Manipulation of DNASE enzymes for manufacture and therapeutic use

Abstract

We provide an engineered human extracellular DNASE protein that is useful for treating conditions characterized by the accumulation and / or release of neutrophil extracellular traps (NETs). [Solution] The present invention provides DNase variants containing specific amino acid sequences (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)). The DNase variants have advantages for therapeutic and / or large-scale production.

Description

Technical Field

[0001] Related Applications This application claims the benefit and priority of U.S. Provisional Application No. 62 / 742,682, filed Oct. 8, 2018; U.S. Provisional Application No. 62 / 775,563, filed Dec. 5, 2018; U.S. Provisional Application No. 62 / 779,104, filed Dec. 13, 2018; U.S. Provisional Application No. 62,808,601, filed Feb. 21, 2019; and U.S. Provisional Application No. 62 / 846,904, filed May 13, 2019, the contents of which are hereby incorporated by reference in their entirety.

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

Background Art

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

[0004] DNASE1(D1), along with DNASE1-like 1(D1L1), DNASE1-like 2(D1L2), and DNASE1-like 3(D1L3), forms the DNASE1 protein family, a group of homologous secreted DNase1 protein enzymes. DNASE2A and DNASE2B form an additional group of homologous extracellular DNase enzymes. The DNASE1- and DNASE2- protein family members are evolutionarily conserved and expressed in various species, including humans. Recombinant human DNASE1 and DNASE2 protein family members offer drug candidates for NET-related diseases. While D1 has been developed for several therapeutic applications in patients, the 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 application. Therefore, it is necessary to define the production processes for the D1L1, D1L2, and D1L3 enzymes and manipulate DNases for therapeutic use, including the degradation of NETs. [Overview of the project]

[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). According to embodiments of the present invention, the DNase variants described herein are more suitable for therapeutic use and / or for large-scale production. In some embodiments, the DNase variants described herein have advantages for medical therapies, including systemic therapies. Such advantages include slower drug efflux, e.g., increased circulating half-life (e.g., serum half-life), extended duration of pharmacodynamic activity, high chromatin degradation activity, and protease resistance.

[0006] In some embodiments, the present invention is a D1L3 variant, and the D1L3 variant is This provides a D1L3 variant that, 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, exhibits increased protein stability, slower drug efflux, increased duration of pharmacodynamic activity, resistance to proteolysis, higher production levels in in vitro expression systems, better suitability for purification, and one or more substantially no lower, identical, or better chromatin and / or NET degradation activity.

[0007] In some embodiments, 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 at the N-terminal end of 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 half-life or serum half-life) and extended duration of pharmacodynamic activity, including in systemic therapies. In some embodiments, the fusion of albumin with a linking sequence to the D1L3 domain substantially does not affect the chromatin degradation activity of the enzyme (e.g., 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 has the deletion of all or part of the C-terminal basic domain present in the wild-type D1L3 enzyme. The 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 for chromatin degradation.

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

[0010] The linkers present may be flexible linkers, rigid linkers, or physiologically cleavable linkers such as protease-cleavable linkers. For example, a linker may be a hydrophilic amino acid sequence and may be constructed mainly from amino acids selected from Gly, Ala, Ser, Thr, and Pro. In some embodiments, the variant is a flexible linker (e.g., (G)) which is mainly glycine and serine residues. y S) n The linker is a linker where y is 1-5 and n is 1-20. In some embodiments, the linker is an α-helix 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 during expression in mammalian and non-mammalian expression systems such as CHO cells or Pichia pastoris. Furthermore, surprisingly, longer linker sequences showed improved chromatin degradation activity in in vitro chromatin degradation assays compared to shorter linker sequences.

[0011] In various embodiments, the D1L3 variant comprises one amino acid sequence from SEQ ID NOs: 17–30, and in each case, has 1–20 amino acid modifications, which are optionally and independently selected from insertions, deletions, or substitutions. These sequences provide exemplary fusion proteins between D1L3 (or D1L3 variants) and albumin sequences, and include various linker designs. In some embodiments, the amino acid modifications are such that the D1L3 domain, albumin, It is located within the bumin domain, or both domains. 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 D1L3 variant comprises, in N-terminus to C-terminus, an albumin amino acid sequence, a medium or long flexible linker, and a D1L3 amino acid sequence (i.e., the D1L3 variant). SEQ ID NO: 28 further includes albumin fusion at the C-terminus via a long flexible peptide linker.

[0012] In other embodiments, the linker is cleavable by a protease, such as a coagulation pathway protease, such as 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 that is targeted for cleavage by a neutrophil-specific protease, such as neutrophil elastase, cathepsin G, or proteinase 3.

[0013] In some embodiments, the present invention provides variants of extracellular DNASE enzymes that are engineered to have advantages in production, providing the production of recombinant enzymes suitable for therapeutic use. In various embodiments, the present invention provides recombinant D1, D1L1, D1L2, and D1L3 variants comprising one or more amino acid substitutions (or PEGylation of Cys residues) at cysteine ​​residues, resulting in a reduction of intramolecular and intermolecular crosslinking via disulfide crosslinking during protein expression.

[0014] In other embodiments, the present invention provides variants of extracellular DNASE enzymes engineered to have advantages in protease resistance to improve in vivo exposure, for example, by delaying efflux, e.g., extending half-life (e.g., serum half-life), and extending the duration of pharmacodynamic activity, and by reducing proteolysis during recombinant enzyme production. The disclosure identifies, for example, D1L3 residues sensitive to proteolysis by plasmin, thrombin, and / or trypsin, as well as residues (e.g., counterbasic amino acids) sensitive to proteases produced by mammalian and non-mammalian cell lines. Engineered mutations of these residues can confer these advantages in protease resistance.

[0015] In other embodiments, the present invention provides a method for the recombinant production of extracellular DNASE proteins, including variants thereof described herein. In some embodiments, the method utilizes a non-mammalian expression system, such as a eukaryotic non-mammalian expression system, like Pichia pastoris. In some embodiments, Pichia pastoris encodes a DNase enzyme having its native signal peptide that enables secretion from host cells. 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 the deletion or inactivation of one or more proteases that cleave at counterbasic amino acids. Exemplary enzymes include furin (expressed by CHO cells), as well as aspartate 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 by protease inhibitors during recombinant protein production.

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

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

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

[0020] In other aspects, the present invention provides a method for treating a subject that requires 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 a variant or composition thereof described herein.

[0021] Other aspects and embodiments of the invention will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] [Figure 1] The mutations Q101R and Q282_S305delinsK of SEQ ID NO: 4 are shown to 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. The supernatant of the transfected cells was 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] Characteristics of two DNASE1L3 variants are shown. Different concentrations of the supernatant of CHO cells transfected with wild-type DNASE1L3 or DNASE1L3 with the Q101R or Q282_S305delinsK mutation were analyzed by Western blot (WB) using an anti-DNASE1L3 antibody. Larger (variant 1) and smaller (variant 2) bands were detected in samples with wild-type DNASE1L3 and the Q101R mutation. Only the smaller band (variant 2) was shown in samples with the Q282_S305delinsK mutation. In parallel, the chromatin degradation activity in different concentrations of the supernatant was analyzed. The figure shows DNA analyzed by agarose gel electrophoresis. Both the Q101R or Q282_S305delinsK mutation increased the chromatin degradation activity compared to wild-type DNASE1L3. [Figure 3]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. Larger (variant 1) and smaller (variant 2) bands were detected in five clones. [Figure 4] To identify high-frequency cleavage sites, the C-terminal amino acid sequence of wild-type D1L3 recombinantly expressed in Pichia pastoris is shown. By amino acid sequencing of purified wild-type D1L3, three C-terminal deletion mutants: K291_S305del, K292_S305del, and S293_S305del were identified. The C-terminal of wild-type D1L3 was not detected. In parallel, the chromatin degradation activity in different concentrations of purified protein was analyzed and compared with purified DNASE1 (D1) and basic domain deletion DNASE1L3 (BDD-D1L3) with the F275Y / F279_K280delinsVM / Q282_S305delinsK mutation. The figure shows DNA analyzed by agarose gel electrophoresis. [Figure 5] Addition of dextran sulfate to CHO medium is shown to improve protein yield. A stable pool of CHO cells expressing wild-type D1L3 was incubated in standard CHO medium or CHO medium supplemented with dextran sulfate. The supernatant was analyzed by Western blot (WB) using an anti-DNASE1L3 antibody. The figure shows that D1L3 is poorly expressed in CHO cells and has a low yield. Addition of dextran sulfate increases the yield but does not prevent production fragmentation. [Figure 6] Shows the use of anion exchange surfaces and cation exchange surfaces for affinity purification of dextran sulfate complexed D1L3. A shows that polyanions such as dextran sulfate (DS) form a complex with D1L3. The D1L3-DS complex is prevented from interacting with and being removed by negatively charged surfaces during the production process. B and C show a two-step process for purifying D1L3 from the DS-D1L3 complex. [Figure 7]This section lists trypsin cleavage site mutation strategies to restrict D1L3 degradation. [Figure 8] This shows the alignment of the amino acid sequences of human D1 (SEQ ID NO: 1) and human D1L3 (SEQ ID NO: 4) with the indicated plasmin-sensitive KR residues. [Figure 9] This document presents a plasmin cleavage site mutation strategy to restrict D1L3 degradation. [Figure 10] This study demonstrates that D1L3 with mutated plasmin cleavage sites retains enzyme 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 agarose gels stained with DNA dyes. [Figure 11] This paper lists plasmin cleavage sites based on plasmin digestion and presents mutational strategies to restrict D1L3 degradation. [Figure 12] This shows that D1L3 tends to misfold when expressed in CHO cells. A shows a simple expression vector for D1L3 expression using a native secretory signal peptide. The supernatant of a stable pool was analyzed by Western blotting using an anti-DNASE1L3 antibody, and B shows the presence of high molecular weight aggregates under non-reducing conditions, which are degraded under reducing conditions. [Figure 13] This shows the alignment of the amino acid sequences of human D1 (SEQ ID NO: 1) and human D1L3 (SEQ ID NO: 4) with the conserved and non-conserved cysteine ​​residues shown. [Figure 14] This paper lists cysteine ​​residues in D1L3 and presents mutational strategies to limit high molecular weight aggregates during protein expression. [Figure 15]This study demonstrates that C68A and C194A mutations in D1L3 do not affect chromatin degradation activity. Mutations C24A and C52A inactivated 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 agarose gels stained with DNA dyes. [Figure 16] This image shows the expression of D1L3 in Pichia pastoris using either a native secretory signal or an α-conjugation factor (αMF) from Saccharomyces cerevisiae. A shows that the N-terminus of D1L3 is induced by a pre-pro-secretion leader of the α-conjugation factor (αMF) from Saccharomyces cerevisiae. B shows that the secretory signal from αMF results in glycosylation and deprocessing of the signal peptide. [Figure 17] This study demonstrates that 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 chromatin degradation activity. A shows the fusion construct of αMF, human serum albumin (HSA), a linker sequence, and D1L3. This fusion construct is not glycosylated in the P. pastoris expression system (B) and retains chromatin degradation activity (C). [Figure 18] This shows the expression levels of human serum albumin (HSA) fusion constructs of basic domain deletion 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 positioned between the HSA and BDD-D1L3 or D1L3. [Figure 19]This shows the expression levels of a human serum albumin (HSA) fusion construct of wild-type DNASE1L3 (D1L3) in Pichia pastoris. HSA was fused to the N-terminus of D1L3. Three different linker sequences (L2, L3, L4) were placed between HSA and D1L3. [Figure 20] This study shows the expression levels and chromatin degradation activity of a human serum albumin (HSA) fusion construct of basic domain deletion DNASE1L3 (BDD-D1L3) produced in Pichia pastoris. HSA was fused to the N-terminus of BDD-D1L3. Three different linker sequences (L5, L6, L7) were placed between the HSA and D1L3. [Figure 21] This shows serum chromatin degradation activity and the circulating half-life of albumin D1L3 fusion protein. A shows that Dnase1- / -Dnase1l3- / - mice injected with SEQ ID NO: 14 and SEQ ID NO: 19 exhibit similar chromatin degradation activity in serum. B shows that SEQ ID NO: 19 has a circulating half-life of 3.3 days in mice expressing the human FcRn receptor. [Figure 22] This study describes the expression levels and chromatin degradation activity of a human serum albumin (HSA) fusion construct of basic domain deletion DNASE1L3 (BDD-D1L3) produced in Pichia pastoris. HSA was fused to the N-terminus and C-terminus of BDD-D1L3. Two different linker sequences (L7 and L8) were placed between the HSA and BDD-D1L3. [Figure 23] The designs of cleavable linkers are shown. A shows a fusion construct of HSA and linker. B shows a fusion construct of a linker cleavable by factor XIIa. Linkers containing the human factor XI sequence (sequence number 42) and human prekallikrein (sequence number 44) are shown. [Figure 24] We present other constructs that utilize a factor XIIa-cleavable linker in half-life extension fusion proteins containing human extracellular DNase, human coagulation factors, and human complement factors. [Modes for carrying out 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). According to embodiments of the present invention, the DNase variants described herein are more therapeutically suitable and / or effective and / or more suitable for large-scale production. In some embodiments, the DNase variants described herein have advantages for 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 herein and in the claims, the singular forms "a," "an," and "the" include singular and plural references unless otherwise explicitly indicated by the context. Thus, for example, a reference to "drugs" includes a single drug and multiple such drugs.

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

[0026] Where used herein in reference to drugs, “half-life” refers to the elimination half-life of the drug concentration in an animal, measured in the matrix of interest, e.g., serum or plasma. Those skilled in the art will understand that not all drugs exhibit primary pharmacokinetics, or do not do so at all stages of elimination. In such cases, those skilled in the art will understand that the terms “extended half-life” or “extended half-life” refer to a slower elimination rate.

[0027] "Isolation" means modification or removal from its natural state. For example, naturally occurring nucleic acids or peptides in living animals are not "isolated," but rather the same nucleic acids or peptides have been partially or completely separated from their naturally occurring coexisting substances. Isolated nucleic acids or proteins may exist in a substantially purified form or in a non-natural environment, such as a host cell.

[0028] As used herein, “neutrophil extracellular trap” and the acronym “NET” refer to a network of extracellular fibers containing nuclear contents, such as DNA bound to histone proteins, released in a programmed manner from immune cells, typically neutrophils.

[0029] Unless otherwise specified, “nucleotide sequences or nucleic acids that code for an amino acid sequence” include all nucleotide sequences that code for the same amino acid sequence in a degenerate form. The phrase “nucleotide sequences that code for a protein or RNA” may also include introns to the extent that nucleotide sequences that code for a protein may include introns in several forms.

[0030] The terms "approximately" and "about" include quantities that are ±10% of the relevant 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 embodiments and aspects, the extracellular DNASE or its variant is optionally fused by an insertion linker to a half-life extension region of albumin, transferrin, Fc or elastin-like protein, or a variant thereof. See, for example, US9,458,218, which is incorporated herein by reference in its entirety. In some embodiments, the extracellular DNASE or its variant is dimerized by an immunoglobulin hinge region. For example, the manipulated enzyme described herein is Fc Fusion domains (e.g., the hinge and CH2 and C H3 domains of immunoglobulins) may also be included. In some embodiments, the DNASE (e.g., the D1L3 variant) is fused to an albumin amino acid sequence or domain, e.g., human albumin or a fragment or variant thereof. See, for example, WO2015 / 066550 and US9,221,896, which are incorporated herein by reference in their entirety. Albumin may optionally be linked to the DNASE at the N-terminus and / or C-terminus of the manipulated extracellular DNASE or variant using an insertion 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 by an 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 may 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 to 100 amino acid long, or 5 to 50 amino acid long. In yet other embodiments, the linker is an organic molecule, group, polymer (e.g., PEG), or chemical moiety covalently bonded to the extracellular DNASE and half-life extension moiety (e.g., albumin).

[0033] In some embodiments, the present invention provides D1L3 variants having one or more of the following characteristics 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: 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 no lower, identical, or better chromatin and / or NET degradation activity. Where used herein, unless otherwise stated, the term "D1L3" includes either isoform 1 or isoform 2.

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

[0035] In some embodiments, the D1L3 variant has nearly 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 higher 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 at the N-terminus of the D1L3 domain. In some embodiments, the albumin domain and optional linker are located at the C-terminus of the D1L3 domain. It is located at the end. In all such embodiments, any 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 may be a variant of wild-type HSA (e.g., represented by SEQ ID NO: 39). In various embodiments, the albumin variant may 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 Sequence ID No. 39. The term “fragment,” when used in the context of albumin, refers to any fragment of full-length albumin or its variant that extends the half-life of the DNASE enzyme it fuses or complexes with compared to the corresponding non-fusion DNASE. In some embodiments, the albumin fragment may refer to an amino acid sequence containing fusions of multiple domains of albumin, such as domains I and III, and domains II and III (see, for example, WO2011 / 124718). Generally, the albumin fragment has at least about 100 amino acids from the full-length sequence, or at least about 200 or at least about 300 amino acids. In various embodiments, the albumin fragment maintains its ability to bind to human FcRn.

[0039] In some embodiments, the D1L3-like domain of the fusion protein is at least about 85%, at least about 90%, at least about 95%, at least about 97%, 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 includes 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 23 amino acids at the C-terminus.

[0040] In some embodiments, the fusion protein includes a D1L3 domain, the amino acid sequence of the D1L3 domain being at least about 80% identical to that of the mature enzyme defined by SEQ ID NO: 4 or SEQ ID NO: 5. The fusion protein may further include 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 includes 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 5, defined by the 23 amino acids at the C-terminus. Fusion proteins containing the D1L3 domain exhibit improved circulating half-life and duration of pharmacodynamic effect, including in systemic therapies. In addition, the fusion of albumin and the linking sequence substantially does not affect (or, in some embodiments, does not negatively affect) chromatin degradation activity, as determined by in vitro chromatin degradation assays, compared to variants without albumin fusion.

[0041] When referring to sequence identity with wild-type DNase enzymes, unless otherwise specified, the sequence is signaling This refers to a mature enzyme lacking the signal peptide. Furthermore, unless otherwise specified, for clarity, amino acid positions are numbered with respect to the fully translated DNase sequence, including the signal peptide. Therefore, for example, a reference to sequence identity for the enzyme in SEQ ID NO: 4 (human D1L3, isoform 1) refers to percent identity with the mature enzyme having M21 at the N-terminus. Similarly, a reference to sequence identity for the enzyme in 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 the deletion of all or part of the C-terminal basic domain. The C-terminal basic domain is defined as the 23 amino acids at the C-terminus 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 the 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 the deletion of the entire C-terminal basic domain, defined by the 23 amino acids at the C-terminus 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 (having a BD deletion) has 1 to 10 or 1 to 5 amino acids at the C-terminus that do not align with the C-terminal BD and 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 NOs: 8 to 16, a linker of a sequence selected from SEQ ID NOs: 31 to 38, and an albumin domain having the sequence of SEQ ID NO: 39 or the variant or fragment described herein. In some embodiments, the D1L3 variant has one or more component substitutions from D1 as described in PCT / US2018 / 04708, which are incorporated herein by reference.

[0044] In some embodiments, the D1L3 sequence or domain includes component substitutions from D1, such as 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 You can choose one or more from N, K259A, K262G, T264_E267delinsSDQL, L269_V271delinsQAI, F275Y, F279_K280delinsVM, and Q282_S205delinsK, and each of the aforementioned substitutions is numbered with respect to sequence number 4.

[0045] For example, the D1L3 variant may have a component substitution from D1, Q282_S305delinsK, which includes a deletion of the C-terminal basic domain, which is absent in D1. In some embodiments, the D1L3 enzyme has an amino acid substitution at the position corresponding to 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] If present, linkers can be selected from flexible, rigid, and cleavable peptide linkers. Flexible linkers consist mainly or entirely of small, nonpolar, or polar residues such as Gly, Ser, and Thr. An example of a flexible linker is (Gly y Ser) n The linkers include y from 1 to 10 (e.g., 1 to 5) and n from 1 to about 10, and in some embodiments from 3 to about 6. In exemplary embodiments, y is from 2 to 4 and n is from 3 to 8. Due to their flexibility, these linkers are non-structural. More rigid linkers include polyproline or polyPro-Ala motifs and α-helix linkers. An exemplary α-helix linker is A(EAAAK) n A is the first amino acid, and n is as defined above (e.g., 1-10 or 2-6). Generally, the linker may consist mainly of amino acids selected from Gly, Ser, Thr, Ala, and Pro. An exemplary linker sequence contains at least 10 amino acids and may range from 15 to 35 amino acids. Exemplary linker designs are provided as SEQ ID NOs. 31-38.

[0047] In some embodiments, the variant includes a linker, the amino acid sequence of which is primarily glycine and serine residues, or essentially composed of glycine and serine residues. In some embodiments, the ratio of Ser to Gly in the linker is approximately 1:1 to approximately 1:10, approximately 1:2 to approximately 1:6, or approximately 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 may have a length of 15 to 30 amino acids. In various embodiments, longer linkers of at least 15 amino acids can provide improved yield during expression in Pichia pastoris. See Figure 20. Furthermore, surprisingly, longer linker sequences showed improved chromatin degradation activity compared to shorter linker sequences. See Figure 20.

[0048] In various embodiments, the D1L3 variant is a fusion protein containing one of the amino acid sequences of SEQ ID NOs: 17-30. In other embodiments, the D1L3 variant is a fusion protein containing one of the amino acid sequences of SEQ ID NOs: 17-30, and has 1-20, 1-10, or 1-5 amino acid modifications, selected from amino acid insertions, deletions, or substitutions, independently of a reference sequence selected from SEQ ID NOs: 17-30. In some embodiments, the amino acid modifications are located 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 NOs: 19, SEQ ID NOs: 22, SEQ ID NOs: 26, SEQ ID NOs: 27, SEQ ID NOs: 28, SEQ ID NOs: 29, or SEQ ID NOs: 30. In these embodiments, the albumin The amino acid sequence is fused to the N-terminus or N-terminal side of D1L3 (or its variant) via a medium- to long flexible linker.

[0049] In other embodiments, the linker is a physiologically cleavable linker, such as a protease-cleavable linker. For example, the protease may 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 part of SEQ ID NO: 42 (e.g., part of SEQ ID NO: 42 including modifications to enable cleavage by factor XIIa). In some embodiments, the linker amino acid sequence from prekallikrein comprises all or part of SEQ ID NO: 44 (e.g., part of SEQ ID NO: 44 including modifications to enable 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] Several exemplary embodiments of the D1L3 fusion protein independently comprise a combination of three amino acid sequences that can be selected from the sequences disclosed herein, such sequences arranged in N-terminus to C-terminus as follows:

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

[0052] In some embodiments, the fusion protein is synthesized with a signal peptide. The signal peptide may be removed during secretion from the host cell. Exemplary signal peptides are shown 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 to 132, and the selected fusion protein may have up to 20 (or up to 10) amino acid modifications, which are optionally and independently selected from amino acid deletions, insertions, and substitutions.

[0054] In some embodiments, the present invention provides variants of extracellular DNASE enzymes engineered to have advantages in production, providing the production of recombinant enzymes suitable for therapeutic use, and, as already described, optionally, can be used in connection with fusion protein embodiments (including albumin fusion embodiments). In various embodiments, the present invention provides recombinant D1, D1L1, D1L2, and D1L3 variants comprising one or more amino acid substitutions or deletions of cysteine ​​residues, resulting in a reduction of intramolecular and intermolecular crosslinking via disulfide crosslinking during protein expression. For example, a DNase variant may lack one, two, or three cysteine ​​residues present in the wild-type sequence (e.g., one, two, or three cysteine ​​residues are deleted), or may have one or more such cysteines substituted with other amino acids. In some embodiments, one or more cysteine ​​residues are independently substituted with amino acids selected from Ala, Gly, and Ser, or one or more cysteine ​​residues are substituted as part of a component substitution. In some embodiments, one or more cysteine ​​residues to be substituted are not conserved among other members of the D1 protein family (e.g., D1, D1L1, D1L2, and D1L3). In some embodiments, the engineered enzyme includes, or further includes, at least one component substitution and / or other point mutation from another member of the D1 protein family, resulting 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 includes only a single modification at a cysteine ​​residue, among other modifications. In some embodiments, the removal of a single cysteine ​​residue is sufficient for significant advantages in manufacturing.

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

[0056] The recombinant extracellular DNASE variants described herein may have combinations of point mutations, including substitutions at cysteine ​​residues and substitutions at protease-sensitive residues, and / or include one or more elemental substitutions. Component protein manipulation (BBPE) is described in PCT / US18 / 47084 and US 62 / 800,790, and these disclosures are incorporated herein by reference. BBPE involves providing protein-protein alignments of donor and recipient extracellular DNASE enzymes and identifying variable amino acid sequences ("components") for translocation. Variable amino acids are These components are adjacent to one or more conserved amino acids in the donor and recipient extracellular DNASE enzymes (upstream and downstream of the components). These constructs can be exchanged between the recipient protein and the donor protein to generate a chimeric enzyme.

[0057] In other embodiments, the present invention provides a method for the recombinant production of extracellular DNASE proteins, including variants thereof described herein. In some embodiments, the method utilizes a non-mammalian expression system such as Pichia pastoris. In some embodiments, Pichia pastoris encodes a DNAse enzyme having a native signal peptide that enables secretion from host cells. 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 intermolecular and intramolecular disulfide bonds by removing cysteine ​​residues that are unnecessary for activity. Otherwise, bonds would form and hinder recombinant production. In some embodiments, a substantial reduction of false intermolecular and intramolecular disulfide bonds can be achieved by the substitution of a single cysteine ​​residue.

[0058] In some embodiments, the recombinant expression system has the deletion or inactivation of one or more proteases that cleave at counterbasic amino acids. Exemplary enzymes include furin (expressed by CHO cells), as well as aspartate 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 by protease inhibitors during recombinant protein production.

[0059] In some embodiments, the growth medium for non-mammalian or mammalian expression systems is supplemented with polyanions such as dextran sulfate, heparin, ferric citrate, or EDTA. In further embodiments, the growth medium 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 their variants from culture media of non-mammalian or mammalian expression systems are purified by a method comprising the dissociation of recombinant extracellular DNASE proteins and variants from polyanions such as dextran sulfate, heparin, or EDTA. In certain embodiments, the purification method comprises a potent anion exchange resin such as triethylaminoethyl. In some embodiments, the extracellular DNASE protein produced according to this method is D1L3 or its variant.

[0061] Accordingly, in some embodiments, the present invention provides D1L3 variants comprising an amino acid sequence having one or more substitutions of cysteine ​​residues and / or one or more substitutions of amino acids, which are 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 are sensitive to proteolysis, e.g., in vivo proteolysis. In some embodiments, the D1L3 protein variant comprises 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 no lower, identical, 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 variant comprises at least one additional component substitution or as disclosed in PCT / US2018 / 47084 (which is incorporated herein by reference in its entirety). This may include point mutations or one or more substitutions described herein to increase protease resistance.

[0062] In some embodiments, the D1L3 variant has a Cys68 substitution, which is optionally substituted 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 (in combination), provided that it does not contain a Cys residue. 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 with reference sequence number 4 or sequence number 5.

[0063] In some embodiments, the present invention provides a D1L3 enzyme having a polyethylene glycol (PEG) moiety compounded at a position corresponding to Cys 68, which is considered to be non-cysteine. In some embodiments, the D1L3 variant has a PEG compound with an amino acid corresponding to C194. In these embodiments, the PEG moiety provides extended half-life properties while avoiding disulfide scrambling and / or protein misfolding. In some embodiments, the PEG moiety is compounded through maleimide chemistry, which can be carried out under mild conditions. Other compound chemistrys, such as vinyl sulfone, dithiopyridine, and iodoacetamide activation chemistry, are known and can be used. The PEG moiety may be linear or branched and may generally be in the range of 10 kDa to 40 kDa, or 20 to 30 kDa.

[0064] Alternatively, or in addition, the present invention provides D1L3 variants comprising one or more substituted arginine and / or lysine residues that result in increased protease resistance. In some embodiments, the D1L3 variant has substitutions at one or more positions corresponding to K180, K200, K259, and / or R285 of SEQ ID NO: 4. According to this disclosure, such lysine and arginine residues are identified as potential protease-sensitive sites. Thus, one or more of these residues (e.g., one, two, three, or four) may be independently modified with uncharged residues such as residues selected from Ala, Gly, Leu, Ile, Val, Thr, Ser, and Pro. In some embodiments, the protease-sensitive lysine or arginine residue is substituted as part of a constituent substitution. For example, the D1L3 variant may comprise 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 at 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 includes one or more mutations in the paired basic residue. In some embodiments, 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. In some embodiments, the D1L3 variant has one or more substitutions selected from substitutions corresponding to R114T, R114A, R114D, R114Q, K227S, and K227E in SEQ ID NO: 4. In some embodiments, one or more mutations in the paired basic residues include amino acid substitutions corresponding to R51K, R81K, R115K, and R304K. This includes the following. In some embodiments, the paired basic residues are substituted using corresponding constituent substitutions. According to these embodiments, the D1L3 variant will be more resistant to proteases expressed by recombinant protein expression systems (e.g., CHO and Pichia pastoris).

[0066] In some embodiments, the present invention provides DNase1(D1) variants comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 1, having one or more substitutions of cysteine ​​residues. In some embodiments, the D1 protein variant has one or more additional modifications that result in increased protein stability, higher production levels in in vitro expression systems, and / or substantially no lower, identical, or better chromatin and / or NET degradation activity compared to the wild-type D1 protein of SEQ ID NO: 1. For example, the D1 variant may comprise at least one additional component substitution or point mutation disclosed in PCT / US2018 / 47084, which is incorporated herein in its entirety by reference.

[0067] In some embodiments, the D1 variant has one or both of the substitutions C123 and C126, which are optionally substituted 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 with SEQ ID NO: 1.

[0068] In some embodiments, the present invention provides a D1 enzyme having a PEG moiety compounded at 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 compounded through maleimide chemistry, which can be carried out under mild conditions. Other compound chemistrys, such as vinyl sulfone, dithiopyridine, and iodoacetamide activation chemistry, are known and can be used. The PEG moiety may be linear or branched and may generally range from 10 kDa to 40 kDa, or from 20 to 30 kDa.

[0069] In other embodiments, the present invention provides D1L1 variants comprising an amino acid sequence having one or more substituted cysteine ​​residues and being at least 80% identical to the enzyme defined by SEQ ID NO: 2. 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 substituted as part of a construct substitution. In some embodiments, the D1L1 variant comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity with SEQ ID NO: 2.

[0070] In some embodiments, the present invention provides a D1L1 enzyme having a PEG moiety compounded at positions corresponding to C22 and / or C50. In these embodiments, the PEG moiety provides extended half-life properties while avoiding disulfide scrambling and / or protein misfolding. In some embodiments, the PEG moiety is compounded through maleimide chemistry, which can be carried out under mild conditions. Other compound chemistrys, such as vinyl sulfone, dithiopyridine, and iodoacetamide activation chemistry, are known and can be used. The PEG moiety may be linear or branched and may generally range from 10 kDa to 40 kDa, or from 20 to 30 kDa.

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

[0072] In some embodiments, the present invention provides a D1L2 enzyme having a PEG moiety compounded at a 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 compounded through maleimide chemistry, which can be carried out under mild conditions. Other compound chemistrys, such as vinyl sulfone, dithiopyridine, and iodoacetamide activation chemistry, are known and can be used. The PEG moiety may be linear or branched and may generally range from 10 kDa to 40 kDa, or from 20 to 30 kDa.

[0073] In other embodiments, the present invention provides isolated polynucleotides encoding the D1, D1L1, D1L2, or D1L3 variants disclosed herein, as well as vectors and host cells. The host cells may be cells of any expression system, including bacteria or eukaryotes, whether non-mammalian such as Pichia pastoris or mammalian such as CHO cells.

[0074] In some embodiments, polynucleotide delivery is used therapeutically. Encoding polynucleotides can be delivered as mRNA or as DNA constructs using known procedures, e.g., electroporation or cell squeezing, and / or vectors (including viral vectors). mRNA polynucleotides may contain known modifications (mRNA) to avoid activation of the innate immune system. See WO2014 / 028429, which is incorporated herein by reference in its entirety. In some embodiments, polynucleotides are delivered to the body of a subject. In some embodiments, polynucleotides are delivered intracellularly in vitro, and the cells are delivered to the body of a subject. The cells may be, for example, leukocytes (e.g., T cells or macrophages), endothelial cells, epithelial cells, hepatocytes, or stem cells.

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

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

[0077] A vector generally contains an isolated nucleic acid that can be used to deliver the isolated nucleic acid into 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 polylysine compounds and liposomes. It should be interpreted as including. Examples of viral vectors, though not limited to them, include adenovirus vectors, adeno-associated virus vectors, and retrovirus vectors.

[0078] Pharmaceutical compositions can be formulated for any route of administration, including topical, parenteral, or pulmonary administration. In various embodiments, compositions are formulated for intradermal, intramuscular, intraperitoneal, intra-articular, intravenous, subcutaneous, intra-arterial, oral, sublingual, pulmonary, or transdermal administration. In some embodiments, compositions are formulated for intravenous or subcutaneous administration.

[0079] In other embodiments, the present invention provides a method for treating subjects requiring 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 the extracellular DNASE described herein or its variants or compositions. Exemplary indications in which subjects require extracellular DNA or chromatin degradation (including ET or NET degradation) are disclosed in PCT / US18 / 47084, which is incorporated herein by reference. The present invention provides a method for treating subjects requiring such degradation, comprising administering a therapeutically effective amount of a protein represented by any one of the sequences of SEQ ID NOs: 8 to SEQ ID NOs: 30.

[0080] In each example describing a method for treating a subject, the present invention similarly provides the use of one or more 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. Such diseases or conditions include, but are not limited to, chronic neutrophilia, neutrophil aggregation and leukocyte stagnation, thrombosis and vascular occlusion, ischemia-reperfusion injury, surgical and traumatic tissue injury, acute or chronic inflammatory reactions or diseases, autoimmune diseases, cardiovascular diseases, metabolic diseases, systemic inflammation, respiratory inflammatory diseases, nephroinflammatory diseases, inflammatory diseases associated with transplanted tissue (e.g., graft-versus-host disease), and diseases associated with cancer (including leukemia).

[0082] In certain embodiments, the present invention relates to the treatment of diseases or conditions characterized by D1L3 deficiency or D1 deficiency. In some cases, subjects have mutations (e.g., loss-of-function mutations) in the Dnase1l3 gene or the Dnase1 gene. Such subjects may have autoimmune diseases (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 urticarial vasculitis). In some cases, subjects have acquired inhibitors of D1 (e.g., anti-DNase1 antibody and actin) and / or D1L3 (e.g., anti-DNase1l3 antibody). Such subjects may also have autoimmune diseases or inflammatory diseases (e.g., SLE, systemic sclerosis).

[0083] In some embodiments, subjects have or are at risk of having NETs that obstruct vascular systems. For example, the DNASE enzymes disclosed herein can be administered to subjects to treat pancreatitis, cholangitis, conjunctivitis, mastitis, dry eye disease, vascular occlusion, or kidney disease.

[0084] In some embodiments, the subject has or is at risk of accumulating NETs on the endothelial surface (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 characterized by the accumulation of NETs on the endothelial surface, including but not limited to surgical adhesions. It can be administered to the subject to treat the condition described above.

[0085] Other NET-related diseases and conditions 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, shock liver, hepatorenal syndrome, myocardial infarction, stroke, ischemic bowel, limb ischemia, testicular torsion, pre-eclampsia, eclampsia, and solid organ transplantation (e.g., kidney, heart, liver, and / or lung transplantation). Furthermore, the DNASE enzymes disclosed herein may be used, for example, by topical application to the skin to prevent scarring or contracture in individuals at risk, such as those with surgical incisions, lacerations, or burns.

[0086] In various embodiments, subjects have a disease that has been treated or is being treated with wild-type DNases including D1 and streptodolase. Such diseases or conditions include thrombosis, stroke, sepsis, lung injury, atherosclerosis, viral infections, sickle cell disease, myocardial infarction, ear infections, wound healing, liver injury, endocarditis, liver infections, pancreatitis, primary graft failure, limb ischemia-reperfusion, kidney injury, blood coagulation, alum-induced inflammation, hepatorenal injury, pleural exudation, hemothorax, cholangiothrombosis, post-pneumatic anemia, ulcers, otolaryngological diseases, and oral infections. This includes conditions such as rhinosinusitis, minor injuries, sinusitis, postoperative rhinoplasty, infertility, bladder catheterization, wound irrigation, skin reaction testing, pneumococcal meningitis, gout, lower extremity ulcers, cystic fibrosis, Kartagener syndrome, asthma, lobar atelectasis, chronic bronchitis, bronchiectasis, lupus, primary ciliary dysfunction, 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. [Examples]

[0088] Approximately 70% of all biological products are produced using Chinese hamster ovary (CHO) cells. In fact, wild-type DNASE1 (D1; dornaise alfa) is typically produced within CHO cells. Despite the significant advantages of developing cell lines and large-scale production using CHO cells, there remain significant challenges in the production of the Dnase enzyme due to considerable variability and the lack of reliable methods for predicting or modeling cell growth characteristics. Importantly, CHO cells cannot consistently 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] Using CHO and microbial expression systems, several challenges in the production of D1L3 were identified, including low production yield, proteolysis, protein misfolding, and incorrect or undesirable glycosylation. This disclosure provides technical solutions to these and other challenges in production, which can also improve the therapeutic properties of D1L3.

[0090] Example 1: Expression and characterization of D1L3 with basic domain deletion (BDD) in Chinese hamster ovary (CHO) cells and Pichia pastoris. DNASE1 and DNASE1L3 preferentially cleave non-proteinaceous 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 involved in the different substrate specificities of both enzymes (Sisirak et al.). .,Cell,2016;Keyel,Developmental Biology,2017).

[0091] Protein engineering techniques, referred to as component protein engineering, are described in PCT / US18 / 47084 and US62 / 800,790, and these disclosures are incorporated herein by reference in their entirety. This approach can be applied to members of the DNASE1 and DNASE2 protein families. The method is based on the steps of: providing protein-protein alignment of donor and recipient DNase enzymes; identifying variable amino acid sequences for migration in which variable amino acids are adjacent to one or more conserved amino acids in the donor and recipient DNase enzymes; creating chimeric DNase by replacing the variable amino acids of the recipient DNase with the variable amino acids of the donor DNase; and recombining and producing the chimeric DNase.

[0092] To characterize the amino acids responsible for chromatin degradation activity ("chromatin enzyme" activity), wild-type D1L3 was substituted with component substitutions from D1, as disclosed in PCT / US2018 / 047084. The component substitutions for D1L3 were selected from human D1 and, with respect to SEQ ID NO: 4, 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 This results in human D1L3 variants characterized by mutations in SA, V225_S228delinsATP, N230H, L238_R239delinsVA, Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256N, K259A, K262G, T264_E267delinsSDQL, L269_V271delinsQAI, F275Y, F279_K280delinsVM, and Q282_S205delinsK.

[0093] These 63 D1L3 variants were screened for loss or increase in chromatin degradation activity. Briefly, D1L3 variants were transiently expressed in CHO cells using an in vitro expression vector. Culture supernatants were collected, and purified nuclei were used as a chromatin source to test for chromatin degradation activity. 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 resulted in the missense mutation Q101R, replacing glutamine at position 101 with arginine (SEQ ID NO: 8). Component substitution #63 resulted in the mutation Q282_S305delinsK, deleting the complete C-terminal BD of D1L3 from amino acids 283-305 and replacing 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, in samples containing both wild-type D1L3 and the Q101R mutation, two D1L3 varian cells of different sizes were found. Samples containing Q282_S305delinsK contained only the smaller D1L3 variant. The data suggest that the BD of wild-type D1L3 is spontaneously removed (e.g., proteolytically) during or after expression in CHO cells. The two D1L3 variants were also detected in the supernatant from CHO cells stably expressing WT-D1L3 (Figure 3). Notably, basic domain deletion D1L3 (BDD-D1L3) showed substantially increased chromatin enzyme activity compared to wild-type D1L3.

[0094] Next, Pichia pastoris was tested as an alternative microbial expression system to CHO cells. In general, higher expression levels were observed with BDD-D1L3 compared to wild-type D1L3. Here, wild-type D1L3 and BDD-D1L3 were purified and characterized from the Pichia pastoris fermentation supernatant (Figure 4). Unexpectedly, wild-type D1L3 was observed to be proteolytically cleaved within 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, Q282_S205delinsK) produced a pure protein.

[0095] Next, we compared the chromatin enzyme activity of both purified D1L3 strains. We observed that heterologous mixtures of D1L3 variants with BD cleavage at the K291, K291, or S293 positions exhibited approximately 10-fold lower chromatin enzyme activity compared to D1L3 variants with complete BD deletion via F275Y / F279_K280delinsVM / Q282_S205delinsK. In summary, the data suggest that BD proteolytic cleavage can occur spontaneously in microbial and mammalian expression systems (i.e., CHO and P. pastoris), and that BD removal manifests as activation of D1L3 activity to degrade chromatin.

[0096] Example 2: Expression of D1L3 in CHO cells in a bioreactor This specification discloses 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. In detail, 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 low levels of D1L3 and D1L3 fragments were detected. The data suggest that the low production yield of D1L3 is a challenge in D1L3 production.

[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, representing the biologically active components in the “anti-cell aggregation reagent.” In detail, 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 the polyanions increased the production yield of D1L3 but did not prevent proteolysis.

[0098] Figure 6A shows that polyanions such as dextran sulfate (DS) form a complex with D1L3. The D1L3-DS complex is protected from interaction and removal of D1L3 by a negatively charged surface during the production process. Such negatively charged surfaces include, but are not limited to, the cell surface of producing 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 detach the DS-D1L3 complex. The objective is dissociation. Dissociation can be achieved by incubating the DS-D1L3 complex with a strong anion exchange surface, which binds to DS and thus releases D1L3. In detail, the purification process may include passing a 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. A second step of the purification process is shown in Figure 6C and includes affinity purification of D1L3 from the DS-free flow via the application of a strong cation exchange resin. In conclusion, the production yield of D1L3 can be significantly increased by adding a polyanion such as dextran sulfate.

[0099] Example 3: D1L3 manipulated 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. These sites can be mutated to produce protease-resistant variants of D1L3.

[0100] In short, 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). It was identified that trypsin cleaved D1L3 at the 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 can be substituted with small amino acids such as alanine, valine, and serine, or with amino acids having similar properties according to Grantham's distance score (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] By applying component protein engineering, the components of 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) were migrated from D1, replacing the components of D1L3, including the trypsin cleavage site (Figure 7).

[0102] Plasmin is a plasma protease produced by the activation of its enzyme precursor, 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 indicated that the amino acid combinations lysine-alanine (KA) or arginine-alanine (RA) are preferably cleaved by plasmin or proteases with plasmin-like activity. D1L3 contains a total of four putative plasmin cleavage sites (Figure 8): (Site 1) K180 / A181 (K160 / A161 without the signal peptide), (Site 2) K200 / A201 (K180 / A181 without the signal peptide), (Site 3) K259 / A260 (K239 / A240 without the signal peptide), and (Site 4) R285 / A286 (R270 / A250 without the signal peptide). Using the pair alignment of D1 and D1L3, We found that none of the plasmin cleavage sites are present in D1 (Figure 8). The data is consistent with the fact that D1 activity is resistant to inactivation by serum proteases such as thrombin and plasmin. By applying component protein engineering, we migrated components (site 1) K180_A181delinsGL, (site 2) P198_A201delinsRPSQ, and (site 3) K259A from D1 and replaced the components of D1L3 that contain plasmin cleavage sites (Figure 9). R285 / A286 (site 4) is located in the C-terminal extension that is absent in D1. Therefore, we created D1L3 variants in which all four putative plasmin cleavage sites—K180_A181delinsGL, P198_A201delinsRPSQ, K259A, and R285A—were mutated. Next, we analyzed chromatin degradation by the D1L3 variant and observed potent chromatin degradation activity in the mutated D1L3 (Figure 10). In summary, the data show that mutations in four arginine and lysine residues, K180, K200, K259, and R285, can reduce the risk of protein degradation without impairing enzyme 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. It was identified that plasmin cleaved D1L3 at the 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 may be substituted with small amino acids such as alanine, valine, and serine, or with amino acids having similar properties according to Grantham's distance score (e.g., histidine, glutamine, and glutamic acid; Figure 11). Protease-resistant D1 is characterized by the lysine residue corresponding to K45, suggesting that this residue is not primarily involved in plasmin-mediated proteolysis of D1L3. By applying component protein engineering, R22 (mutation: M21_R22delinsLK), R29 (V28_S30delinsIQT), K47 (K47_K50delinsQILS), K74 (M72_K74delinsLDN), R81 (R77_I83delinsQDAPD), R92 (S91_R92delinsEP), K107 (K107_L110delinsRPDQ), K The components of 176 (K176_R178delinsQEK), R212 (R212T), K226 (V225_S228delinsATP), K227 (V225_S228delinsATP), K250 (K250D), K259 (K259A), and K262 (K262G) were migrated from D1, replacing the components of D1L3, including the trypsin cleavage sites as determined by computer analysis (Figure 11).

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

[0106] Example 4: D1L3 manipulated to prevent decomposition We observed the fragmentation of D1L3 after heterologous expression in Pichia pastoris. Analysis of the fragments characterized the pars basic amino acids, arginine (R), and lysine (K) residues as proteolytic cleavage sites. A similar degradation pattern was observed after D1L3 expression in CHO cells. These observations suggest that Pichia pastoris and CHO cells share homologous proteases that cleave D1L3 at pars basic amino acids, but this effect was more pronounced in CHO cells.

[0107] We determined that the parsing nucleotide cleavage enzyme (PACE) contributes to DNASE1L3 fragmentation. PACE is also known as furin (Uniprot ID: P09958). These enzymes are expressed in humans and mammals. Pichia pastoris expresses two enzymes that target counterbasic amino acids: aspartate 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, resulting in pharmacological inhibition or genetic depletion of furin, aspartate proteinase 3, and kexin.

[0108] In addition, mutations in the counterbasic amino acids in DNASE1L3 and DNASE1L3 variants enable their expression in CHO and Pichia pastoris with reduced fragmentation. The identified DNASE1L3 fragments, upon analysis, are characterized by counterbasic amino acids at the K50 / R51, R80 / R81, K114 / R115, K199 / K200, K226 / K227, K291 / K292, R297 / K298 / K299, and K303 / R304 positions in SEQ ID NO: 2.

[0109] As disclosed in U.S. Provisional Patent Application No. 62 / 800,790 (which is 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 (cattle), K227S (dog), and K227E (elephant). Applying these amino acid substitutions to human DNASE1L3 can make the enzyme resistant to proteolysis, including during expression in CHO cells and Pichia pastoris.

[0110] Kexin is preferably cleaved after KR and RR residues. The DNASE1L3 features at K50 / R51, R80 / R81, K114 / R115, and K303 / R304 are four KEX2 cleavage sites. Amino acid substitutions of these residues make DNASE1L3 resistant to KEX2, enabling the expression of DNASE1L3 and DNASE1L3 variants in Pichia pastoris and CHO cells. These amino acid substitutions may be conserved, for example, R51K, R81K, R115K, and R304K.

[0111] Example 5: D1L3 variant manipulated to prevent high molecular weight aggregates During cGMP-compliant expression of D1L3 in CHO cells (Figure 12A), we observed accumulation of high molecular weight aggregates of D1L3, highlighting additional challenges for the analytical production of D1L3. High molecular weight aggregates were observed to a much lower degree in Pichia pastoris.

[0112] The application of reducing conditions to the bioreactor material dissolved D1L3 aggregates. The data indicate that D1L3 aggregate formation is caused by intramolecular and / or intermolecular crosslinking via disulfide crosslinks during protein expression. In detail, as shown in Figure 12B, the gel was run under non-reducing conditions, showing the accumulation of high molecular weight D1L3 aggregates over time. The gel was run under reducing conditions, and no aggregates were detected. The data suggest that incorrect intramolecular and intermolecular disulfide bonds cause misfolding of human D1L3 under manufacturing conditions.

[0113] Figure 13 shows the amino acid sequence alignments of human D1 (SEQ ID NO: 1) and human D1L3 (SEQ ID NO: 4). Signal peptides, conserved amino acids, variable amino acids, non-conserved cysteine ​​residues, and conserved cysteine ​​residues are highlighted. Mutations in non-conserved cysteine ​​residues reduce the likelihood of intramolecular and intermolecular disulfide bonds during protein expression. Analysis of the amino acid sequence of D1L3 (SEQ ID NO: 4) revealed C24, C52, C68, C194, and C2 Figure 14 shows the presence of five cysteine ​​(C) residues. Cysteine ​​residues C194 and C231 are conserved among all members of the DNASE1 protein family and form the disulfide bond necessary for the enzymatic activity of DNASE1. The function of the cysteine ​​residues in D1L3 was unknown prior to this disclosure. Therefore, as disclosed herein, mutations in these cysteine ​​residues reduce crosslinking via disulfide bridges and thus increase 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. In addition, components containing conserved cysteine ​​residues can be replaced by 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 the D1L3 variants was quantified as described in PCT / US18 / 4708. Both conventional amino acid substitutions (C24A, C52A) and component 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, mutations in cysteine ​​C68 by either 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 highly conserved cysteine ​​C194 was substituted with alanine (C194A), but highly conserved cysteine ​​C231 was not mutated with alanine (C231A), resulting in enzymatically active D1L3 variants (Figure 15). Therefore, by mutating cysteine ​​C68 and C194, the risk of incorrect disulfide bond formation during D1L3 production can be reduced.

[0115] A similar approach can be applied to mutate non-conserved cysteine ​​residues in other members of the DNase1 protein family, including D1, DNase1-like 1 (D1L1), and DNase1-like 2 (D1L2). D1 has two non-conserved cysteines, C123 and C126. D1L1 shows that it has only one non-conserved cysteine ​​residue: C43, instead of the two non-conserved cysteine ​​residues (C22, C50) corresponding to C24 and C52 in D1L2. Mutations of non-conserved cysteine ​​residues in members of the DNase1 protein family reduce crosslinking via incorrect disulfide crosslinks 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 protein in Pichia pastoris Pichia pastoris expression of recombinant human extracellular DNASES containing D1L3 was tested. As shown in Figure 16A, the N-terminus of D1L3 was led by an alpha junction factor (aMF) pre-pro secretory reader from Saccharomyces cerevisiae (SEQ ID NO: 46), a common tool for heterologous protein expression in Pichia pastoris. As disclosed herein, the combination of aMF and 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 the analytical production of D1L3. When the N-terminus was led by the native secretory signal peptide of D1L3, D1L3 was processed appropriately [Figure 16B, (SEQ ID NO: 48)]. Importantly, aMF compared to the native signal peptide of D1L3 D1L3 expression was increased 3-5 times. Therefore, processing of the D1L3 fusion protein was tested. In preliminary tests, N-terminal fusions of aMF and human serum albumin [HSA (SEQ ID NO: 39)] with D1L3 were generated (Figure 17A). Several variants contained a linker peptide [e.g., (GSSSS)3] between HSA and D1L3. As shown in Figures 17B and 17C, expression of the fusion protein in P. pastoris generated non-glycosylated and enzymatically active D1L3. Furthermore, the expression level was increased 5-10 times compared to the innate secretory signal peptide-driven expression of D1L3. In summary, the data indicate 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 the expression levels of target proteins (SEQ ID NOs: 17-28). As shown in Figure 18, N-terminal fusion of human serum albumin (SEQ ID NO: 17) with the BDD-D1L3 variant (SEQ ID NO: 16) was observed to not substantially increase the expression level. However, a strong increase in expression levels was detected when a flexible linker consisting of glycine (G) and serine (S) residues was inserted between the HSA and BDD-D1L3. Furthermore, the length of the linker sequence correlated with the increase in expression. For example, expression of 12 ± 1.9 relative units was obtained with a 5-amino acid linker (SEQ ID NO: 18), while expression with a 15-amino acid linker (SEQ ID NO: 19) was 32 ± 3.2 relative units, an improvement of approximately 7.5 times compared to HSA fusion without a linker. Furthermore, since the C-terminal fusion of the linker-HSA construct was expressed at a low level, the N-terminal position was important for improving expression levels (SEQ ID NO: 20, SEQ ID NO: 21). Notably, N-terminal fusion of HSA via the flexible linker increased the expression of wild-type D1L3 (SEQ ID NO: 22) by approximately 20 times compared to native D1L3 (SEQ ID NO: 4). In conclusion, fusion of HSA with the N-terminus via the linker enables the production of D1L3 and BDD-D1L3 variants.

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

[0119] Next, the relationship between linker length, expression level, and enzyme activity was analyzed. For these tests, expression vectors were designed containing an N-terminal fusion of HSA with a GS-linker for 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 SGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSS[31 amino acids, (SEQ ID NOs. 37)]. As shown in Figure 20, elongation of the linker sequence from 7 amino acids to 16 amino acids resulted in an increase in expression level. Further elongation of amino acids from 16 to 31 did not increase protein expression, but increased enzyme activity, as detected by the degradation of HMW-chromatin to LMW-chromatin. Biologics fused to albumin fusions often show decreased activity because albumin sterically inhibits the interaction with substrates and ligands. Therefore, peptide linkers 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 improved 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). In short, DNase1 - / -Dnase1l3 - / - Mice were injected with SEQ ID NO: 4 or SEQ ID NO: 19. Serum was collected 15 minutes after injection. Similar serum chromatin degradation activity was observed in both animals, as shown in Figure 21A. Importantly, the fusion of albumin to the N-terminus of D1L3 and other human extracellular DNASESs 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 15-amino acid flexible 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 circulating albumin. While non-complex D1L3 (e.g., SEQ ID NO: 4) has a very short half-life in circulation (less than 30 minutes), the albumin fusion extends the half-life to 3.3 days, thereby substantially improving systemic exposure and simultaneously reducing the half-life by 5 minutes. max This resulted in rapid absorption (Figure 21B). In summary, the data show that N-terminal fusion of HSA to D1L3 via the 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-terminus of D1L3. First, we analyzed the C-terminus of D1L3 for potential attachment sites. We identified two serine residues at positions 283 and 284 that provide flexible attachment of BD(RAFTNSKKSVTLRKKTKSKRS) to the core body of D1L3. Therefore, we chose to delete BD and attach the HSA to S284 via a flexible GS linker (SEQ ID NO: 38). As shown in Figure 22, the fusion of HSA to the N and C-terminus 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 a severable linker array The findings disclosed herein have implications beyond manufacturing. For example, D1L3 variants having C-terminal amino acid deletions that preserve their enzymatic activity for degrading chromatin and / or NETs, ​​as exemplified by SEQ ID NOs: 9 to 12, can be used in D1L3 therapy. In addition, site-directed alkylation of non-paired cysteine ​​thiols is commonly used to produce half-life extended biological products for therapeutic use. In particular, the non-essential cysteines C68 and C194 of D1L3 can be used for site-directed PEGylation (PEG, polyethylene glycol). Furthermore, D1L3 variants resistant to plasmin inactivation resulting from 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, fusion of albumin to the N-terminus of D1L3 and other human extracellular DNASESs provides DNASE therapeutics with extended half-lives (Figure 23A). Several linker sequences were used to reduce albumin-induced steric inhibition of D1L3. Furthermore, physiologically cleavable peptide linkers were developed. These were designed to cleave the linker peptide when the fusion protein is in proximity to a neutrophil extracellular trap (NET). Peptide sequences targeted by neutrophil-specific proteases such as neutrophil elastase, cathepsin G, and proteinase 3 are candidate cleavable linker sequences.

[0124] We developed a cleavable linker sequence optimized for DNASE therapeutics that are cleaved intravascularly and therefore applied intravenously and intraarterially. To design the peptide, we hypothesized that NET has 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, the cleavage site is located between arginine 387 and isoleucine 388. In PK, the cleavage site is located between arginine 390 and isoleucine 391. In fact, FXI and PK are homologous proteins. As disclosed herein, several linker peptides were designed that include all or part of positions 380-403 of the FXI sequence (SEQ ID NO: 42, SEQ ID NO: 43) or positions 383-406 of the PK sequence (SEQ ID NO: 44).

[0125] Finally, the FXIIa-cleavable linker can be used to produce forms (Figure 24) that extend the half-lives 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 incorporated herein by reference in their entirety. Wild-type human DNASE Sequence ID 1 TIFF2026082949000002.tif43169 Sequence ID 2 TIFF2026082949000003.tif52169 Sequence ID 3 TIFF2026082949000004.tif54169 Sequence ID 4 TIFF2026082949000005.tif53169 Sequence ID 5 TIFF2026082949000006.tif47169 Sequence ID 6 TIFF2026082949000007.tif59169 Sequence ID 7 TIFF2026082949000008.tif57169 Human DNA SE1L3 variant Sequence ID 8 TIFF2026082949000009.tif47169 Sequence ID 9 TIFF2026082949000010.tif42169 Sequence ID 10 TIFF2026082949000011.tif44169 Sequence ID 11 TIFF2026082949000012.tif48169 Sequence ID 12 TIFF2026082949000013.tif50169 Sequence ID 13 TIFF2026082949000014.tif47169 Sequence ID 14 TIFF2026082949000015.tif52169 Sequence ID 15 TIFF2026082949000016.tif49169 Sequence ID 16 TIFF2026082949000017.tif47169DNASE1L3 and albumin fusion with variants Sequence ID 17 TIFF2026082949000018.tif111169 Sequence ID 18 TIFF2026082949000019.tif110169 Sequence ID 19 TIFF2026082949000020.tif108169 Sequence ID 20 TIFF2026082949000021.tif110169 Sequence ID 21 TIFF2026082949000022.tif113169 Sequence ID 22 TIFF2026082949000023.tif114169 Sequence ID 23 TIFF2026082949000024.tif114169 Sequence ID 24 TIFF2026082949000025.tif118169 Sequence ID 25 TIFF2026082949000026.tif108169 Sequence ID 26 TIFF2026082949000027.tif109169 Sequence ID 27 TIFF2026082949000028.tif118169 Sequence ID 28 TIFF2026082949000029.tif192169 Sequence ID 29 TIFF2026082949000030.tif124169 Sequence ID 30 TIFF2026082949000031.tif113169 Linker Array Sequence ID 31 GGGGS Sequence ID 32 GGGGSGGGGGGGGGS Sequence ID 33 APAPAPAPAPAPAP Sequence ID 34 AEAAAKEAAAKA Sequence ID 35 SGGSGSS Sequence ID 36 SGGSGGSGGSGGSGSS Sequence ID 37 SGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGSGS Sequence ID 38 GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGS Other arrays Sequence ID 39 Human serum albumin (mature protein): DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL Sequence number 40 Human Factor XI: MIFLYQVVHFILFTSVSGECVTQLLKDTCFEGGDITTVFTPSAKYCQVVCTYHPRCLLFTFTAESPSEDPTRWFTCVLKDSVTETLPRVNRTAAISGYSFKQCSHQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYATRQF PSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLACIRDIFPNTVFADSNIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIPVFCHSSFYHDTDFLGEELDIVAAK SHEACQKLCTNAVRCQFFTYTPAQASCNEGKGKCYLKLSSNGSPTKILHGRGGISGYTLRLCKMDNECTTKIKPRIVGGTASVRGEWPWQVTLHTTSPTQRHLCGGSIIGNQWILTAAHCFYGVESPKILRVYSGILNQSEIKEDTSFFGVQEIII HDQYKMAESGYDIALLKLETTVNYTDSQRPICLPSKGDRNVIYTDCWVTGWGYRKLRDKIQNTLQKAKIPLVTNEECQKRYRGHKITHKMICAGYREGGKDACKGDSGGPLSCKHNEVWHLVGITSWGEGCAQRERPGVYTNVVEYVDWILEKTQAV Sequence ID 41 Human prekallikrein: MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSINDMEKRFGCFLKDSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTNNIRCQFFSYATQTFHK AEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYSLLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQ ETCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTRIVGGTNSSWGEWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVS EGNHDIALIKLQAPLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYMDWILEKTQSSDGKAQMQSPA Activated linker sequence Sequence ID 42 FXIIa-sensitive linker (factor XI peptide): CTTKIKPRIVGGTASVRGEWPWQVT Sequence ID 43 FXIIa Sensitivity Linker GGGGSPRIGGGS Sequence ID 44 FXIIa-sensitive linker (prekallikrein peptide): VCTTKTSTRIVGTNSSWGEWPWQVS Sequence ID 45 FXIIa-sensitive linker (prekallikrein peptide): STRIVGG Signal peptide Sequence ID 46 Alpha zygosity factor (P01149): MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNGLLFINTTIASIAAKEEGVS Sequence ID No. 47 Human albumin secretion signaling peptide + propeptide (P02768): MKWVTFISLLFLFSSAYSRGVFRR Sequence ID 48 Human DNASE1L3 signal peptide (Q13609): MSRELAPLLLLLLSIHSALA

Claims

1. A DNase1-like 3 (D1L3) variant, wherein the D1L3 protein variant, in each case, has one or more of the following compared to the wild-type D1L3 protein of SEQ ID NO: 4 and / or SEQ ID NO: 5: increased resistance to proteolysis, increased cyclic half-life, higher production levels in in vitro expression systems, and substantially no lower, identical, or better chromatin and / or NET degradation activity in vitro.

2. The D1L3 variant according to claim 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, the N-terminal albumin amino acid sequence 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.

3. The D1L3 variant according to claim 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.

4. The D1L3 variant according to claim 2, wherein the D1L3 variant has a deletion of at least five 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.

5. The D1L3 variant according to claim 4, wherein the D1L3 has a deletion of at least 10 amino acids in the C-terminal basic domain.

6. The D1L3 variant according to claim 4, wherein the D1L3 has a deletion of at least 15 amino acids in the C-terminal basic domain.

7. The D1L3 variant according to claim 6, wherein the D1L3 has a deletion of the entire C-terminal basic domain.

8. The D1L3 variant according to any one of claims 1 to 7, wherein the D1L3 has an amino acid substitution at the position corresponding to position 101 of SEQ ID NO:

4.

9. The D1L3 variant according to claim 8, wherein the D1L3 has an amino acid substitution in which D1L3 is Q101R.

10. The D1L3 variant according to claim 2, wherein the linker is flexible, rigid, or includes a protease cleavage site.

11. The D1L3 variant according to claim 10, wherein the linker is a flexible linker.

12. The D1L3 variant according to claim 10 or 11, wherein the linker has at least 10 amino acids or at least 15 amino acids.

13. The D1L3 variant according to claim 12, wherein the linker has 15 to 35 amino acids.

14. The variant contains one amino acid sequence from sequence numbers 8 to 30, and in each case, 1 to 20 amino acids are optionally and independently selected from insertions, deletions, or substitutions. The D1L3 variant according to claim 1, having no acid modification.

15. The D1L3 variant according to claim 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.

16. A D1L3 variant according to any one of claims 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.

17. The D1L3 variant according to claim 16, wherein the amino acid corresponding to C68 in SEQ ID NO: 4 is substituted.

18. The D1L3 variant according to claim 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.

19. The D1L3 variant according to claim 16, wherein the variant comprises the substitution N64_I70delinsHLTAVGK, which is optionally modified by one, two, or three amino acid substitutions, deletions, or insertions, provided that the constituent substitution is not modified to include a cysteine ​​residue.

20. The D1L3 variant according to claim 1, comprising a PEG complex to an amino acid corresponding to C68 and / or C194.

21. A D1L3 variant according to claim 1 or 2, having one or more substitutions of arginine and lysine residues present in SEQ ID NO: 4 and / or SEQ ID NO: 5, wherein the substitutions reduce the D1L3 variant's sensitivity 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.

22. The D1L3 variant according to claim 21, wherein one or more of the arginine and lysine substitutions correspond to the positions(s) of Sequence ID No. 4: K180, K200, K259, and R285.

23. The D1L3 variant according to claim 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.

24. The D1L3 variant according to claim 22, wherein the variant includes substitutions selected from K180_A181delinsGL, P198_A201delinsRPSQ, and K259A with respect to sequence number 4.

25. The D1L3 variant according to claim 21, wherein the variant comprises one or more mutations in a pair of basic residues, the pair of basic residues corresponding 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.

26. The D1L3 variant according to claim 25, wherein one or more mutations in the paired basic residues include an amino acid substitution selected from R114T, R114A, R114D, R114Q, K227S, and K227E with respect to SEQ ID NO:

4.

27. The D1L3 variant according to claim 25, wherein one or more mutations in the paired basic residues include an amino acid substitution selected from R51K, R81K, R115K, and R304K with respect to SEQ ID NO:

4.

28. The D1L3 variant according to claim 10, wherein the linker is cleavable by a coagulation pathway protease.

29. The D1L3 variant according to claim 28, wherein the linker is cleavable by factor XII or neutrophil protease.

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, having one or more deletions or substitutions of cysteine ​​residues.

31. The D1 variant according to claim 30, wherein one or more amino acids corresponding to C123 and C126 of SEQ ID NO: 1 are deleted or substituted.

32. The D1 variant according to claim 31, wherein the cysteine(s) are substituted with an amino acid independently selected from Ala, Ser, and Gly.

33. The D1 variant according to claim 30, wherein the variant includes the substitution G122_N128delinsYQGDA.

34. The D1 variant according to any one of claims 30 to 33, wherein the D1 variant is a fusion protein comprising an albumin amino acid sequence and optionally a peptide linker.

35. The D1 variant according to claim 34, wherein the albumin domain of the fusion protein is located on the N-terminal side of the D1 domain.

36. The D1 variant protein according to claim 34 or 35, wherein the peptide linker is inserted between the albumin domain and the D1 domain.

37. The D1 variant according to claim 36, wherein the peptide linker is a flexible linker, a rigid linker, or a protease-cleavable linker.

38. The D1 variant according to claim 37, wherein the linker has at least 10 amino acids or at least 15 amino acids.

39. The D1 variant according to claim 37, wherein the linker has 15 to 35 amino acids.

40. The D1 variant according to any one of claims 34 to 39, wherein the D1 domain of the fusion protein comprises an amino acid sequence that is at least 80% identical to that of the mature enzyme defined by SEQ ID NO: 4 or SEQ ID NO:

5.

41. The D1 variant according to 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.

42. The D1 variant according to claim 40 or 41, wherein the D1 variant comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 98% identical to the enzyme of SEQ ID NO:

1.

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 a PEG complex to the amino acid corresponding to C123 and / or C126.

44. A DNase1-like-1 (D1L1) variant comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 2, having one or more deleted or substituted cysteine ​​residues.

45. The D1L1 variant according to claim 44, wherein at least one substituted or deleted cysteine ​​residue is at the position corresponding to C22 or C50 of SEQ ID NO:

2.

46. The D1L1 variant according to claim 45, wherein one or more of the substituted cysteine ​​residues are substituted with an amino acid that is Gly, Arg, or Ser.

47. The D1L1 variant according to any one of claims 44 to 46, wherein the D1L1 variant comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 98% identical to the enzyme of SEQ ID NO:

2.

48. The D1L1 variant according to any one of claims 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.

49. The D1L1 variant according to claim 48, wherein the albumin domain is located on the N-terminal side of the D1L1 domain.

50. The D1L1 variant according to claim 48 or 49, wherein the fusion protein comprises a peptide linker, the peptide linker being a flexible linker, a rigid linker, or a protease-cleavable linker.

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 a PEG complex to the amino acid corresponding to C22 and / or C50.

52. A DNase1-like-2 (D1L2) variant comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 3, having one or more substituted or deleted cysteine ​​residues.

53. The D1L2 variant according to claim 52, wherein the cysteine ​​residue corresponding to C43 of SEQ ID NO: 3 is substituted or deleted.

54. The D1L2 variant according to claim 52 or 53, wherein one or more of the substituted cysteine ​​residues are substituted with an amino acid that is Gly, Arg, or Ser.

55. The D1L2 variant according to any one of claims 52 to 54, wherein the D1L2 variant comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 98% identical to the enzyme of SEQ ID NO:

2.

56. The D1L2 variant according to any one of claims 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.

57. The D1L2 variant according to claim 56, wherein the albumin domain is located on the N-terminal side of the D1L2 domain.

58. The D1L2 variant according to claim 56 or 57, wherein a peptide linker is inserted between the albumin domain and the D1L2 domain.

59. The D1L2 variant according to claim 58, wherein the peptide linker is a flexible linker, a rigid linker, or a protease-cleavable linker.

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 a PEG complex to the amino acid corresponding to C43.

61. An isolated polynucleotide encoding a D1, D1L1, D1L2, or D1L3 variant according to any one of claims 1 to 60.

62. The isolated polynucleotide according to claim 61, wherein the polynucleotide is mRNA.

63. The isolated polynucleotide according to claim 61, wherein the polynucleotide is DNA.

64. A vector comprising a polynucleotide according to any one of claims 61 to 63.

65. A host cell comprising the vector according to claim 64.

66. A host cell modified to express a D1, D1L1, D1L2, or D1L3 variant as described in any one of claims 1 to 60.

67. A pharmaceutical composition comprising a D1, D1L1, D1L2, or D1L3 variant according to any one of claims 1 to 60, a polynucleotide according to any one of claims 61 to 63, a vector according to claim 64, or a host cell according to claim 65 or 66, and a pharmaceutically acceptable carrier.

68. The pharmaceutical composition according to claim 67, which is formulated for topical, parenteral, or pulmonary administration.

69. The pharmaceutical composition according to claim 68, formulated for intradermal, intramuscular, intraperitoneal, intra-articular, intravenous, subcutaneous, intra-arterial, oral, sublingual, pulmonary, or transdermal administration.

70. A method for treating a subject requiring extracellular DNA degradation, extracellular chromatin degradation, extracellular trap (ET) degradation, and / or neutrophil extracellular trap (NET) degradation, wherein the method comprises administering a therapeutically effective amount of the composition according to any one of claims 67 to 69.

71. The method according to claim 70, wherein the subject has a loss-of-function mutation in the D1L3 gene.

72. The method according to claim 70 or 71, wherein the subject has a condition selected from chronic neutrophilia, neutrophil aggregation or leukocyte stagnation, thrombosis or vascular occlusion, ischemia-reperfusion injury, surgical or traumatic tissue injury, acute or chronic inflammatory reactions or diseases, autoimmune diseases, cardiovascular diseases, metabolic diseases, systemic inflammation, respiratory inflammatory diseases, nephroinflammatory diseases, inflammatory diseases associated with transplanted tissue, and cancer.

73. The method according to claim 70 or 71, wherein the subject has or is at risk of having a NET that obstructs a duct system, and the condition is optionally selected from pancreatitis, cholangitis, conjunctivitis, mastitis, dry eye disease, vascular occlusion, and kidney disease.

74. The method according to claim 70 or 71, wherein the subject has or is at risk of NET accumulation on the endothelial surface.

75. The method according to any one of claims 70 to 74, wherein the D1L3 variant is a fusion protein containing an albumin amino acid sequence, and is administered parenterally to the subject, and the administration is approximately once a week.

76. A method for producing a recombinant DNase variant according to any one of claims 1 to 60, comprising culturing cells expressing a polynucleotide encoding the DNase variant, and recovering a recombinant protein.

77. The method according to claim 76, wherein the cell is Pichia pastoris.

78. The method according to claim 77, wherein the Pichia pastoris cells have a genetic deletion or inactivation, or pharmacological inhibition, of aspartate proteinase 3 and / or kexin.

79. The method according to claim 76, wherein the cells are CHO cells.

80. The method according to claim 79, wherein the CHO cells have a genetic deletion or inactivation, or pharmacological inhibition, of furin protease.

81. The method according to any one of claims 76 to 80, wherein the DNase variant is a D1L3 variant comprising one or more mutations in a pair of basic residues, and the pair of basic residues corresponds to a position 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.

82. The method according to claim 81, wherein one or more mutations in the paired basic residue include an amino acid substitution selected from R114T, R114A, R114D, R114Q, K227S, and K227E.

83. The method according to claim 81, wherein one or more mutations in the paired basic residues include an amino acid substitution selected from R51K, R81K, R115K, and R304K.

84. A method for producing a recombinant D1 protein family member or a variant thereof, comprising culturing cells expressing a polynucleotide encoding a DNase variant in the presence of a polyanionic compound, and recovering the recombinant protein.

85. The method according to claim 84, wherein the recombinant D1 protein family member is a variant of D1, D1L3, D1L1, or D1L2.

86. The method according to claim 84 or 85, wherein the polyanion is selected from one or more of dextran sulfate, heparin, ferric citrate, and EDTA.

87. The method according to claim 86, wherein the polyanion is dextran sulfate.

88. The method according to any one of claims 84 to 87, wherein the cells are prokaryotic or eukaryotic.

89. The method according to claim 88, wherein the DNase is expressed using a non-mammalian expression system and is optionally Pichia pastoris, Saccharomyces species, or Escherichia coli.

90. The method according to claim 89, wherein the expression system is Pichia pastoris.

91. The method according to claim 90, wherein the Pichia pastoris cells have a genetic deletion or inactivation, or pharmacological inhibition, of aspartate proteinase 3 and / or kexin.

92. The method according to claim 88, wherein the cells are CHO cells.

93. The method according to claim 92, wherein the CHO cells have a genetic deletion or inactivation, or pharmacological inhibition, of furin protease.

94. The method according to any one of claims 84 to 93, wherein the recombinant D1 family member comprises albumin fusion at the N-terminus via a peptide linker.

95. The method according to claim 94, wherein the linker is a flexible linker.

96. The method according to claim 95, wherein the linker is a cleavable linker and can optionally be cleaved by a coagulation pathway protease or a neutrophil protease.

97. The method according to claim 96, wherein the coagulation factor protease is factor XII.

98. The method according to claim 97, wherein the peptide linker is selected from all or part of the FXII cleavage sites in factor XI or prekallikrein.

99. The method according to any one of claims 84 to 98, wherein the recombinant D1 family member or variant is expressed in a construct that encodes a signal peptide that directs the secretion of the recombinant protein from the cell.

100. The method according to any one of claims 84 to 99, wherein the recombinant D1 family member or variant is purified from the polyanionic compound using an anion exchange resin.

101. The method according to claim 100, wherein the recombinant D1 family member or variant is further purified using a cation exchange resin.

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