ ID NO: 16, SEQ ID NO: 44, SEQ ID NO: 39; Fusion 122: SEQ ID NO:4, SEQ ID NO:45, SEQ ID NO:39; Fusion 123: SEQ ID NO:5, SEQ ID NO:45, SEQ ID NO:39; Fusion 124: SEQ ID NO:8, SEQ ID NO:45, SEQ ID NO:39; Fusion 125: SEQ ID NO:9, SEQ ID NO:45, SEQ ID NO:39; Fusion 126: SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 39; Fusion 127: SEQ ID NO:11, SEQ ID NO:45, SEQ ID NO:39; Fusion 128: SEQ ID NO: 12, SEQ ID NO: 45, SEQ ID NO: 39; Fusion 129: SEQ ID NO: 13, SEQ ID NO: 45, SEQ ID NO: 39; Fusion 130: SEQ ID NO: 14, SEQ ID NO: 45, SEQ ID NO: 39; Fusion 131: SEQ ID NO: 15, SEQ ID NO: 45, SEQ ID NO: 39; Fusion 132: SEQ ID NO: 16, SEQ ID NO: 45, SEQ ID NO: 39;

[0052] In some embodiments, the fusion protein is synthesized with a signal peptide that can be removed during secretion from the host cell. Exemplary signal peptides are set forth in SEQ ID NOS: 4-16 and 44-46. In some embodiments, the fusion protein is a mature protein, i.e., lacks a signal peptide.

[0053] In various embodiments, the fusion protein is selected from fusion proteins 1-132, and the selected fusion protein may optionally have up to 20 (or up to 10) amino acid modifications independently selected from amino acid deletions, insertions, and substitutions.

[0054] In some aspects, the present invention provides variants of extracellular DNASE enzymes engineered to have manufacturing advantages, providing for the production of recombinant enzymes suitable for therapeutic use, and can optionally be used in conjunction with fusion protein embodiments (including albumin fusion embodiments), as previously described. In various embodiments, the present invention provides recombinant D1, D1L1, D1L2, and D1L3 variants that contain one or more amino acid substitutions or deletions of cysteine ​​residues, resulting in reduced intramolecular and intermolecular cross-linking via disulfide bridges during protein expression. For example, the DNase variants can lack one, two, or three cysteine ​​residues present in the wild-type sequence (e.g., one, two, or three cysteine ​​residues are deleted) or have one or more of such cysteine ​​residues substituted with another amino acid(s). In some embodiments, one or more cysteine ​​residues are independently substituted with an amino acid selected from Ala, Gly, and Ser, or one or more of the cysteine ​​residues are substituted as part of a component substitution. In some embodiments, the one or more substituted cysteine ​​residues are not conserved among other members of the D1 protein family (e.g., D1, D1L1, D1L2, and D1L3). In some embodiments, the engineered enzyme comprises, or further comprises, at least one component substitution and / or other point mutation from another member of the D1 protein family that results in increased protein stability, increased resistance to protease degradation, increased bioavailability, and substantially the same or better DNA and / or chromatin and / or NET degradation activity (in vitro or in vivo) compared to the wild-type enzyme. In some embodiments, the substitution and / or modification comprises only a single modification at a cysteine ​​residue, among other modifications. In some embodiments, removal of a single cysteine ​​residue is sufficient for significant advantages in manufacturing.

[0055] In another aspect, the present invention provides variants of extracellular DNASE enzymes engineered to have advantages in protease resistance to improve in vivo half-life and reduce proteolysis during recombinant enzyme production. The present disclosure identifies D1L3 residues that are susceptible to proteolysis by, for example, plasmin, thrombin, and / or trypsin, as well as residues (e.g., paired basic amino acids) that are susceptible to proteases produced by mammalian and non-mammalian cell lines.

[0056] The recombinant extracellular DNASE variants described herein may have a combination of point mutations, including substitutions in cysteine ​​residues, substitutions in protease-sensitive residues, and / or may contain one or more element substitutions. Building block protein engineering (BBPE) is described in PCT / US18 / 47084 and US 62 / 800,790, the disclosures of which are incorporated herein by reference. BBPE involves providing a protein-protein alignment of the donor and recipient extracellular DNASE enzymes and identifying the variable amino acid sequence ("building block") for transfer. The variable amino acid(s) may be: , flanked by one or more conserved amino acids in the donor and recipient extracellular DNASE enzymes (upstream and downstream of the building blocks). These building blocks can be exchanged between the recipient and donor proteins to generate chimeric enzymes.

[0057] In another aspect, the present invention provides methods for the recombinant production of extracellular DNASE proteins, including variants thereof described herein. In some embodiments, the methods utilize a non-mammalian expression system, such as Pichia pastoris. In some embodiments, the Pichia pastoris encodes a DNAse enzyme with a native signal peptide that allows for secretion from the host cell. In some embodiments, the expression system is a mammalian cell expression system, such as Chinese hamster ovary (CHO) cells. In some embodiments, the present invention avoids inter- and intramolecular disulfide bonds by removing cysteine ​​residues unnecessary for activity, which would otherwise form and interfere with recombinant production. In some embodiments, substantial reduction of erroneous inter- and intramolecular disulfide bonds can be achieved by substitution of a single cysteine ​​residue.

[0058] In some embodiments, the recombinant expression system has deletion or inactivation of one or more proteases that cleave at paired basic amino acids. Exemplary enzymes include furin (expressed by CHO cells) and aspartic proteinase 3 (Ysp1) and kexin (Kex2) expressed by Pichia pastoris. In some embodiments, these enzymes are not genetically deleted or inactivated, but their activity is inhibited with protease inhibitors during recombinant protein production.

[0059] In some embodiments, growth media for non-mammalian or mammalian expression systems are supplemented with polyanions such as dextran sulfate, heparin, ferric citrate, or EDTA. In further embodiments, growth media for Pichia pastoris or other expression systems is supplemented with dextran sulfate having an average molecular weight of 5 kDa to 100 kDa. In some embodiments, the dextran sulfate has an average molecular weight of about 10 kDa or less, or about 20 kDa or less, or about 30 kDa or less, or about 40 kDa or less, or about 50 kDa or less, or about 75 kDa or less, or about 100 kDa or less. In various embodiments, the polyanion is added to the culture in an amount sufficient to complex with the recombinant protein produced.

[0060] In some embodiments, recombinant extracellular DNASE proteins and variants thereof from culture media of non-mammalian or mammalian expression systems are purified through a method comprising dissociation of the recombinant extracellular DNASE proteins and variants from polyanions such as dextran sulfate, heparin, EDTA, etc. In certain embodiments, the purification method comprises a strong anion exchange resin such as triethylaminoethyl. In some embodiments, the extracellular DNASE protein produced according to the method is D1L3 or a variant thereof.

[0061] Thus, in some embodiments, the present invention provides D1L3 variants comprising an amino acid sequence at least 80% identical to the enzyme defined by SEQ ID NO:4 (human D1L3, isoform 1) or SEQ ID NO:5 (human D1L3, isoform 2) and having one or more substitutions of cysteine ​​residues and / or one or more substitutions of amino acids that are susceptible to proteolysis, e.g., in vivo proteolysis. In some embodiments, the D1L3 protein variants comprise one or more additional modifications that result in increased protein stability (e.g., protease resistance), higher production levels in in vitro expression systems, and / or substantially less, but the same, or better chromatin and / or NET degradation activity compared to the wild-type D1L3 protein of SEQ ID NO:4 or SEQ ID NO:5. For example, the D1L3 variants comprise at least one additional component substitution or substitution as disclosed in PCT / US2018 / 47084 (incorporated herein by reference in its entirety). may contain point mutations or may contain one or more substitutions described herein to increase protease resistance.

[0062] In some embodiments, the D1L3 variant has a substitution of Cys68, optionally with an amino acid selected from Ala, Ser, and Gly. In some embodiments, this variant includes the substitution N64_I70delinsHLTAVGK. In some embodiments, the sequence HLTAVGK may be further modified by one, two, or three substitutions, deletions, and / or insertions (combined), provided that no Cys residues are included. In some embodiments, the D1L3 variant includes an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, or at least about 98% identity to reference SEQ ID NO:4 or SEQ ID NO:5.

[0063] In some embodiments, the present invention provides a D1L3 enzyme having a polyethylene glycol (PEG) moiety conjugated to a position corresponding to Cys68, which is considered to be the unpaired cysteine. In some embodiments, a D1L3 variant has PEG conjugated to the amino acid corresponding to C194. In these embodiments, the PEG moiety provides half-life extension properties while avoiding disulfide scrambling and / or protein misfolding. In some embodiments, the PEG moiety is conjugated through maleimide chemistry, which can be performed under mild conditions. Other conjugation chemistries, such as vinyl sulfone, dithiopyridine, and iodoacetamide activation chemistries, are known and can be used. The PEG moiety can be linear or branched and generally ranges from 10 kDa to 40 kDa, or from 20 to 30 kDa.

[0064] Alternatively, or in addition, the present invention provides D1L3 variants containing one or more substituted arginine and / or lysine residues that confer increased protease resistance. In some embodiments, the D1L3 variants have substitutions at one or more positions corresponding to K180, K200, K259, and / or R285 of SEQ ID NO: 4. According to the present disclosure, such lysine and arginine residues are identified as potential protease-susceptible sites. Accordingly, one or more (e.g., one, two, three, or four) of these residues can be independently modified with an uncharged residue, such as a residue selected from Ala, Gly, Leu, Ile, Val, Thr, Ser, and Pro. In some embodiments, the protease-susceptible lysine or arginine residue is substituted as part of a component substitution. For example, the D1L3 variant can include one or more substitutions selected from K180_A181delinsGL, P198_A201delinsRPSQ, and K259A. In some embodiments, the D1L3 variant comprises one or both of the substitutions K180_A181delinsGL and / or P198_A201delinsRPSQ, either of which is optionally modified by one or two amino acid substitutions, deletions, or insertions, provided that the constituent substitutions are not modified by substitutions or insertions with R or K residues. In some embodiments, the D1L3 variant has increased resistance to proteolysis by one or more proteases selected from plasmin, thrombin, and / or trypsin.

[0065] Alternatively, or in addition, the D1L3 variant comprises one or more mutations of pairing basic residues. In some embodiments, the pairing basic residues correspond to positions selected from K50 / R51, R80 / R81, K114 / R115, K199 / K200, K226 / K227, K291 / K292, R297 / K298 / K299, and K303 / R304 of SEQ ID NO: 4. In some embodiments, the D1L3 variant has one or more substitutions selected from substitutions corresponding to R114T, R114A, R114D, R114Q, K227S, and K227E of SEQ ID NO: 4. In some embodiments, the one or more mutations of pairing basic residues include amino acid substitutions corresponding to R51K, R81K, R115K, and R304K. In some embodiments, the paired basic residues are replaced using corresponding building block substitutions. According to these embodiments, the D1L3 variants will be more resistant to proteases expressed by recombinant protein expression systems (e.g., CHO and Pichia pastoris).

[0066] In some aspects, the present invention provides DNase 1 (D1) variants comprising an amino acid sequence at least 80% identical to the enzyme defined by SEQ ID NO: 1, with one or more substitutions of cysteine ​​residues. In some embodiments, the D1 protein variants have one or more additional modifications that result in increased protein stability, higher production levels in in vitro expression systems, and / or substantially less, but the same, or better chromatin and / or NET degradation activity compared to the wild-type D1 protein of SEQ ID NO: 1. For example, the D1 variants may include at least one additional component substitution or point mutation disclosed in PCT / US2018 / 47084, the entire contents of which are incorporated herein by reference.

[0067] In some embodiments, the D1 variant has a substitution of one or both of C123 and C126, optionally with Ala, Ser, and Gly. In some embodiments, the D1 variant includes the substitution G122_N128delinsYQGDA. In some embodiments, the D1 variant includes an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:1.

[0068] In some embodiments, the present invention provides a D1 enzyme having a PEG moiety conjugated to positions corresponding to C123 and / or C126. In these embodiments, the PEG moiety provides half-life extension properties while avoiding disulfide scrambling and / or protein misfolding. In some embodiments, the PEG moiety is conjugated through maleimide chemistry, which can be performed under mild conditions. Other conjugation chemistries, such as vinyl sulfone, dithiopyridine, and iodoacetamide activation chemistries, are known and can be used. The PEG moiety can be linear or branched and generally ranges from 10 kDa to 40 kDa, or from 20 to 30 kDa.

[0069] In another aspect, the invention provides D1L1 variants comprising an amino acid sequence at least 80% identical to the enzyme defined by SEQ ID NO:2 with one or more substituted cysteine ​​residues. The cysteine ​​residue(s) are optionally non-conserved within the D1 family (e.g., C22 and / or C50) and are optionally substituted with Gly, Arg, or Ser, or as part of a building block substitution. In some embodiments, the D1L1 variant comprises an amino acid sequence at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:2.

[0070] In some embodiments, the present invention provides D1L1 enzymes with PEG moieties conjugated to positions corresponding to C22 and / or C50. In these embodiments, the PEG moieties provide half-life extension properties while avoiding disulfide scrambling and / or protein misfolding. In some embodiments, the PEG moieties are conjugated through maleimide chemistry, which can be performed under mild conditions. Other conjugation chemistries, such as vinyl sulfone, dithiopyridine, and iodoacetamide activation chemistries, are known and can be used. The PEG moieties can be linear or branched and generally range from 10 kDa to 40 kDa, or from 20 to 30 kDa.

[0071] In some embodiments, the present invention provides a D1L2 variant comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO:3, with one or more substituted cysteine ​​residues. The cysteine ​​residue may be non-conserved within the D1 family (e.g., C43) and is optionally substituted with Gly, Arg, or Ser, or as part of a building block substitution. In some embodiments, the D1L2 variant comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:3.

[0072] In some embodiments, the present invention provides a D1L2 enzyme having a PEG moiety conjugated to the position corresponding to C43. In these embodiments, the PEG moiety provides half-life extension properties while avoiding disulfide scrambling and / or protein misfolding. In some embodiments, the PEG moiety is conjugated through maleimide chemistry, which can be performed under mild conditions. Other conjugation chemistries, such as vinyl sulfone, dithiopyridine, and iodoacetamide activation chemistries, are known and can be used. The PEG moiety can be linear or branched and generally range from 10 kDa to 40 kDa, or from 20 to 30 kDa.

[0073] In other aspects, the invention provides isolated polynucleotides encoding the D1, D1L1, D1L2, or D1L3 variants disclosed herein, as well as vectors and host cells. Host cells can be cells of any expression system, including bacterial or eukaryotic, whether non-mammalian, such as Pichia pastoris, or mammalian, such as CHO cells.

[0074] In some embodiments, delivery of polynucleotides is used for therapy. The encoding polynucleotide can be delivered as mRNA or as a DNA construct using known procedures, such as electroporation or cell squeezing, and / or vectors (including viral vectors). mRNA polynucleotides can include known modifications (mRNA) to avoid activation of the innate immune system. See WO 2014 / 028429, incorporated herein by reference in its entirety. In some embodiments, the polynucleotide is delivered to the subject's body. In some embodiments, the polynucleotide is delivered intracellularly in vitro, and the cell is delivered to the subject's body. The cell can be, for example, a leukocyte (e.g., a T cell or macrophage), an endothelial cell, an epithelial cell, a hepatocyte, or a stem cell.

[0075] In another aspect, the present invention provides a method for producing an extracellular DNASE variant described herein. The method includes culturing cells expressing a polynucleotide encoding the extracellular DNASE and recovering the recombinant DNase protein. The cells can be prokaryotic or eukaryotic. In some embodiments, the DNase is optionally expressed using a non-mammalian expression system, such as Pichia pastoris or a Saccharomyces species. In some embodiments, a mammalian expression system, such as CHO cells, is used.

[0076] The present invention further provides a pharmaceutical composition comprising an extracellular DNASE or variant thereof described herein, or optionally a polynucleotide or vector described, and a pharmaceutically acceptable carrier.

[0077] A vector generally comprises an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Exemplary vectors include autonomously replicating plasmids or viruses. The term also refers to non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0078] The pharmaceutical composition can be formulated for any administration route, including topical, parenteral, or pulmonary administration.In various embodiments, the composition is formulated for intradermal, intramuscular, intraperitoneal, intraarticular, intravenous, subcutaneous, intraarterial, oral, sublingual, pulmonary, or transdermal administration.In some embodiments, the composition is formulated for intravenous or subcutaneous administration.

[0079] In another aspect, the present invention provides a method for treating a subject in need of extracellular DNA degradation, extracellular chromatin degradation, extracellular trap (ET) degradation, and / or neutrophil extracellular trap (NET) degradation. The method comprises administering a therapeutically effective amount of an extracellular DNASE or variant thereof, or a composition described herein. Exemplary indications in which a subject requires extracellular DNA or chromatin degradation (including ET or NET degradation) are disclosed in PCT / US18 / 47084, the disclosure of which is incorporated herein by reference. The present invention provides a method for treating a subject in need thereof, the method comprising administering a therapeutically effective amount of a protein represented by any one of the sequences set forth in SEQ ID NOs: 8 to 30.

[0080] In each instance where a method for treating a subject is described, the invention also provides the use of one or more of the extracellular DNASE proteins for the treatment or prevention of diseases associated with ET and / or NET.

[0081] In various embodiments, the present invention provides methods for treating, preventing, or managing diseases or conditions characterized by the presence or accumulation of NETs, ​​including, but not limited to, chronic neutrophilia, neutrophil aggregation and leukostasis, thrombosis and vascular occlusion, ischemia-reperfusion injury, surgical and traumatic tissue injury, acute or chronic inflammatory responses or diseases, autoimmune diseases, cardiovascular diseases, metabolic diseases, systemic inflammation, respiratory inflammatory diseases, renal inflammatory diseases, inflammatory diseases associated with transplanted tissues (e.g., graft-versus-host disease), and diseases associated with cancer (including leukemia).

[0082] In certain embodiments, the present invention relates to treating a disease or condition characterized by a deficiency in D1L3 or a deficiency in D1. In some cases, the subject has a mutation (e.g., a loss-of-function mutation) in the Dnase1l3 gene or the Dnase1 gene. Such a subject may exhibit an autoimmune disease (e.g., systemic lupus erythematosus (SLE) (including lupus nephritis), scleroderma or systemic sclerosis, rheumatoid arthritis, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), and urticaria vasculitis). In some cases, the subject has an acquired inhibitor of D1 (e.g., anti-DNase1 antibodies and actin) and / or D1L3 (e.g., anti-DNase1l3 antibodies). Such a subject may also have an autoimmune disease or an inflammatory disease (e.g., SLE, systemic sclerosis).

[0083] In some embodiments, the subject has or is at risk for having a NET that obstructs the ductal system. For example, the DNASE enzymes disclosed herein can be administered to a subject to treat pancreatitis, cholangitis, conjunctivitis, mastitis, dry eye disease, vascular obstruction, or renal disease.

[0084] In some embodiments, the subject has or is at risk for NETs accumulating on endothelial surfaces (e.g., surgical adhesions), skin (e.g., wounds / scars), or synovial joints (e.g., gout and arthritis, e.g., rheumatoid arthritis). The DNASE enzymes described herein are useful for detecting and treating NETs that are characterized by the accumulation of NETs on endothelial surfaces, such as, but not limited to, surgical adhesions. The compounds can be administered to a subject to treat a condition in which

[0085] Other diseases and conditions associated with NETs that can be treated or prevented using the DNASE enzymes disclosed herein include ANCA-associated vasculitis, asthma, chronic obstructive pulmonary disease, neutrophilic dermatosis, dermatomyositis, burns, cellulitis, meningitis, encephalitis, otitis media, pharyngitis, tonsillitis, pneumonia, endocarditis, cystitis, pyelonephritis, appendicitis, cholecystitis, pancreatitis, uveitis, keratitis, disseminated intravascular coagulation, acute kidney injury, acute respiratory distress syndrome, liver shock, hepatorenal syndrome, myocardial infarction, stroke, ischemic bowel, limb ischemia, testicular torsion, preeclampsia, eclampsia, and solid organ transplants (e.g., kidney, heart, liver, and / or lung transplants). Additionally, the DNASE enzymes disclosed herein can be used to prevent scarring or contracture in at-risk individuals, e.g., individuals with surgical incisions, lacerations, or burns, for example, by topical application to the skin.

[0086] In various embodiments, the subject has a disease or condition that has been or is being treated with wild-type DNase, including D1 and streptodornase, including thrombosis, stroke, sepsis, lung injury, atherosclerosis, viral infection, sickle cell disease, myocardial infarction, ear infection, wound healing, liver damage, endocarditis, liver infection, pancreatitis, primary graft dysfunction, limb ischemia-reperfusion, kidney damage, blood clotting, alum-induced inflammation, hepatorenal injury, pleural effusion, hemothorax, intrabiliary thrombus, post-pneumococcal anemia, ulcers, ENT disease, and oral infection. These include: minor injuries, sinusitis, post-operative rhinoplasty, infertility, bladder catheterization, wound irrigation, skin testing, pneumococcal meningitis, gout, leg ulcers, cystic fibrosis, Kartagener's syndrome, asthma, lobar atelectasis, chronic bronchitis, bronchiectasis, lupus, primary ciliary dyskinesia, bronchiolitis, empyema, pleural infection, cancer, dry eye disease, lower respiratory tract infection, chronic hematoma, Alzheimer's disease, and obstructive pulmonary disease.

[0087] Other aspects and embodiments of the present invention will become apparent from the following examples. [Example]

[0088] Approximately 70% of all biologics are produced using Chinese hamster ovary (CHO) cells. In fact, wild-type DNASE1 (D1; dornase alfa) is typically produced in CHO cells. Despite significant advantages in cell line development and large-scale production using CHO cells, significant challenges remain in the production of Dnase enzymes due to considerable variability and the lack of reliable methods for predicting or modeling cell growth characteristics. Importantly, CHO cells are unable to stably produce hyperactive variants of D1, hindering clinical production, and prior to this disclosure, the production characteristics of other DNASE1 protein family members, including DNASE1-like 3 (D1L3), were unknown.

[0089] Several challenges have been identified in the production of D1L3 using CHO and microbial expression systems, including low production yields, protein degradation, protein misfolding, and incorrect or undesired glycosylation. The present disclosure provides technical solutions to these and other manufacturing challenges, which may also improve the therapeutic properties of D1L3.

[0090] Example 1: Expression and characterization of D1L3 with a basic domain deletion (BDD) in Chinese hamster ovary (CHO) cells and Pichia pastoris DNASE1 and DNASE1L3 preferentially cleave protein-free DNA and DNA-histone complexes (i.e., chromatin), respectively. Previous studies have suggested that the C-terminal basic domain (BD) of DNASE1L3, which is absent in DNASE1, is responsible for the different substrate specificities of both enzymes (Sisirak et al. .,Cell,2016;Keyel,Developmental Biology,2017).

[0091] A protein engineering technique, referred to as building block protein engineering, is described in PCT / US18 / 47084 and US62 / 800,790, the disclosures of which are incorporated herein by reference in their entireties. This approach can be applied to members of the DNASE1 and DNASE2 protein families. The method is based on the steps of providing a protein-protein alignment of the donor and recipient Dnase enzymes; identifying a variable amino acid sequence for transfer, in which the variable amino acid is adjacent to one or more conserved amino acids in the donor and recipient Dnase enzymes; substituting the variable amino acid of the recipient Dnase with the variable amino acid of the donor Dnase to create a chimeric Dnase; and recombinantly producing the chimeric Dnase.

[0092] To characterize the amino acids responsible for chromatin degradation activity ("chromatin enzyme" activity), wild-type D1L3 was replaced with building block substitutions from D1, as disclosed in PCT / US2018 / 047084. Building block substitutions for D1L3 were selected from human D1 and, with respect to SEQ ID NO: 4, were M21_R22delinsLK, C24_S25delinsAA, V28_S30delinsIQT, S34T, Q36_V44delinsMSNATLVSY, K47_K50delinsQILS, C52Y, I55_M58delinsIALVQE, I60_K61delinsVR, N64_I70delinsHLTAVGK, M72_K74delinsLDN, R77_I83d elinsQDAPD, N86H, I89V, S91_R92delinsEP, T97S, Q101R, A103L, L105V, K107_L110delinsRPDQ, V113_R115delinsAVD, H118Y, H12 0D, Y122_A127delinsGCEPCGN, V129T, S131N, F135_V136delinsAI, W138R, Q140_H143delinsFSRF, A145_D148delinsEVRE, V150A, I1 52V, T156_T157delinsAA, E159_S161delinsGDA, K163A, E167A, V169_E170delinsYD, T173L, K176_R178delinsQEK, K180_A181del insGL, N183_F186delinsDVML, P198_A201delinsRPSQ, K203_N204delinsSS, R208W, D210S, R212T, V214Q, G218P, Q220_E221delins resulting in a human D1L3 variant characterized by the following mutations: SA, V225_S228delinsATP, N230H, L238_R239delinsVA, Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256N, K259A, K262G, T264_E267delinsSDQL, L269_V271delinsQAI, F275Y, F279_K280delinsVM, Q282_S205delinsK.

[0093] These 63 D1L3 variants were screened for loss or gain of chromatin degradation activity. Briefly, D1L3 variants were transiently expressed in CHO cells using an in vitro expression vector. Culture supernatants were collected and tested for chromatin degradation activity using purified nuclei as a chromatin source. As shown in Figure 1, component substitutions #17 and #63 from D1 significantly improved the degradation of high-molecular-weight (HMW) chromatin into small fragments compared to wild-type D1L3. Component substitution #7 causes a missense mutation, Q101R, replacing glutamine at position 101 with arginine (SEQ ID NO: 8). Component substitution #63 causes a mutation, Q282_S305delinsK, which deletes the complete C-terminal BD of D1L3 from amino acids 283 to 305 and replaces glutamine (Q) at position 282 with lysine (SEQ ID NO: 9). Next, Western blot analysis of the supernatant was performed to detect the expression levels of wild-type D1L3 and both mutations (Figure 2). Surprisingly, two D1L3 variants of different sizes were detected in samples with wild-type D1L3 and the Q101R mutation. The sample with Q282_S305delinsK contained only the smaller D1L3 variant. The data suggest that the BD of wild-type D1L3 is spontaneously removed (e.g., proteolyzed) during expression in CHO cells or after secretion. Two D1L3 variants were also detected in the supernatant from CHO cells stably expressing WT-D1L3 (Figure 3). Notably, the basic domain-deleted D1L3 (BDD-D1L3) exhibited substantially increased chromatin enzymatic activity compared to wild-type D1L3.

[0094] Next, we tested Pichia pastoris as an alternative microbial expression system to CHO cells. In general, we observed higher expression levels with BDD-D1L3 compared to wild-type D1L3. Here, we purified and characterized wild-type D1L3 and BDD-D1L3 from Pichia pastoris fermentation supernatant (Figure 4). Unexpectedly, we observed that wild-type D1L3 was proteolytically cleaved within the BD at amino acid positions K291, K291, or S293, resulting in a heterogeneous mixture of D1L3 variants after purification. Unlike wild-type D1L3, expression of BDD-D1L3 with three component substitutions (F275Y, F279_K280delinsVM, and Q282_S205delinsK) produced pure protein.

[0095] Next, we compared the chromatin enzyme activity of both D1L3 purifications. We observed that a heterologous mixture of D1L3 variants with BD truncations at K291, K291, or S293 had approximately 10-fold lower chromatin enzyme activity compared to a D1L3 variant with a complete BD deletion by F275Y / F279_K280delinsVM / Q282_S205delinsK. Collectively, our data demonstrate that proteolytic cleavage of the BD can occur naturally in microbial and mammalian expression systems (i.e., CHO and P. pastoris) and that removal of the BD activates D1L3 activity to degrade chromatin.

[0096] Example 2: Expression of D1L3 in CHO cells in a bioreactor Disclosed herein is the development of a stable CHO cell line producing wild-type D1L3 (SEQ ID NO: 4). The cell line was cultured in a bioreactor using standard CHO culture medium. Specifically, Figure 5 shows a Western blot of human D1L3 expressed and secreted by CHO cells in a bioreactor under cGMP-compliant conditions. Samples were collected at different time points (t1-t3). Only trace levels of D1L3 and D1L3 fragments were detected. The data suggest that low production yields of D1L3 are a challenge in D1L3 manufacturing.

[0097] As disclosed herein, high production levels of wild-type D1L3 were achieved by adding polyanions to the culture medium. Such polyanions may include one or more of heparin, dextran sulfate, ferric citrate, and ethylenediaminetetraacetic acid, and represent the biologically active components in "anti-cell agglutination reagents." Specifically, dextran sulfate was added to CHO culture medium, and a strong increase in D1L3 and D1L3 fragments was observed (Figure 5). The data indicate that polyanions increased the production yield of D1L3 but did not prevent proteolysis.

[0098] Figure 6A shows that polyanions such as dextran sulfate (DS) form complexes with D1L3. The D1L3-DS complex is prevented from interacting with and being removed from negatively charged surfaces during the production process. Such negatively charged surfaces include, but are not limited to, the cell surfaces of production cells (e.g., CHO cells, Pichia pastoris, Saccharomyces species), DNA exposed by stained cells, and bioreactor surfaces. Figures 6B and 6C show a two-step purification process for D1L3 from the DS-D1L3 complex. As shown in Figure 6, the first step is to dissociate the DS-D1L3 complex. The goal is to dissociate the DS-D1L3 complex. Dissociation can be achieved by incubating the DS-D1L3 complex with a strong anion exchange surface, which binds DS and thus releases D1L3. Specifically, the purification process involves passing the culture medium containing DS-D1L3 through a chromatography column filled with a strong anion exchange resin, followed by collection of the flow-through containing DS-free D1L3. The second step of the purification process is shown in Figure 6C and involves affinity purification of D1L3 from the DS-free flow through the application of a strong cation exchange resin. In conclusion, the production yield of D1L3 can be significantly increased by the addition of polyanions such as dextran sulfate.

[0099] Example 3: D1L3 engineered for protease resistance Wild-type D1L3 contains 50 arginine and lysine residues, which make the enzyme particularly sensitive to proteases such as trypsin, thrombin, and plasmin. In this example, trypsin and plasmin cleavage sites were identified in D1L3. The sites can be mutated to generate protease-resistant variants of D1L3.

[0100] Briefly, purified D1L3 was digested with trypsin. D1L3 fragments were isolated and their amino acid sequences were determined using a combination of liquid chromatography (LC) and mass spectrometry (MS). We determined that trypsin cleaved D1L3 at the following arginine and lysine residues: R22, R29, R51, R66, R80, R81, R95, K99, R115, K147, K163, K180, R208, R212, R235, R239, K250, and K262. These arginine and lysine residues could be substituted with small amino acids such as alanine, valine, and serine, or with amino acids with similar properties according to Grantham distance scores (e.g., histidine, glutamine, and glutamic acid; Figure 7). The protease-resistant D1 is characterized by arginine and lysine residues corresponding to R51, R95, K99, and R235, suggesting that these residues are not primarily involved in the proteolysis of D1L3.

[0101] Component protein engineering was applied to transfer the R22 (mutation: M21_R22delinsLK), R29 (V28_S30delinsIQT), R66 (N64_I70delinsHLTAVGK), R80 (R77_I83delinsQDAPD), R81 (R77_I83delinsQDAPD), R115 (V113_R115delinsAVD), K163 (K163A), K180 (K180_A181delinsGL), R208 (R208W), MR212 (R212T), R239 (L238_R239delinsVA), K250 (K250D), and K262 (K262G) components from D1 to replace the D1L3 components containing trypsin cleavage sites (Figure 7).

[0102] Plasmin is a plasma protease generated by activation of its zymogen, plasminogen. Plasminogen activator inhibitor 1 (PAI-1) inhibits plasmin activation. Interestingly, PAI-1 increases the enzymatic activity of D1L3 in serum, suggesting that plasmin may proteolytically inactivate D1L3. However, the plasmin cleavage site in D1L3 has not been identified.

[0103] Computer analysis showed that the amino acid combinations lysine-alanine (KA) or arginine-alanine (RA) are thought to be preferentially cleaved by proteases with plasmin or plasmin-like activity. D1L3 contains a total of four putative plasmin cleavage sites (Figure 8): (site 1) K180 / A181 (K160 / A161 without signal peptide), (site 2) K200 / A201 (K180 / A181 without signal peptide), (site 3) K259 / A260 (K239 / A240 without signal peptide), and (site 4) R285 / A286 (R270 / A250 without signal peptide). Using pairwise alignment of D1 and D1L3, We found that none of the plasmin cleavage sites were present in D1 (Figure 8). This data is consistent with the fact that D1 activity is resistant to inactivation by serum proteases such as thrombin and plasmin. Using component protein engineering, we transferred the following components from D1: (site 1) K180_A181delinsGL, (site 2) P198_A201delinsRPSQ, and (site 3) K259A, replacing the plasmin cleavage site-containing components of D1L3 (Figure 9). R285 / A286 (site 4) is located in the C-terminal extension that is missing in D1. Therefore, we generated a D1L3 variant in which all four putative plasmin cleavage sites, K180_A181delinsGL, P198_A201delinsRPSQ, K259A, and R285A, were mutated. We next analyzed chromatin degradation by D1L3 variants and observed potent chromatin degradation activity in the mutated D1L3 (Figure 10). Collectively, the data indicate that four arginine and lysine residues, K180, K200, K259, and R285, can be mutated to reduce the risk of proteolysis without compromising enzymatic activity.

[0104] Next, purified D1L3 was digested with purified plasmin. D1L3 fragments were isolated and their amino acid sequences were determined using a combination of LC and MS. We determined that plasmin cleaved D1L3 at arginine and lysine residues R22, R29, K45, K47, K74, R81, R92, K107, K176, R212, R226, R227, K250, K259, and K262. These arginine and lysine residues could be substituted with small amino acids such as alanine, valine, and serine, or with amino acids with similar properties according to Grantham distance scores (e.g., histidine, glutamine, and glutamic acid; Figure 11). Protease-resistant D1 is characterized by a lysine residue corresponding to K45, suggesting that this residue is not primarily involved in the proteolysis of D1L3 by plasmin. Applying component protein engineering, we isolated R22 (mutation: M21_R22delinsLK), R29 (V28_S30delinsIQT), K47 (K47_K50delinsQILS), K74 (M72_K74delinsLDN), R81 (R77_I83delinsQDAPD), R92 (S91_R92delinsEP), K107 (K107_L110delinsRPDQ), K The elements 176 (K176_R178delinsQEK), R212 (R212T), K226 (V225_S228delinsATP), K227 (V225_S228delinsATP), K250 (K250D), K259 (K259A), and K262 (K262G) were transferred from D1 to replace the elements of D1L3 that contained the trypsin cleavage site in computational analysis (Figure 11).

[0105] Finally, recombinantly expressed wild-type D1L3 was isolated and its C-terminus sequenced. Three distinct amino acid sequences were identified, ending at S290 (SEQ ID NO: 10), K291 (SEQ ID NO: 11), and K292 (SEQ ID NO: 12), respectively (Figure 4, Example 1). The data identify lysine residues 291 and 292 as the predominant proteolytic cleavage sites of D1L3 during large-scale production.

[0106] Example 4: D1L3 engineered to prevent degradation We observed fragmentation of D1L3 after heterologous expression in Pichia pastoris. Analysis of the fragments characterized paired basic amino acids, arginine (R), and lysine (K) residues, as proteolytic cleavage sites. We observed a similar degradation pattern after expressing D1L3 in CHO cells. These observations suggest that Pichia pastoris and CHO cells share a homologous protease that cleaves D1L3 at paired basic amino acids, although the effect was more pronounced in CHO cells.

[0107] We determined that paired basic amino acid cleaving enzyme (PACE) contributes to DNASE1L3 fragmentation. PACE, also known as furin (Uniprot ID: P09958), is expressed in humans and mammals. Pichia pastoris expresses two enzymes that target paired basic amino acids: aspartic proteinase 3 (gene: Ysp1; Uniprot ID: P32329) and kexin (gene: Kex2; Uniprot ID: P13134). Therefore, DNASE1L3 and DNASE1L3 variants can be expressed in Pichia pastoris and CHO cells, and furin, aspartic proteinase 3, and kexin can be pharmacologically inhibited or genetically depleted.

[0108] Additionally, mutation of paired basic amino acids in DNASE1L3 and DNASE1L3 variants allows their expression in CHO and Pichia pastoris with reduced fragmentation. Analysis of the identified DNASE1L3 fragments reveals that they are characterized by paired basic amino acids at positions K50 / R51, R80 / R81, K114 / R115, K199 / K200, K226 / K227, K291 / K292, R297 / K298 / K299, and K303 / R304 of SEQ ID NO:2.

[0109] As disclosed in U.S. Provisional Patent Application No. 62 / 800,790 (incorporated herein by reference in its entirety), DNASE1L3 from other species is characterized by amino acid substitutions at these cleavage sites, including R114T (mouse), R114A (rat), R114D (guinea pig), R114Q (cow), K227S (dog), and K227E (elephant). These amino acid substitutions can be applied to human DNASE1L3 to make the enzyme resistant to proteolysis, including during expression in CHO cells and Pichia pastoris.

[0110] Kexin preferably cleaves after the KR and RR residues. DNASE1L3 features four KEX2 cleavage sites at K50 / R51, R80 / R81, K114 / R115, and K303 / R304. Amino acid substitutions at these residues render DNASE1L3 resistant to KEX2 and allow expression of DNASE1L3 and DNASE1L3 variants in Pichia pastoris and CHO cells. These amino acid substitutions can be conservative, such as R51K, R81K, R115K, and R304K.

[0111] Example 5: D1L3 variants engineered to prevent high molecular weight aggregates During cGMP-competent expression of D1L3 in CHO cells (Figure 12A), we observed the accumulation of high molecular weight aggregates of D1L3, pointing to an additional challenge for analytically producing D1L3. High molecular weight aggregates were observed to a much lesser extent in Pichia pastoris.

[0112] Applying reducing conditions to the protein in the bioreactor material dissolved the D1L3 aggregates. The data indicate that D1L3 aggregate formation is caused by intramolecular and / or intermolecular cross-linking via disulfide bridges during protein expression. Specifically, as shown in Figure 12B, a gel run under non-reducing conditions shows the accumulation of high molecular weight aggregates of D1L3 over time. The gel was run under reducing conditions, and no aggregates were detected. The data indicate that incorrect intramolecular and intermolecular disulfide bonds cause misfolding of human D1L3 under production conditions.

[0113] Figure 13 shows an amino acid sequence alignment of human D1 (SEQ ID NO: 1) and human D1L3 (SEQ ID NO: 4). The signal peptide, conserved amino acids, variable amino acids, non-conserved cysteine ​​residues, and conserved cysteine ​​residues are highlighted. Mutations at non-conserved cysteine ​​residues reduce the likelihood of intra- and intermolecular disulfide bonds during protein expression. Analysis of the amino acid sequence of D1L3 (SEQ ID NO: 4) revealed that mutations at C24, C52, C68, C194, and C2 The present study demonstrated the presence of five cysteine ​​(C) residues in D1L3 (Figure 14). Cysteine ​​residues C194 and C231 are conserved among all members of the DNASE1 protein family and form disulfide bonds necessary for DNASE1 enzymatic activity. The function of the cysteine ​​residues in D1L3 was unknown prior to this disclosure. Therefore, as disclosed herein, mutation of these cysteine ​​residues reduces cross-linking via disulfide bridges, thus increasing the yield of protein production.

[0114] Cysteine ​​residues can be substituted with other small amino acids, such as alanine (A), serine (S), and glycine (G). Such substitutions result in the amino acid mutations C24A / S / G, C52A / S / G, C68A / S / G, C194A / S / G, and C231A / S / G. Additionally, components containing conserved cysteine ​​residues can be replaced with components from donor DNases of the DNASE1 protein family (e.g., D1 and D1L3). The following components from D1 were used to replace components of D1L3 containing non-conserved cysteine ​​residues C24, C52, and C68: C24_S25delinsAA, C52Y, and N64_I70delinsHLTAVGK. The chromatin degradation activity of D1L3 variants was quantified as described in PCT / US18 / 4708. Both conventional amino acid substitutions (C24A, C52A) and structural substitutions (C24_S25delinsAA, C52Y) resulted in a complete lack of chromatin degradation, indicating that C24 and C52 are required for D1L3 activity (Figure 15). Importantly, mutation of cysteine ​​C68 by either the conventional amino acid substitution [C68A, (SEQ ID NO: 13)] or the BB mutation (N64_I70delinsHLTAVGK) resulted in D1L3 variants with chromatin degradation activity (Figure 15). Amino acid sequence alignment showed that cysteine ​​C68 is not conserved among other DNASE1 protein family members, supporting the idea that C68 is not required for enzymatic activity. Furthermore, we observed that amino acid substitution of the highly conserved cysteine ​​C194 with alanine (C194A), but not mutation of the highly conserved cysteine ​​C231 with alanine (C231A), resulted in enzymatically active D1L3 variants (Figure 15). Therefore, cysteines C68 and C194 can be mutated to reduce the risk of incorrect disulfide bonds during D1L3 production.

[0115] A similar approach can be applied to mutate non-conserved cysteine ​​residues in other members of the DNase1 protein family: D1, DNase1-like 1 (D1L1), and DNase1-like 2 (D1L2). D1 has two non-conserved cysteines, C123 and C126. D1L1 has only one non-conserved cysteine ​​residue: C43, whereas two non-conserved cysteine ​​residues (C22, C50) correspond to C24 and C52 in D1L2. Mutation of non-conserved cysteine ​​residues in members of the DNASE1 protein family reduces cross-linking via incorrect disulfide bridges during protein expression, thus enabling the production of D1, D1L1, D1L2, and D1L3 for therapeutic applications.

[0116] Example 6: Construction and expression of D1L3 and albumin-D1L3 fusion proteins in Pichia pastoris Pichia pastoris expression of recombinant human extracellular DNA SES containing D1L3 was tested. As shown in Figure 16A, the N-terminus of D1L3 was driven by the alpha mating factor (aMF) pre-pro secretory leader from Saccharomyces cerevisiae (SEQ ID NO: 46), a common tool for heterologous protein expression in Pichia pastoris. As disclosed herein, the combination of aMF with D1L3 resulted in unexpected non-processing of aMF due to glycosylation (Figure 16B). Glycosylation of the D1L3 protein prevented the use of P. pastoris for analytical production of D1L3. D1L3 was properly processed when the N-terminus was driven by the native secretory signal peptide of D1L3 [Figure 16B, (SEQ ID NO: 48)]. Importantly, aMF was significantly more potent than the native signal peptide of D1L3. The expression of D1L3 was increased 3- to 5-fold. Therefore, processing of D1L3 fusion proteins was tested. In preliminary studies, N-terminal fusions of aMF and human serum albumin [HSA (SEQ ID NO: 39)] with D1L3 were generated (Figure 17A). Some variants contained a linker peptide [e.g., (GSSSS)3] between HSA and D1L3. As shown in Figures 17B and 17C, expression of the fusion proteins in P. pastoris produced non-glycosylated and enzymatically active D1L3. Furthermore, expression levels were increased 5- to 10-fold compared to expression driven by the native secretory signal peptide of D1L3. Collectively, the data demonstrate that fusion of D1L3 with albumin enables its production in Pichia pastoris.

[0117] Based on these preliminary studies, various HSA fusion constructs of wild-type D1L3 and BDD-D1L3 were designed and screened for expression levels of target proteins (SEQ ID NOS: 17-28). As shown in Figure 18, we observed that N-terminal fusion of human serum albumin (SEQ ID NOS: 17) with the BDD-D1L3 variant (SEQ ID NOS: 16) did not substantially increase expression levels. However, a strong increase in expression levels was detected when a flexible linker consisting of glycine (G) and serine (S) residues was inserted between HSA and BDD-D1L3. Furthermore, the length of the linker sequence correlated with increased expression. For example, expression of 12 ± 1.9 relative units was obtained with a five-amino acid linker (SEQ ID NOS: 18), while expression with a 15-amino acid linker (SEQ ID NOS: 19) was 32 ± 3.2 relative units, approximately 7.5-fold improvement over the HSA fusion without a linker. Furthermore, the N-terminal position was important for improving expression levels, as C-terminal fusions of linker-HSA constructs were expressed at low levels (SEQ ID NO: 20, SEQ ID NO: 21). Notably, N-terminal fusion of HSA via a flexible linker potently increased the expression of wild-type D1L3 (SEQ ID NO: 22) by approximately 20-fold compared to native D1L3 (SEQ ID NO: 4). In conclusion, fusion to the N-terminus of HSA via a linker enables the production of D1L3 and BDD-D1L3 variants.

[0118] Next, we tested whether the nature of the linker sequence was important for improving D1L3 expression. Two additional sequences were tested: APAPAPAPAPAPAP (SEQ ID NO: 33, 14 amino acids, rigid linker) and AEAAAKEAAAKA (SEQ ID NO: 34, 12 amino acids, rigid helical linker). As shown in Figure 19, we observed a strong increase in expression with both test constructs (SEQ ID NO: 23, SEQ ID NO: 24), although the rigid helical linker did not achieve the same strong expression level as observed with the GGGGSGGGSGGGGGS linker. Thus, the length and acid composition of the linker affected the level of D1L3 expression.

[0119] Next, we analyzed the relationship between linker length, expression level, and enzyme activity. For these studies, we designed expression vectors containing N-terminal fusions of HSA with a GS-linker to BDD-D1L3 variants (SEQ ID NOS: 25-27). Three different linker lengths were tested: SGGSGSS [7 amino acids, (SEQ ID NOS: 35)], SGGSGGSGGSGGSGSS [16 amino acids, (SEQ ID NOS: 36)], and SGGSGGSGGSGGSGGSGGSGGSGGSGSS [31 amino acids, (SEQ ID NOS: 37)]. As shown in Figure 20, extending the linker sequence from 7 amino acids to 16 amino acids resulted in increased expression levels. Further extension from 16 to 31 amino acids did not increase protein expression but did increase enzyme activity, as detected by degradation of HMW-chromatin to LMW-chromatin. Biologics fused to albumin fusions often exhibit reduced activity due to steric inhibition of albumin interactions with substrates and ligands. Therefore, a peptide linker can be used to increase the distance between albumin and the fusion protein or peptide. However, the observation that inserting a linker sequence between HSA and D1L3 simultaneously improves enzyme activity and expression levels was unexpected.

[0120] The chromatin degradation activity of BDD-D1L3 (SEQ ID NO: 14) was compared with that of its albumin fusion counterpart (SEQ ID NO: 19). - / -Dnase1l3 - / - Mice were injected with SEQ ID NO: 4 or SEQ ID NO: 19. Serum was collected 15 minutes after injection. As shown in Figure 21A, similar serum chromatin degradation activity was observed in both animals. Importantly, fusion of albumin to the N-terminus of D1L3 and other human extracellular DNASEs provides DNASE therapeutics with extended half-lives. As disclosed herein, the half-life of SEQ ID NO: 19, an HSA-BDD-D1L3 fusion protein with a flexible 15-amino acid GS linker, was determined in a commercially available rodent model. The animal model features transgenic expression of human FcRn, which is responsible for the long half-life of albumin in circulation. While unconjugated D1L3 (e.g., SEQ ID NO: 4) has a very short half-life in circulation (<30 minutes), the albumin fusion extended the half-life to 3.3 days, thereby substantially improving systemic exposure and simultaneously achieving a t value of 5 minutes. max This resulted in rapid absorption at 1000 kJ / kg (Figure 21B). Collectively, the data indicate that N-terminal fusion of HSA to D1L3 via a linker sequence not only facilitates manufacturing but also improves the in vivo pharmacokinetic properties of D1L3.

[0121] Finally, we tested double fusion of HSA to the N- and C-termini of D1L3. First, we analyzed the C-terminus of D1L3 for potential attachment sites. We identified two serine residues at positions 283 and 284, which provide a flexible connection of the BD (RAFTNSKKSVTLRKKTKSKRS) to the core body of D1L3. Therefore, we chose to delete the BD and connect HSA to S284 via a flexible GS linker (SEQ ID NO: 38). As shown in Figure 22, fusion of HSA to the N- and C-termini of BDD-D1L3 (SEQ ID NO: 28) maintained the high expression levels observed with the N-terminal HSA fusion (SEQ ID NO: 27).

[0122] Example 7: Design of cleavable linker sequences The discoveries disclosed herein have implications beyond manufacturing. For example, as exemplified by SEQ ID NOs: 9-12, D1L3 variants with C-terminal amino acid deletions that retain their enzymatic activity for degrading chromatin and / or NETs can be used for D1L3 therapy. In addition, site-specific alkylation of unpaired cysteine ​​thiols is commonly used to generate half-life-extended biologics for therapeutic applications. Specifically, the non-essential cysteines C68 and C194 of D1L3 can be used for site-specific PEGylation (PEG, polyethylene glycol). Furthermore, D1L3 variants that are resistant to inactivation by plasmin due to mutations such as K180_A181delinsGL, P198_A201delinsRPSQ, K259A, and R285A have improved half-lives and are therefore expected to be effective in therapeutic applications.

[0123] Importantly, the fusion of albumin to the N-terminus of D1L3 and other human extracellular DNASEs provides DNASE therapeutics with extended half-lives (Figure 23A). Several linker sequences were used to reduce the steric inhibition of D1L3 by albumin. Furthermore, physiologically cleavable peptide linkers were developed. The linker peptide was designed to be cleaved when the fusion protein was in close proximity to neutrophil extracellular traps (NETs). Peptide sequences targeted by neutrophil-specific proteases, such as neutrophil elastase, cathepsin G, and proteinase 3, are candidates for cleavable linker sequences.

[0124] We developed a cleavable linker sequence that is cleaved intravascularly and therefore optimal for DNASE therapeutics administered intravenously and intraarterially. To design the peptide, we considered that NETs have the ability to activate blood coagulation factors, particularly coagulation factor XII (FXII). Activated FXII (FXIIa) has two major substrates: coagulation factor XI (FXI, SEQ ID NO: 40) and prekallikrein (PK, SEQ ID NO: 41). Amino acid sequence alignment showed that the FXIIa cleavage site is conserved in FXI and PK (Figure 23B). In FXI, In FXI, the cleavage site is between arginine 387 and isoleucine 388. In PK, the cleavage site is between arginine 390 and isoleucine 391. Indeed, FXI and PK are homologous proteins. As disclosed herein, several linker peptides were designed that contain all or part of the FXI sequence at positions 380-403 (SEQ ID NO: 42, SEQ ID NO: 43) or the PK sequence at positions 383-406 (SEQ ID NO: 44).

[0125] Finally, the FXIIa-cleavable linker can be used to produce half-life-extending forms (Figure 24) of other biologics, including, but not limited to, variants of other extracellular DNASEs, human coagulation factors (e.g., Factor VII, Factor VIII, and Factor IX), and complement factors (e.g., Factor H).

[0126] All patents and patent publications cited herein are hereby incorporated by reference in their entirety. Wild-type human DNASE SEQ ID NO: 1 TIFF0007818850000001.tif43169 SEQ ID NO: 2 TIFF0007818850000002.tif52169 SEQ ID NO: 3 TIFF0007818850000003.tif54169 SEQ ID NO: 4 TIFF0007818850000004.tif53169 SEQ ID NO: 5 TIFF0007818850000005.tif47169 SEQ ID NO: 6 TIFF0007818850000006.tif59169 SEQ ID NO: 7 TIFF0007818850000007.tif57169 Human DNASE1L3 variants SEQ ID NO:8 TIFF0007818850000008.tif47169 SEQ ID NO: 9 TIFF0007818850000009.tif42169 SEQ ID NO: 10 TIFF0007818850000010.tif44169 SEQ ID NO: 11 TIFF0007818850000011.tif48169 SEQ ID NO: 12 TIFF0007818850000012.tif50169 SEQ ID NO: 13 TIFF0007818850000013.tif47169 SEQ ID NO: 14 TIFF0007818850000014.tif52169 SEQ ID NO: 15 TIFF0007818850000015.tif49169 SEQ ID NO: 16 TIFF0007818850000016.tif47169 Albumin fusions with DNASE1L3 and variants SEQ ID NO: 17 TIFF0007818850000017.tif111169 SEQ ID NO: 18 TIFF0007818850000018.tif110169 SEQ ID NO: 19 TIFF0007818850000019.tif108169 SEQ ID NO: 20 TIFF0007818850000020.tif110169 SEQ ID NO: 21 TIFF0007818850000021.tif113169 SEQ ID NO: 22 TIFF0007818850000022.tif114169 SEQ ID NO: 23 TIFF0007818850000023.tif114169 SEQ ID NO: 24 TIFF0007818850000024.tif118169 SEQ ID NO: 25 TIFF0007818850000025.tif108169 SEQ ID NO: 26 TIFF0007818850000026.tif109169 SEQ ID NO: 27 TIFF0007818850000027.tif118169 SEQ ID NO: 28 TIFF0007818850000028.tif192169 SEQ ID NO: 29 TIFF0007818850000029.tif124169 SEQ ID NO: 30 TIFF0007818850000030.tif113169 Linker sequence SEQ ID NO: 31 GGGGS SEQ ID NO: 32 GGGGSGGGGGGGGGS SEQ ID NO: 33 APAPAPAPAPAPAP SEQ ID NO: 34 AEAAAKEAAAKA SEQ ID NO: 35 SGGSGSS SEQ ID NO: 36 SGGSGGSGGSGGSGSS SEQ ID NO: 37 SGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGSGS SEQ ID NO: 38 GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGS Other arrays SEQ ID NO: 39 Human serum albumin (mature protein): DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL Sequence number 40 Human Factor XI: MIFLYQVVHFILFTSVSGECVTQLLKDTCFEGGDITTVFTPSAKYCQVVCTYHPRCLLFTFTAESPSEDPTRWFTCVLKDSVTETLPRVNRTAAISGYSFKQCSHQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYATRQF PSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLACIRDIFPNTVFADSNIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIPVFCHSSFYHDTDFLGEELDIVAAK SHEACQKLCTNAVRCQFFTYTPAQASCNEGKGKCYLKLSSNGSPTKILHGRGGISGYTLRLCKMDNECTTKIKPRIVGGTASVRGEWPWQVTLHTTSPTQRHLCGGSIIGNQWILTAAHCFYGVESPKILRVYSGILNQSEIKEDTSFFGVQEIII HDQYKMAESGYDIALLKLETTVNYTDSQRPICLPSKGDRNVIYTDCWVTGWGYRKLRDKIQNTLQKAKIPLVTNEECQKRYRGHKITHKMICAGYREGGKDACKGDSGGPLSCKHNEVWHLVGITSWGEGCAQRERPGVYTNVVEYVDWILEKTQAV SEQ ID NO: 41 Human prekallikrein: MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSINDMEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTNNIRCQFFSYATQTFHK AEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYSLLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQ ETCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTRIVGGTNSSWGEWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVS EGNHDIALIKLQAPLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYMDWILEKTQSSDGKAQMQSPA Activatable Linker Sequence SEQ ID NO: 42 FXIIa-sensitive linker (Factor XI peptide): CTTKIKPRIVGGTASVRGEWPWQVT SEQ ID NO: 43 FXIIa-sensitive linker GGGGSPRIGGGS SEQ ID NO: 44 FXIIa-sensitive linker (prekallikrein peptide): VCTTKTSTRIVGTNSSWGEWPWQVS SEQ ID NO: 45 FXIIa-sensitive linker (prekallikrein peptide): STRIVGG signal peptide SEQ ID NO: 46 Alpha mating factor (P01149): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNGLLFINTTIASIAAKEEGVS SEQ ID NO: 47 Human albumin secretory signal peptide + propeptide (P02768): MKWVTFISLLFLFSSAYSRGVFRR SEQ ID NO: 48 Human DNASE1L3 signal peptide (Q13609): MSRELAPLLLLLLSIHSALA

Claims

1. 1. A DNase 1-like 3 (D1L3) enzyme having chromatin degrading activity, comprising an amino acid sequence having at least 95% sequence identity to amino acids 21-282 of SEQ ID NO:4 or amino acids 21-252 of SEQ ID NO:5, wherein the D1L3 enzyme comprises a modification at the amino acid corresponding to C68 in SEQ ID NO:4, wherein the modification is a substitution with C68 in SEQ ID NO:4, or wherein the modification is PEGylation at C68 in SEQ ID NO:

4.

2. 2. The D1L3 enzyme of claim 1, wherein the modification is an amino acid substitution at C68 relative to SEQ ID NO:

4.

3. The D1L3 enzyme of claim 2, wherein the amino acid substitution is C68A, C68S, or C68G with respect to SEQ ID NO:

4.

4. The D1L3 enzyme of claim 1, wherein the amino acid corresponding to C68 in SEQ ID NO: 4 is PEGylated.

5. The D1L3 enzyme of claim 4, wherein the amino acid corresponding to C68 of SEQ ID NO: 4 is PEGylated with a linear or branched PEG having a molecular weight ranging from about 10 kDa to about 40 kDa.

6. The D1L3 enzyme of claim 5, wherein the amino acid corresponding to C68 of SEQ ID NO: 4 is PEGylated with a linear or branched PEG having a molecular weight ranging from about 20 kDa to about 30 kDa.

7. The D1L3 enzyme of claim 4, wherein the PEG is conjugated through a maleimide group.

8. The D1L3 enzyme of claim 1, wherein the D1L3 enzyme is a fusion protein with a half-life extending polypeptide.

9. 9. The D1L3 enzyme of claim 8, wherein the half-life extending polypeptide is selected from albumin, transferrin, an Fc domain, and an elastin-like protein.

10. The D1L3 enzyme of claim 8, further comprising an inserted amino acid linker connecting the amino acid sequence of the D1L3 and the half-life extending polypeptide.

11. The D1L3 enzyme of claim 10, wherein the amino acid linker is a flexible linker comprising glycine and serine amino acid residues, and the ratio of serine to glycine residues in the linker is from about 1:1 to about 1:

10.

12. The D1L3 enzyme of claim 10, wherein the amino acid linker is a rigid linker.

13. The D1L3 enzyme of claim 10, wherein the amino acid linker comprises a protease cleavage site.

14. The D1L3 enzyme of claim 10, wherein the linker has at least 15 amino acids.

15. The D1L3 enzyme of claim 10, wherein the half-life extending polypeptide is albumin.

16. The D1L3 enzyme according to claim 15, wherein the albumin is located on the N-terminal side of the amino acid sequence of the D1L3.

17. The D1L3 enzyme according to claim 15, wherein the albumin is located on the C-terminal side of the amino acid sequence of the D1L3.

18. The D1L3 enzyme of claim 15, wherein the albumin comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

39.

19. 16. The D1L3 enzyme of claim 15, wherein the albumin comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

39.

20. The D1L3 enzyme of claim 9 , wherein the half-life extending polypeptide comprises an Fc domain.

21. 2. The D1L3 enzyme of claim 1, wherein the D1L3 enzyme has a deletion of at least 5 amino acids in the C-terminal basic domain, the C-terminal basic domain being defined by the C-terminal 23 amino acids of SEQ ID NO: 4 or SEQ ID NO:

5.

22. 22. The D1L3 enzyme of claim 21, wherein the D1L3 enzyme has a deletion of the entire C-terminal basic domain.

23. The D1L3 enzyme of claim 1, wherein the enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

4.

24. The D1L3 enzyme of claim 1, wherein the enzyme comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:5.

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