EA046216B1 - DEVELOPMENT OF DNases FOR PRODUCTION AND THERAPY - Google Patents

Description

Related applications

The application for this invention claims priority to US Provisional Patent Application No. 62/742682, filed October 8, 2018; 62/775563, filed December 5, 2018; 62/779104, filed December 13, 2018; 62808601, filed February 21, 2019; and 62/846904, filed May 13, 2019, the contents of each of which are incorporated herein by reference in their entirety.

Field of technology

This invention relates to the field of development of enzymes - DNases.

State of the art

Inflammation is a necessary host response to control invading microbes and heal damaged tissue. Uncontrolled and persistent inflammation causes tissue damage in a wide range of inflammatory diseases. In acute inflammation, neutrophils are the dominant leukocytes. During infection, neutrophils generate neutrophil extracellular traps (NETs), lattices of DNA filaments decorated with toxic histones and enzymes that immobilize and neutralize bacteria. However, mistakenly released NETs can cause harm to host cells through their cytotoxic, anti-inflammatory, and prothrombotic activities.

DNase 1 (D1) forms, together with DNase 1-like protein 1 (D1L1), DNase 1-like protein 2 (D1L2), and DNase 1-like protein 3 (D1L3), the DNase 1 protein family, a group of homologous secreted DNase enzymes. DNase 2A and DNase 2B form an additional group of homologous extracellular DNase enzymes. Members of the DNase 1 and DNase 2 protein families are evolutionarily conserved and are expressed in a variety of species, including humans. Recombinant members of the human DNase 1 and DNase 2 protein families are drug candidates for the treatment of NET-related diseases. Although the D1 protein has been developed for some therapeutic applications in patients, the conditions for large-scale production of other members of the DNase 1 protein family have not been described. In addition, the physical, enzymatic and pharmacokinetic properties of these enzymes are not ideal for clinical applications. Thus, there is a need to determine the production process of the D1L1, D1L2 and D1L3 enzymes, as well as to develop DNases for use in therapeutics, including NET degradation.

The essence of the invention

This invention provides engineered human extracellular DNase proteins (e.g., variants of DNase 1 (D1), DNase1-like protein 1 (D1L1), DNase1-like protein 2 (D1L2), DNase1-like protein 3 (D1L3) isoform 1, isoform 2 DNase1-like protein 3 (D1L3-2), DNase 2A (D2A) and DNase 2B (D2B)), which can be used to treat pathological conditions characterized by the accumulation and/or release of extracellular DNA, extracellular chromatin and neutrophil extracellular trap (NET) . In accordance with aspects of the invention, the DNase variants described herein are more useful for therapy and/or more suitable for large-scale production. In some embodiments, the DNase variants described herein are advantageous for drug therapy, including systemic therapy. Such advantages include slower drug elimination, such as increased half-life in the bloodstream (eg, serum half-life), increased duration of pharmacodynamic activity, high chromatin degradation activity, and resistance to proteases.

In some aspects of the invention, a D1L3 variant is provided, wherein the D1L3 variant has one or more of the following: increased protein stability, slower drug clearance and increased duration of pharmacodynamic activity, resistance to proteolytic degradation, higher production levels in in vitro expression systems, better suitability for purification, but not significantly less, the same or better chromatin and/or NET degradation activity compared to the wild-type D1L3 enzyme isoform 1 of SEQ ID NO:4 or the wild-type D1L3 enzyme isoform 2 of SEQ ID NO:5.

In some embodiments, the D1L3 variant is a fusion protein that contains 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 amino acid sequence of albumin at the N-terminus of the mature enzyme, and an optionally linking amino acid sequence between the amino acid sequence of albumin (albumin domain) and the amino acid sequence of D1L3 (D1L3 domain). In these embodiments, D1L3 exhibits slower elimination (eg, improved circulation half-life or serum half-life) and increased duration of pharmacodynamic activity, including for systemic therapy. In some embodiments, the fusion of albumin to the D1L3 domain binding sequence does not significantly affect the chromatin degradation activity of the enzyme (eg, measured by an in vitro assay) compared to the enzyme without the albumin fusion.

In these embodiments, the D1L3 domain of the fusion protein has all or part of it deleted

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The C-terminal basic domain that is present in the wild-type D1L3 enzyme. Deletion or inactivation of the C-terminal basic domain significantly improves chromatin degradation activity. That is, removal of the C-terminal basic domain (BD) activates the wild-type D1L3 enzyme to degrade chromatin.

In some embodiments, embodiment D1L3 has one or more substitutions of building blocks from D1. For example, the D1L3 variant may have a substitution of the Q282_S305delinsK building block, which involves the removal of the C-terminal core domain that is missing in D1. In some embodiments, the D1L3 variant has an amino acid substitution at a position corresponding to position 101 of SEQ ID NO: 4. The substitution may be an Arg based on the corresponding building block from D1, or in some embodiments may be a Lys. Substitutions at this position may enhance the chromatin degradation activity of the D1L3 variant.

The linker, if present, may be a flexible linker, a rigid linker, or a physiologically cleavable linker, such as a protease cleavable linker. For example, the linker may be a hydrophilic amino acid sequence and may be constructed from predominantly amino acids selected from Gly, Ala, Ser, Thr and Pro. In some embodiments, the variant is a flexible linker that consists primarily of glycine and serine residues (eg, a ( _GyS ) _n linker, where y is a number from 1 to 5 and n is from 1 to 20). In some embodiments, the linker is an α-helical linker. In some embodiments, the linker contains at least 15 amino acids or at least 25 amino acids. In various embodiments, longer linkers containing at least 15 amino acids may provide improved productivity when expressed in mammalian and non-mammalian expression systems, such as CHO or Pichia pastoris cells. Additionally, and unexpectedly, longer linker sequences showed improved chromatin degradation activity in an in vitro chromatin degradation assay compared to shorter linker sequences.

In various embodiments, variant D1L3 comprises the amino acid sequence of any of SEQ ID NO: 17-30, in each case optionally having from one to twenty amino acid modifications independently selected from insertions, deletions or substitutions. These sequences provide examples of D1L3 fusion proteins (or D1L3 variants) with albumin sequences, including various linker designs. In some embodiments, the amino acid modifications are in the D1L3 domain, the albumin 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 from the N-terminus to the C-terminus: an albumin amino acid sequence, a medium or long flexible linker, and a D1L3 amino acid sequence (ie, including D1L3 variants). SEQ ID NO: 28 further includes an albumin fusion at the C-terminus through a long flexible peptide linker.

In other embodiments, the linker is cleaved by a protease, such as a coagulation system protease, such as activated factor XII. In some 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 cleaved by a neutrophil-specific protease, such as neutrophil elastase, cathepsin G, or proteinase 3.

In some aspects of the invention, variants of extracellular DNases are provided that are designed to have manufacturing advantages by providing the production of a recombinant enzyme suitable for use in therapy. In various embodiments, the invention provides recombinant variants of D1, D1L1, D1L2 and D1L3 containing one or more amino acid substitutions at cysteine residues (or PEGylation of Cys residues), resulting in reduced intra- and intermolecular cross-linking via disulfide bridges during protein expression.

Other aspects of the invention provide variants of extracellular DNases designed to have protease resistance advantages to improve in vivo effects, such as slowing elimination, e.g., extending half-life (e.g., serum half-life), and increasing the duration of pharmacodynamic activity. , as well as a decrease in proteolysis during the production of recombinant enzymes. The present invention identifies, for example, D1L3 residues that are sensitive to proteolysis by plasmin, thrombin and/or trypsin, as well as residues (eg, paired basic amino acids) that are sensitive to proteases produced by mammalian and other cell lines. Engineered mutation of these residues may provide these advantages in protease resistance.

In other aspects of the invention, a method is provided for the recombinant production of extracellular DNase proteins, including variants thereof described herein. In some embodiments, the method uses a non-mammalian expression system, such as a non-mammalian eukaryotic expression system such as Pichia pastoris. In some embodiments, Pichia pastoris encodes a DNase enzyme with its native signal

- 2 046216 peptide that ensures 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 recombinant expression system has a deletion or inactivation of one or more proteases that cleave paired basic amino acids. Examples of enzymes include furin (expressed by CHO cells) and aspartic proteinase 3 (Ysp1) and kexin (Kex2) expressed by Pichia pastoris. In some embodiments, these enzymes are not genetically deleted or inactivated, but their activity is inhibited by a protease inhibitor during production of the recombinant protein.

In some embodiments, the non-mammalian or mammalian expression system cell growth medium is supplemented with polyanions such as dextran sulfate, heparins, ferric citrate, and EDTA. In further embodiments, the growth medium for Pichia pastoris or other expression system is supplemented with dextran sulfate, which has an average molecular weight of 5 to 100 kDa. For example, the polyanion can be added to the culture in an amount sufficient to form a complex with the recombinant protein being produced. In some embodiments, recombinant extracellular DNase proteins and variants thereof from the culture medium of a non-mammalian expression system or mammalian expression system are purified by a method that includes dissociating the recombinant extracellular proteins and DNase variants from polyanions such as dextran sulfate, heparins, and EDTA.

Other aspects of the invention provide isolated polynucleotides encoding D1, D1L1, D1L2, or D1L3 variants, as well as vectors and host cells. Polynucleotides can code for mRNA or DNA. The host cells may be cells of the recombinant expression system, including bacterial or eukaryotic, whether non-mammalian, such as Pichia pastoris, or mammalian, such as CHO cells. In other embodiments, the host cell may be delivered for DNase therapy. For example, some embodiments of the invention provide host cells, such as human cells, such as white blood cells, modified to secrete one or more extracellular DNase proteins described herein for administration as a drug.

The invention also provides pharmaceutical compositions comprising an extracellular DNase protein or variant thereof, as described herein, or, optionally, a polynucleotide or vector, as described, and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated for any route of administration.

In other aspects of the invention, there is provided a method of treating a subject in need of extracellular DNA degradation, extracellular chromatin degradation, extracellular trap (ET) degradation, and/or neutrophil extracellular trap (NET) degradation by administering a therapeutically effective amount of an extracellular DNase or a variant or composition thereof described in this document.

Other aspects and embodiments of the present invention will become apparent from the following detailed description.

Description of graphic materials

In fig. 1 shows that the mutations Q101R and Q282_S305delinsK in SEQ ID NO: 4 increase the activity of DNase1E3 for the degradation of high molecular weight chromatin. CHO cells were transiently transfected with DNase1E3 or wild-type DNase1b3 with building block substitutions. Supernatants of transfected cells were incubated with purified nuclei (high molecular weight chromatin) or buffer. DNA was isolated and analyzed by agarose gel electrophoresis. The figure shows an agarose gel stained with a dye to detect DNA.

In fig. Figure 2 shows the characteristics of two variants of DNase1b3. Various concentrations of supernatants from CHO cells transfected with wild-type DNaseGE3 or DNase1E3 with the Q101R or Q282_S305delinsK mutation were analyzed by Western blot (WB) analysis using anti-DNase1b3 antibodies. Wider (variant 1) and thinner (variant 2) bands were detected in samples with wild-type DNase1b3 and the Q101R mutant. In samples with the Q282_S305delinsK mutant, only a thinner band was detected (variant 2). In parallel, chromatin degradation activity was analyzed at different supernatant concentrations. The figure shows DNA analyzed by agarose gel electrophoresis. Both mutations, Q101R or Q282_S305delinsK, increased chromatin degradation activity compared to wild-type DNase1b3.

In fig. Figure 3 shows the presence of DNase1b3 variants 1 and 2 in the supernatants of CHO cells that were stably transfected with wild-type DNase1b3. Samples were analyzed by Western blot (WB) analysis using anti-DNase1b3 antibodies. Wider (variant 1) and thinner (variant 2) bands were found in 5 clones.

In fig. 4 shows the C-terminal amino acid sequences of recombinantly expressed wild-type D1L3 in Pichia pastoris to identify common cleavage sites. Amino acid sequencing of purified wild-type D1L3 reveals three deletion mutants

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C-terminal: K291_S305del, K292_S305del and S293_S305del. The C terminus of wild-type D1L3 was not detected. In parallel, chromatin degradation activity was analyzed at different concentrations of purified protein and compared with purified DNase1 (D1) and DNase1L3 with the basic domain deleted (BDD-D1L3) with the mutation F275Y/F279_K280delinsVM/Q282_S305delinsK. The figure shows DNA analyzed by agarose gel electrophoresis.

In fig. Figure 5 shows that the addition of dextran sulfate to CHO growth medium improves protein yield. Stable pools of CHO cells expressing wild-type D1L3 were incubated in standard CHO growth medium or CHO growth medium supplemented with dextran sulfate. The supernatant was analyzed by Western blot (WB) using anti-DNase1b3 antibodies. The figure shows that D1L3 is poorly expressed in CHO cells with low protein yield. The addition of dextran sulfate increases the yield but does not prevent product fragmentation.

In fig. Figure 6 shows the use of an anion exchange surface and a cation exchange surface for the affinity purification of D1L3 complex with dextran sulfate.

In fig. 7 lists strategies for mutation of the trypsin cleavage site to limit D1L3 degradation.

In fig. 8 shows a comparison of the amino acid sequences of human D1 (SEQ ID NO: 1) and human D1L3 (SEQ ID NO: 4) with the plasmin-sensitive KR residues shown.

In fig. Figure 9 shows strategies for mutating the plasmin cleavage site to limit D1L3 degradation.

In fig. Figure 10 shows that D1L3 with mutated plasmin cleavage sites retains enzymatic activity. Supernatants from cells that had been transiently transfected with DNase1b3 containing mutations at four putative plasmin cleavage sites (K180_A181delinsGL, P198_A201delinsRPSQ, K259A, R285A) were incubated with purified nuclei (high molecular weight chromatin) or buffer. DNA was isolated and analyzed by agarose gel electrophoresis. The figure shows an agarose gel stained with a dye to detect DNA.

In fig. 11 lists plasmin cleavage sites based on their plasmin cleavage and shows mutation strategies to limit D1L3 degradation.

In fig. 12A,B shows that D1L3 is prone to misfolding when expressed in CHO cells. In fig. 12A shows a simple expression vector for expressing D1L3 using a natural secretory signal peptide. Supernatants from stable pools were analyzed by Western blot analysis using an anti-DNase1b3 antibody. and in fig. 12B shows the presence of high molecular weight aggregates under non-reducing conditions, which are separated under reducing conditions.

In fig. 13 shows a comparison of the amino acid sequences of human D1 (SEQ ID NO: 1) and human D1L3 (SEQ ID NO: 4) with conserved and non-conserved cysteine residues shown.

In fig. 14 lists the cysteine residues in D1L3 and shows mutation strategies to limit the formation of high molecular weight aggregates during protein expression.

In fig. 15 shows that the C68A and C194A mutations in D1L3 do not affect chromatin degradation activity. Mutations C24A and C52A suppressed chromatin degradation activity. Supernatants from cells that had been transiently transfected with mutated variants of DNAseL3 were incubated with purified nuclei or buffer. DNA was isolated and analyzed by agarose gel electrophoresis. The figure shows an agarose gel stained with a dye to detect DNA.

In fig. 16A shows the expression of D1L3 in Pichia pastoris using either the native secretory signal or α-mating factor from Saccharomyces cerevisiae (aMF). The aMF secretory signal resulted in glycosylation and unprocessing of the signal peptide (Fig. 16B).

In fig. 17A shows a fusion construct of aMF, human serum albumin (HSA), linker sequence and D1L3. The hybrid construct was not susceptible to glycosylation in the P. pastoris expression system (Fig. 17B) and retained chromatin degradation activity (Fig. 17C).

In fig. 18 shows the expression levels of human serum albumin (HSA) fusion constructs with basic domain deleted DNase3 (BDD-D1L3) or wild-type DNase33 (D1L3) in Pichia pastoris. HSA is fused to either the N- or C-terminus of BDD-D1L3 or D1L3. Two linker sequences, L1 and L2, were placed between HSA and BDD-D1L3 or D1L3.

In fig. 19 shows the expression levels of wild-type human serum albumin (HSA) DNaseIII (D1L3) fusion constructs in Pichia pastoris. HSA is fused to the N-terminus of D1L3. Three different linker sequences (L2, L3, L4) were placed between HSA and D1L3.

In fig. 20 shows the expression levels and chromatin degradation activity of human serum albumin (HSA) DNASE1L3 core domain fusion constructs (BDD-D1L3) produced in Pichia pastoris. HSA is fused to the N-terminus of BDD-D1L3. Three different linker sequences (L5, L6, L7) were placed between HSA and D1L3.

In fig. 21A shows that Dnase1 ^-/- Dnase1l3 ^-/- mice injected with SED ID NO: 14 and SEQ ID NO: 19 exhibited similar serum chromatin degradation activity.

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In fig. 21B shows that the half-life of SEQ ID NO: 19 in the circulation of mice expressing the human FcRn receptor is 3.3 days.

In fig. 22 shows the expression levels and chromatin degradation activity of human serum albumin (HSA) DNaseH3 basic domain fusion constructs (BDD-D1L3) produced in Pichia pastoris. HSA is fused to the N- and C-terminus of BDD-D1L3. Two different linker sequences (L7 and L8) were placed between HSA and BDD-D1L3.

In fig. 23 shows a hybrid construct of HSA and a factor XIIa cleavable linker. Shown are the sequences of a linker containing the sequence of human factor XI (SEQ ID NO: 42) and a linker containing the sequence of human prekallekrein (SEQ ID NO: 44).

In fig. 24 shows other designs that use factor XIIa cleavable linkers for extended half-life fusion proteins, including human extracellular DNases, human coagulation factors, and human complement factors.

Description of the invention

This invention provides candidates for the design of extracellular human DNase proteins (for example, variants of DNase 1 (D1), DNase1-like protein 1 (D1L1), DNase1-like protein 2 (D1L2), DNase1-like protein 3 isoform 1 (D1L3), DNase1-like protein 3 isoform 2 (D1L3-2), DNase 2A (D2A) and DNase 2B (D2B)), which can be used to treat pathological conditions characterized by the accumulation and/or release of extracellular DNA, extracellular chromatin and neutrophil extracellular trap ( NET). In accordance with aspects of the invention, the DNase variants described herein are more useful/or effective for therapy and/or more suitable for large-scale production. In some embodiments, the DNase variants described herein are advantageous for systemic therapy. Such benefits include longer duration of action (eg, slower clearance, longer circulation half-life), increased duration of pharmacodynamic action, improved chromatin degradation activity, and resistance to proteases.

Definitions

The use of the singular form in this specification and in the claims includes reference to the singular and plural unless the context clearly indicates otherwise. Thus, for example, the term agent includes both a single agent and a plurality of such agents.

The term chromatinase refers to a class of deoxyribonuclease enzymes that exhibit more than minor ability to cut, degrade or recycle chromatin, i.e. DNA bound to one or more histone proteins. Human DNAse1b3 is a chromatinase. Usually different variants of the DNAse1b3 protein. described herein are chromatinases. Not all DNase enzymes are chromatinases. For example, human DNase1 has virtually no ability to cut, degrade, or recycle chromatin and is not a chromatinase.

As used herein, the term drug half-life refers to the half-life of the drug concentration in an animal measured in a matrix of interest, such as serum or plasma. One skilled in the art will appreciate that not all drugs exhibit first order kinetics or exhibit them at all stages of elimination. In such cases, one skilled in the art will understand that the terms extended half-life or extended half-life are expressions that refer to a slower rate of elimination.

Separated means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living being is not isolated, but the same nucleic acid or peptide, partially or completely separated from coexisting materials in its natural state, will be isolated. The isolated nucleic acid or protein may exist in substantially purified form or may exist in a foreign environment, such as, for example, a host cell.

As used herein, the term neutrophil extracellular trap and the abbreviation NET refer to a network of extracellular fibers containing nuclear contents, such as DNA, associated with histone proteins, which are released from an immune cell, usually a neutrophil, in a programmed manner.

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

The terms about and about include an amount that is ±10% of the

- 5 046216 corresponding numeric value.

The term extracellular DNase refers to the extracellular DNase proteins of the DNase 1 and DNase 2 families (e.g., DNase 1 (D1), DNase1-like protein 1 (D1L1), DNase1-like protein 2 (D1L2), DNase1-like protein 3 isoform 1 (D1L3 ), DNase1-like protein 3 isoform 2 (D1L3-2), DNase 2A (D2A), and DNase 2B (D2B)).

In some aspects and embodiments, the extracellular DNase or a variant thereof is fused, optionally through an inserted linker, with a half-life extending moiety such as albumin, transferrin, Fc or elastin-like protein, or a variant thereof. See, for example, US Pat. No. 9,458,218, which is incorporated herein by reference in its entirety. In some embodiments, the extracellular DNase or a variant thereof dimerizes at the hinge region of the immunoglobulin. For example, the engineered enzymes described herein may also include a fusion to an Fc domain (eg, the hinge domain and the CH2 and CH3 domains of an immunoglobulin). In some embodiments, the DNase (eg, D1L3 variant) is fused to an albumin amino acid sequence or domain, such as human albumin or a fragment or variant thereof. See, for example, WO 2015/066550 and US Patent No. 9221896, which are incorporated herein by reference in their entirety. Albumin can be attached to the DNase, optionally by an inserted linker, at the N-terminus and/or C-terminus of the engineered extracellular DNase or variant thereof. The exemplary amino acid sequence of albumin is shown as SEQ ID NO: 39. In some embodiments, D1L3 and D1 or variants described herein are dimerized together by the Fc hinge region, creating a dimeric molecule with synergistic functionality for NET degradation. In some embodiments, the extracellular DNase or a variant thereof is fused at the N-terminus to the amino acid sequence of albumin through a peptide linker. The peptide linker can be a flexible linker, a rigid linker, or in some embodiments, a physiologically cleavable linker (eg, a protease cleavable linker). In some embodiments, the linker has a length of from 5 to 100 amino acids, or from 5 to 50 amino acids. In other embodiments, the linker is an organic molecule, moiety, polymer (eg, PEG), or chemical moiety that is covalently linked to an extracellular DNase and a half-life extending moiety (eg, albumin).

In some aspects, the invention provides a D1L3 variant, wherein the D1L3 variant has one or more of the following: increased protein stability, slower drug clearance and increased duration of pharmacodynamic activity, resistance to proteolytic degradation, higher production levels in in vitro expression systems, better suitability for purification, but not significantly less, equal or better chromatin and/or NET degradation activity compared to wild type D1L3 isoform 1 of SEQ ID NO:4 or wild type D1L3 isoform 2 of SEQ ID NO:5 . As used herein, unless otherwise indicated, the term D1L3 includes both isoform 1 and isoform 2.

The activity of an enzyme to degrade DNA and/or chromatin and/or NETs, for example the D1L3 variant, can be measured in vitro, for example by incubating the enzyme with DNA, chromatin or NETs obtained, for example, from purified nuclei, DNA or blood ex vivo or neutrophils induced to form NETs. Alternatively, the activity of an enzyme to degrade DNA and/or chromatin and/or NETs, for example, the D1L3 variant, can be measured in vivo, for example, by administering the enzyme to a subject, wherein the subject produces or induces the production of extracellular DNA, chromatin or NETs and measuring the effect of the enzyme on concentrations of DNA, chromatin, or NET levels in a matrix, such as serum, preferably with a parallel negative control, or by temporally comparing concentrations before and after enzyme administration.

In some embodiments, the D1L3 variant has approximately the same chromatin and/or NET degradation activity as wild-type D1L3 isoform 1 SEQ ID NO: 4 or wild-type D1L3 isoform 2 SEQ ID NO: 5. In some embodiments embodiment, the D1L3 variant has higher chromatin and/or NET degradation activity compared to wild-type D1L3 isoform 1 SEQ ID NO: 4 or wild-type D1L3 isoform 2 SEQ ID NO: 5.

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 on the N-terminal side of the D1L3 domain. In some embodiments, the albumin domain and optional linker are located on the C-terminal side of the D1L3 domain. In all such embodiments, the optional linker is located between the albumin domain and the D1L3 domain.

In some embodiments, the amino acid sequence or domain of the albumin 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 sequence

- 6 046216 albumin, as defined by SEQ ID NO: 39. In some embodiments, the amino acid sequence or domain of albumin contains or consists of a reference albumin sequence, as defined by SEQ ID NO: 39. In various embodiments, the amino acid sequence of albumin binds to the neonatal Fc receptor ( FcRn), for example human FcRn. The amino acid sequence of albumin may be a wild type HSA variant (eg, represented by SEQ ID NO: 39). In various embodiments, the albumin variants may have from one to twenty or one to ten 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 albumin amino acid sequence mammal.

In some embodiments, the amino acid sequence or domain of albumin is a fragment of full-length albumin represented by SEQ ID NO: 39. The term fragment, when used in the context of albumin, refers to any fragment of full-length albumin or variant thereof (as described above) that prolongs the half-life the DNase enzyme to which it is fused or conjugated, relative to the corresponding unfused DNase. In some embodiments, an albumin fragment may refer to an amino acid sequence comprising a fusion of several albumin domains (see, for example, WO 2011/124718), such as domains I and III, and domains II and III. Typically, the albumin fragment contains at least about 100 amino acids, or at least about 200 or at least about 300 amino acids of full-length sequence. In various embodiments, the albumin moiety retains the ability to bind human FcRn.

In some embodiments, the D1L3-like domain of the fusion protein is at least about 85%, or at least about 90%, or at least about 95%, at least about 97%, or at least about 98%, or at least about 99% identical to the reference sequence of the mature D1L3 enzyme defined by SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the D1L3 domain contains 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 contain the C-terminal basic domain of SEQ ID NO: 4 or 5 defined by the C-terminal 23 amino acids.

In some embodiments, the fusion protein comprises a D1L3 domain, wherein the amino acid sequence of the D1L3 domain is at least about 80% identical to the mature enzyme defined by SEQ ID NO: 4 or SEQ ID NO: 5. The fusion protein may further comprise an amino acid sequence or domain of albumin The N-terminus of the mature enzyme and the linking amino acid sequence between the amino acid sequence of albumin and the amino acid sequence of the mature enzyme. In some embodiments, the D1L3 domain contains 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 contain a C-terminal core domain SEQ ID NO: 4 or 5, defined by the C-terminal 23 amino acids. The D1L3 domain-containing fusion protein exhibits improved circulating half-life and duration of pharmacodynamic effect, including for systemic therapy. In addition, the fusion of albumin to the binding sequence does not significantly affect (or in some embodiments does not have any detrimental effect on) chromatin degradation activity, as determined by an in vitro chromatin degradation assay, compared to a variant without the albumin fusion .

Sequence identity is implied to be with wild-type DNase enzymes, and unless otherwise stated, refers to mature enzymes lacking the signal peptide. In addition, unless otherwise noted, for clarity, amino acid positions are numbered relative to the fully translated DNase sequence, including the signal peptide. Accordingly, for example, reference to the sequence identity of the enzyme SEQ ID NO: 4 (human D1L3, isoform 1) refers to the percentage identity with the mature enzyme having M21 at the N-terminus. Similarly, reference to the sequence identity of the enzyme SEQ ID NO: 1 (human D1) refers to the percentage identity with the mature enzyme having L23 at the N-terminus.

In some embodiments, D1L3 has a deletion of all or part of the C-terminal core domain. The C-terminal basic domain is defined as the C-terminal 23 amino acids of SEQ ID NO: 4 or SEQ ID NO: 5. Deletion or inactivation of the C-terminal basic domain of D1L3 significantly improves chromatin degradation activity. See fig. 1, 2, and 4. In some embodiments, the D1L3 variant has a deletion of the C-terminal amino acids of the core domain, 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 of the C-terminal basic domain. In some embodiments, the D1L3 variant has a deletion of the entire C-terminal core domain defined by 23 C-terminal

- 7,046,216 amino acids SEQ ID NO: 4 or SEQ ID NO: 5. Examples of 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) contains 1 to 10 or 1 to 5 amino acids at the C terminus that are not the same as the C terminus of the BD and do not adversely affect chromatin degradation activity in the in vitro.

In some embodiments, the D1L3 variant is an engineered fusion protein comprising: a DNAL3 domain of a sequence selected from SEQ ID NO: 8 SEQ ID NO: 16; a linker sequence selected from SEQ ID NO: 31 - SEQ ID NO: 38; and an albumin domain having the sequence of SEQ ID NO: 39 or variants or fragment as described. In some embodiments, embodiment D1L3 has one or more replacements with D1 building blocks that are described in PCT/US2018/04708, which is incorporated herein by reference.

In some embodiments, the D1L3 sequence or domain contains a replacement with a D1 building block, which may be selected from one or more of: M21_R22delinsLK, C24_S25delinsAA, V28_S30delinsIQT, S34T, Q36_V44delinsMSNATLVSY, K47_K50delinsQILS, C52Y, I55_M58delinsIALVQE, I60 _K61delinsVR, N64_I70delinsHLTAVGK, M72_K74delinsLDN, R77_I83delinsQDAPD, N86H, I89V, S91_R92delinsEP, T97S, Q101R, A103L, L105V,

K107_L110delinsRPDQ, V113_R115delinsAVD, H118Y, H120D, Y122_A127delinsGCEPCGN, V129T, S131N, F135_V136delinsAI, W138R, Q140_H143delinFSRF, A145_D148delinsEVRE, V150A, I1 52V, T156_T157delinsAA, E159_S161delinsGDA, K163A, E167A, V169_E170delinsYD, T173L,

K176_R178delinsQEK, K180_A181delinsGL, N183_F186delinsDVML, P198_A201delinsRPSQ, K203_N204delinsSS, R208W, D210S, R212T, V214Q, G218P, Q220_E221delinsSA, V225_S228delins ATP, N230H, L238_R239delinsVA, Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256N, K259A, K262G, T264_E267delinsSDQL, L269_V271delinsQAI, F275Y, F279_K280delinsVM, Q2 82_S205delinsK, wherein each of the above substitutions is numbered according to SEQ ID NO: 4.

For example, the D1L3 variant may have a substitution with the D1 building block Q282_S305delinsK, which involves the removal of the C-terminal core domain that is missing in D1. In some embodiments, the D1L3 enzyme has an amino acid substitution at a position corresponding to position 101 of SEQ ID NO: 4. The substitution may be Arg based on the corresponding building block from D1 or in some embodiments may be Lys. Substitutions at this position may enhance the chromatin degradation activity of D1L3. Other replacements in this position are likely to have similar properties.

Linkers, if present, can be selected from flexible, rigid and cleavable peptide linkers. Flexible linkers consist predominantly or entirely of small, non-polar or polar amino acid residues such as Gly, Ser and Thr. A typical flexible linker comprises (Gly _y Ser) _n linkers, where y is a number from 1 to 10 (e.g., 1 to 5) and n is a number from 1 to about 10, and in some embodiments, a number from 3 to about 6 In exemplary embodiments, y is a number from 2 to 4 and n is a number from 3 to 8. Because of their flexibility, these linkers are unstructured. More rigid linkers include polyproline or poly Pro-Ala motifs and α-helical linkers. A typical example of an α-helical linker is A(EAAAK) _nA , where n is as defined above (eg, 1 to 10 or 2 to 6). Typically, linkers may consist predominantly of amino acids selected from Gly, Ser, Thr, Ala and Pro. Typical linker sequences contain at least 10 amino acids and can range from 15 to 35 amino acids. Exemplary linker designs are provided as SEQ ID NO: 31-38.

In some embodiments, the variant comprises a linker, wherein the amino acid sequence of the linker consists primarily of glycine and serine residues or consists primarily of glycine and serine residues. In some embodiments, the ratio of Ser to Gly in the linker is, respectively, from about 1:1 to about 1:10, from about 1:2 to about 1:6, or about 1:4. Typical linker sequences include S(GGS)4GSS (SEQ ID NO: 36), S(GGS)9GS (SEQ ID NO: 37), (GGS) _9GS (SEQ ID NO: 39). In some embodiments, the linker contains 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 be between 15 and 30 amino acids in length. In various embodiments, longer linkers containing at least 15 amino acids may provide improved productivity when expressed in Pichia pastoris. See fig. 20. Additionally, and unexpectedly, longer linker sequences showed improved chromatin degradation activity compared to shorter linker sequences. See fig. 20.

In various embodiments, the D1L3 variant is a fusion protein comprising the amino acid sequence of any of SEQ ID NO: 17-30. In other embodiments, the D1L3 variant is a fusion protein comprising the amino acid sequence of any of SEQ ID NO: 17-30 and having from one to twenty, or one to ten, or one to five amino acid modifications independently selected from amino acid insertions, deletions or substitutions relative to the reference sequence selected from SEQ ID NO: 17-30. In some countries

- 8 046216 ants implementation amino acid modifications are in the D1L3 domain, the albumin domain or both domains of the fusion protein. In some embodiments, the variant has the amino acid sequence of SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, or SEQ ID NO: 30. In these embodiments, the amino acid sequence of albumin is fused to the N-terminus or N-terminal side of D1L3 (or variant) via a medium or long flexible linker.

In other embodiments, the linker is a physiologically cleavable linker, such as a protease cleavable linker. For example, the protease may be a coagulation system protease such as activated factor XII. In some 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 portions of SEQ ID NO: 42 (eg, portions of SEQ ID NO: 42, including modifications of SEQ ID NO: 42 that are cleavable by Factor XIIa). In some embodiments, the linker amino acid sequence from prekallikrein comprises all or portions of SEQ ID NO: 44 (e.g., portions of SEQ ID NO: 44, including modifications of SEQ ID NO: 44 that are cleavable by factor XIIa). In other embodiments, the linker comprises a peptide sequence that is cleaved by a neutrophil-specific protease, such as neutrophil elastase, cathepsin G, and proteinase 3.

Some exemplary embodiments of D1L3 fusion proteins comprise a combination of three amino acid sequences that may be independently selected from the sequences disclosed herein, and such sequences, in order from N-terminus to C-terminus:

Merge 1: SEQ ID NO: 4, SEQ ID NO: 31, SEQ ID NO:39;

Merger 2: SEQ ID NO: 5, SEQ ID NO: 31, SEQ ID NO:39;

Merger 3: SEQ ID NO: 8, SEQ ID NO: 31, SEQ ID NO:39;

Merge 4: SEQ ID NO: 9, SEQ ID NO: 31, SEQ ID NO:39;

Merge 5: SEQ ID NO: 10, SEQ ID NO: 31, SEQ ID NO:39;

Merge 6: SEQ ID NO: 11, SEQ ID NO: 31, SEQ ID NO:39;

Merge 7: SEQ ID NO: 12, SEQ ID NO: 31, SEQ ID NO:39;

Merge 8: SEQ ID NO: 13, SEQ ID NO: 31, SEQ ID NO:39;

Merge 9: SEQ ID NO: 14, SEQ ID NO: 31, SEQ ID NO:39;

Merge 10: SEQ ID NO: 15, SEQ ID NO: 31, SEQ ID NO: 39;

Merge 11: SEQ ID NO: 16, SEQ ID NO: 31, SEQ ID NO: 39;

Merge 12: SEQ ID NO: 4, SEQ ID NO: 32, SEQ ID NO: 39;

Merge 13: SEQ ID NO: 5, SEQ ID NO: 32, SEQ ID NO: 39;

Merge 14: SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 39;

Merge 15: SEQ ID NO: 9, SEQ ID NO: 32, SEQ ID NO: 39;

Merge 16: SEQ ID NO: 10, SEQ ID NO: 32, SEQ ID NO: 39;

Merge 17: SEQ ID NO: 11, SEQ ID NO: 32, SEQ ID NO: 39;

Merge 18: SEQ ID NO: 12, SEQ ID NO: 32, SEQ ID NO: 39;

Merge 19: SEQ ID NO: 13, SEQ ID NO: 32, SEQ ID NO: 39;

Merge 20: SEQ ID NO: 14, SEQ ID NO: 32, SEQ ID NO: 39;

Merge 21: SEQ ID NO: 15, SEQ ID NO: 32, SEQ ID NO: 39;

Merge 22: SEQ ID NO: 16, SEQ ID NO: 32, SEQ ID NO: 39;

Merge 23: SEQ ID NO: 4, SEQ ID NO: 33, SEQ ID NO: 39;

Merge 24: SEQ ID NO: 5, SEQ ID NO: 33, SEQ ID NO: 39;

Merge 25: SEQ ID NO: 8, SEQ ID NO: 33, SEQ ID NO: 39;

Merge 26: SEQ ID NO: 9, SEQ ID NO: 33, SEQ ID NO: 39;

Merge 27: SEQ ID NO: 10, SEQ ID NO: 33, SEQ ID NO: 39;

Merge 28: SEQ ID NO: 11, SEQ ID NO: 33, SEQ ID NO: 39;

Merge 29: SEQ ID NO: 12, SEQ ID NO: 33, SEQ ID NO: 39;

Merge 30: SEQ ID NO: 13, SEQ ID NO: 33, SEQ ID NO: 39;

Merger 31: SEQ ID NO: 14, SEQ ID NO: 33, SEQ ID NO: 39;

Merge 32: SEQ ID NO: 15, SEQ ID NO: 33, SEQ ID NO: 39;

Merge 33: SEQ ID NO: 16, SEQ ID NO: 33, SEQ ID NO: 39;

Merge 34: SEQ ID NO: 4, SEQ ID NO: 34, SEQ ID NO: 39;

Merge 35: SEQ ID NO: 5, SEQ ID NO: 34, SEQ ID NO: 39;

Merge 36: SEQ ID NO: 8, SEQ ID NO: 34, SEQ ID NO: 39;

Merge 37: SEQ ID NO: 9, SEQ ID NO: 34, SEQ ID NO: 39;

Merge 38: SEQ ID NO: 10, SEQ ID NO: 34, SEQ ID NO: 39;

Merge 39: SEQ ID NO: 11, SEQ ID NO: 34, SEQ ID NO: 39;

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Merge 40 SEQ ID NO 12, SEQ ID NO: 34, SEQ ID NO: 39; Merge 41 SEQ ID NO 13, SEQ ID NO: 34, SEQ ID NO: 39; Merge 42 SEQ ID NO 14, SEQ ID NO: 34, SEQ ID NO: 39; Merge 43 SEQ ID NO 15, SEQ ID NO: 34, SEQ ID NO: 39; Merge 44 SEQ ID NO 16, SEQ ID NO: 34, SEQ ID NO: 39; Merge 45 SEQ ID NO 4, SEQ ID NO: 35, SEQ ID NO: 39; Merge 46 SEQ ID NO 5, SEQ ID NO: 35, SEQ ID NO: 39; Merge 47 SEQ ID NO 8, SEQ ID NO: 35, SEQ ID NO: 39; Merge 48 SEQ ID NO 9, SEQ ID NO: 35, SEQ ID NO: 39; Merge 49 SEQ ID NO 10, SEQ ID NO: 35, SEQ ID NO: 39; Merge 50 SEQ ID NO 11, SEQ ID NO: 35, SEQ ID NO: 39; Merge 51 SEQ ID NO 12, SEQ ID NO: 35, SEQ ID NO: 39; Merge 52 SEQ ID NO 13, SEQ ID NO: 35, SEQ ID NO: 39; Merge 53 SEQ ID NO 14, SEQ ID NO: 35, SEQ ID NO: 39; Merge 54 SEQ ID NO 15, SEQ ID NO: 35, SEQ ID NO: 39; Merge 55 SEQ ID NO 16, SEQ ID NO: 35, SEQ ID NO: 39; Merge 56 SEQ ID NO 4, SEQ ID NO: 36, SEQ ID NO: 39; Merge 57 SEQ ID NO 5, SEQ ID NO: 36, SEQ ID NO: 39; Merge 58 SEQ ID NO 8, SEQ ID NO: 36, SEQ ID NO: 39; Merge 59 SEQ ID NO 9, SEQ ID NO: 36, SEQ ID NO: 39; Merge 60 SEQ ID NO 10, SEQ ID NO: 36, SEQ ID NO: 39; Merge 61 SEQ ID NO 11, SEQ ID NO: 36, SEQ ID NO: 39; Merge 62 SEQ ID NO 12, SEQ ID NO: 36, SEQ ID NO: 39; Merge 63 SEQ ID NO 13, SEQ ID NO: 36, SEQ ID NO: 39; Merge 64 SEQ ID NO 14, SEQ ID NO: 36, SEQ ID NO: 39; Merge 65 SEQ ID NO 15, SEQ ID NO: 36, SEQ ID NO: 39; Merge 66 SEQ ID NO 16, SEQ ID NO: 36, SEQ ID NO: 39; Merge 67 SEQ ID NO 4, SEQ ID NO: 37, SEQ ID NO: 39; Merge 68 SEQ ID NO 5, SEQ ID NO: 37, SEQ ID NO: 39; Merge 69 SEQ ID NO 8, SEQ ID NO: 37, SEQ ID NO: 39; Merge 70 SEQ ID NO 9, SEQ ID NO: 37, SEQ ID NO: 39; Merger 71 SEQ ID NO 10, SEQ ID NO: 37, SEQ ID NO: 39; Merge 72 SEQ ID NO 11, SEQ ID NO: 37, SEQ ID NO: 39; Merger 73 SEQ ID NO 12, SEQ ID NO: 37, SEQ ID NO: 39; Merge 74 SEQ ID NO 13, SEQ ID NO: 37, SEQ ID NO: 39; Merge 75 SEQ ID NO 14, SEQ ID NO: 37, SEQ ID NO: 39; Merge 76 SEQ ID NO 15, SEQ ID NO: 37, SEQ ID NO: 39; Merge 77 SEQ ID NO 16, SEQ ID NO: 37, SEQ ID NO: 39; Merger 78 SEQ ID NO 4, SEQ ID NO: 38, SEQ ID NO: 39; Merge 79 SEQ ID NO 5, SEQ ID NO: 38, SEQ ID NO: 39; Merge 80 SEQ ID NO 8, SEQ ID NO: 38, SEQ ID NO: 39; Merger 81 SEQ ID NO 9, SEQ ID NO: 38, SEQ ID NO: 39; Merger 82 SEQ ID NO 10, SEQ ID NO: 38, SEQ ID NO: 39; Merger 83 SEQ ID NO 11, SEQ ID NO: 38, SEQ ID NO: 39; Merger 84 SEQ ID NO 12, SEQ ID NO: 38, SEQ ID NO: 39; Merge 85 SEQ ID NO 13, SEQ ID NO: 38, SEQ ID NO: 39; Merger 86 SEQ ID NO 14, SEQ ID NO: 38, SEQ ID NO: 39; Merger 87 SEQ ID NO 15, SEQ ID NO: 38, SEQ ID NO: 39; Merger 88 SEQ ID NO 16, SEQ ID NO: 38, SEQ ID NO: 39; Merger 89 SEQ ID NO 4, SEQ ID NO: 42, SEQ ID NO: 39; Merge 90 SEQ ID NO 5, SEQ ID NO: 42, SEQ ID NO: 39; Merger 91 SEQ ID NO 8, SEQ ID NO: 42, SEQ ID NO: 39; Merger 92 SEQ ID NO 9, SEQ ID NO: 42, SEQ ID NO: 39; Merger 93 SEQ ID NO 10, SEQ ID NO: 42, SEQ ID NO: 39; Merger 94 SEQ ID NO 11, SEQ ID NO: 42, SEQ ID NO: 39; Merge 95 SEQ ID NO 12, SEQ ID NO: 42, SEQ ID NO: 39; Merger 96 SEQ ID NO 13, SEQ ID NO: 42, SEQ ID NO: 39; Merger 97 SEQ ID NO 14, SEQ ID NO: 42, SEQ ID NO: 39; Merger 98 SEQ ID NO 15, SEQ ID NO: 42, SEQ ID NO: 39; Merge 99 SEQ ID NO 16, SEQ ID NO: 42, SEQ ID NO: 39;

Merge 100: SEQ ID NO: 4, SEQ ID NO: 43, SEQ ID NO: 39;

Merge 101: SEQ ID NO: 5, SEQ ID NO: 43, SEQ ID NO: 39;

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Merger 102: SEQ ID NO: 8, SEQ ID NO: 43, SEQ ID NO: 39;

Merger 103: SEQ ID NO: 9, SEQ ID NO: 43, SEQ ID NO: 39;

Merger 104: SEQ ID NO: 10, SEQ ID NO: 43, SEQ ID NO: 39;

Merger 105: SEQ ID NO: 11, SEQ ID NO: 43, SEQ ID NO: 39;

Merger 106: SEQ ID NO: 12, SEQ ID NO: 43, SEQ ID NO: 39;

Merger 107: SEQ ID NO: 13, SEQ ID NO: 43, SEQ ID NO: 39;

Merger 108: SEQ ID NO: 14, SEQ ID NO: 43, SEQ ID NO: 39;

Merger 109: SEQ ID NO: 15, SEQ ID NO: 43, SEQ ID NO: 39;

Merger 110: SEQ ID NO: 16, SEQ ID NO: 43, SEQ ID NO: 39;

Merger 111: SEQ ID NO: 4, SEQ ID NO: 44, SEQ ID NO: 39;

Merger 112: SEQ ID NO: 5, SEQ ID NO: 44, SEQ ID NO: 39;

Merger 113: SEQ ID NO: 8, SEQ ID NO: 44, SEQ ID NO: 39;

Merger 114: SEQ ID NO: 9, SEQ ID NO: 44, SEQ ID NO: 39;

Merger 115: SEQ ID NO: 10, SEQ ID NO: 44, SEQ ID NO: 39;

Merger 116: SEQ ID NO: 11, SEQ ID NO: 44, SEQ ID NO: 39;

Merge 117: SEQ ID NO: 12 SEQ ID NO: 44, SEQ ID NO: 39;

Merger 118: SEQ ID NO: 13, SEQ ID NO: 44, SEQ ID NO: 39;

Merger 119: SEQ ID NO: 14, SEQ ID NO: 44, SEQ ID NO: 39;

Merger 120: SEQ ID NO: 15, SEQ ID NO: 44, SEQ ID NO: 39;

Merger 121: SEQ ID NO: 16, SEQ ID NO: 44, SEQ ID NO: 39;

Merger 122: SEQ ID NO: 4, SEQ ID NO: 45, SEQ ID NO: 39;

Merger 123: SEQ ID NO: 5, SEQ ID NO: 45, SEQ ID NO: 39;

Merge 124: SEQ ID NO: 8, SEQ ID NO: 45, SEQ ID NO: 39;

Merge 125: SEQ ID NO: 9, SEQ ID NO: 45, SEQ ID NO: 39;

Merger 126: SEQ ID NO: 10, SEQ ID NO: 45, SEQ ID NO: 39;

Merger 127: SEQ ID NO: 11, SEQ ID NO: 45, SEQ ID NO: 39;

Merger 128: SEQ ID NO: 12, SEQ ID NO: 45, SEQ ID NO: 39;

Merger 129: SEQ ID NO: 13, SEQ ID NO: 45, SEQ ID NO: 39;

Merger 130: SEQ ID NO: 14, SEQ ID NO: 45, SEQ ID NO: 39;

Merger 131: SEQ ID NO: 15, SEQ ID NO: 45, SEQ ID NO: 39;

Merger 132: SEQ ID NO: 16, SEQ ID NO: 45, SEQ ID NO: 39;

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 NO: 4-16 and SEQ ID NO: 44-46. In some embodiments, the fusion protein is a mature protein, i.e. without signal peptide.

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

Some aspects of the invention provide variants of extracellular DNase enzymes designed to have manufacturing advantages by providing the production of a recombinant enzyme suitable for use in therapy and which may optionally be used in connection with embodiments of a fusion protein (including embodiments of a fusion protein) , as already described. In various embodiments, the invention provides recombinant variants of D1, D1L1, D1L2 and D1L3 containing one or more amino acid substitutions or deletions at cysteine residues, resulting in reduced intra- and intermolecular cross-linking via disulfide bridges during protein expression. For example, a DNase variant may lack one, two, or three cysteine residues present in the wild-type sequence (eg, one, two, or three cysteine residues deleted), or may contain one or more such cysteine residues replaced by other amino acid(s). In some embodiments, one or more cysteine residues are replaced with an amino acid independently selected from Ala, Gly, and Ser, or one or more cysteine residues are replaced as part of a building block replacement. In some embodiments, the one or more substituted cysteine residues are not conserved between other members of the D1 protein family (eg, D1, D1L1, D1L2, and D1L3). In some embodiments, the engineered enzyme comprises or further comprises at least one building block substitution from another member of the D1 protein family and/or another point mutation that increases protein stability, resistance to protease degradation, bioavailability, and is 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 substitutions and/or modifications include, among other modifications, only a single modification of cysteine residues. In some embodiments, removal of a single cysteine residue is sufficient to provide significant manufacturing benefits.

In other aspects of the invention, variants of extracellular DNases designed

- 11 046216 so as to have advantages in protease resistance, to improve half-life in vivo, as well as to reduce proteolysis during the production of the recombinant enzyme. The present invention identifies, for example, D1L3 residues that are sensitive to proteolysis by plasmin, thrombin and/or trypsin, as well as residues (eg, paired basic amino acids) that are sensitive to proteases produced by mammalian and other cell lines.

The recombinant extracellular DNase variants described herein may have a combination of point mutations, including substitutions at cysteine residues, substitutions at protease-sensitive residues, and/or may contain one or more block-based substitutions. The construction of building blocks of proteins (BBPE) is described in PCT/US18/47084 and US 62/800790, the descriptions of which are incorporated herein by reference. BBPE involves ensuring protein-protein alignment of donor and recipient extracellular DNase enzymes and identifying variable amino acid sequences for transfer (building block). The variable amino acid(s) are flanked by one or more conserved amino acids in the donor and recipient extracellular DNase enzymes (upstream and downstream of the building block). These building blocks can be swapped between the recipient and donor proteins to produce a chimeric enzyme.

In other aspects of the invention, a method is provided for the recombinant production of extracellular DNase proteins, including variants thereof described herein. In some embodiments, the method uses a non-mammalian expression system such as Pichia pastoris. In certain embodiments, Pichia pastoris encodes a DNase enzyme with a native signal peptide, allowing 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, by removing cysteine residues unnecessary for activity, the invention avoids intermolecular and intramolecular disulfide bonds that would otherwise form and interfere with recombinant production. In some embodiments, a significant reduction in erroneous intermolecular and intramolecular disulfide bonds can be achieved by replacing a single cysteine residue.

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

In some embodiments, the non-mammalian or mammalian expression system cell growth medium is supplemented with polyanions such as dextran sulfate, heparins, ferric citrate, and EDTA. In further embodiments, the growth medium for Pichia pastoris or other expression system is supplemented with dextran sulfate, which has an average molecular weight of 5 to 100 kDa. In some embodiments, the dextran sulfate has an average molecular weight that is 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 form a complex with the recombinant protein being produced.

In some embodiments, recombinant extracellular DNase proteins and variants thereof from the culture medium of a non-mammalian expression system or mammalian expression system are purified by a method that includes dissociating the recombinant extracellular proteins and DNase variants from polyanions such as dextran sulfate, heparins, EDTA. In some embodiments, the purification method includes strong anion exchange resins such as triethylaminoethyl. In some embodiments, the extracellular DNase protein produced in accordance with the method is D1L3 or a variant thereof.

Accordingly, some embodiments of the invention provide a D1L3 variant comprising an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 4 (human D1L3 isoform 1) or SEQ ID NO: 5 (human D1L3 isoform 2 ), and having one or more cysteine residue substitutions and/or one or more amino acid substitutions that are susceptible to proteolysis, such as in vivo proteolysis. In some embodiments, the D1L3 protein variant contains one or more additional modifications that result in increased protein stability (e.g., protease resistance), higher levels of production in in vitro expression systems, and/or non-substantially less, equal, or better protein activity. DNA and/or chromatin and/or NET degradation compared to the wild-type D1L3 protein of SEQ ID NO: 4 or SEQ ID NO: 5. For example, the D1L3 variant may contain at least one additional building block substitution or point mutation described in PCT/US2018/47084 (which

- 12 046216 is incorporated herein by reference in its entirety) or may contain one or more of the substitutions described herein to improve protease resistance.

In some embodiments, the D1L3 variant has a Cys 68 substitution that is optionally substituted with an amino acid selected from Ala, Ser, and Gly. In some embodiments, the variant comprises the substitution N64_I70delinsHLTAVGK. In some embodiments, the HLTAVGK sequence may be further modified by one, two, or three substitutions, deletions, and/or insertions (together) as long as the Cys residue is not included. In some embodiments, variant D1L3 contains an amino acid sequence that is at least about 85%, at least about 90%, at least about 95%, or at least about 98% identical to reference SEQ ID NO:4 or SEQ ID NO: 5.

In some embodiments, the invention provides a D1L3 enzyme containing a polyethylene glycol (PEG) moiety conjugated at a position corresponding to Cys 68, which is believed to be an unpaired cysteine. In some embodiments, the D1L3 variant has a PEG conjugation to an amino acid corresponding to C194. In these embodiments, the PEG moiety will provide extended half-life while avoiding disulfide scrambling and/or protein misfolding. In some embodiments, the PEG moiety is conjugated using a maleimide chemical reaction, which can be performed under mild conditions. Other conjugation chemistries are known and can be used, such as the activation chemistries of vinyl sulfone, dithiopyridine and iodoacetamide. The PEG moiety can be linear or branched and can typically be in the range of 10 to 40 kDa or in the range of 20 to 30 kDa.

Alternatively or in addition to this, the invention provides a variant of D1L3 containing one or more substituted arginine and/or lysine residues resulting in increased resistance to proteases. In some embodiments, the D1L3 variant has a substitution at one or more positions corresponding to K180, K200, K259 and/or R285 in SEQ ID NO: 4. According to this disclosure, such lysine and arginine residues are identified as potential protease responsive sites. Thus, one or more (eg, 1, 2, 3 or 4) of these residues may be modified with an uncharged residue, such as a residue independently selected from Ala, Gly, Leu, Ile, Val, Thr, Ser and Pro. In some embodiments, protease-sensitive lysine or arginine residues are replaced as part of a building block replacement. For example, variant D1L3 may contain one or more substitutions selected from K180_A181delinsGL, P198_A201delinsRPSQ and K259A. In some embodiments, the D1L3 variant contains one or both of K180_A181delinsGL and/or P198_A201delinsRPSQ substitutions, each of which is optionally modified by one or two amino acid substitutions, deletions, or insertions, provided that the building block substitution is not modified by a substitution or insertion of an R or K residue. In some embodiments, the D1L3 variant has increased resistance to proteolysis by one or more proteases selected from plasmin, thrombin, and/or trypsin.

Alternatively or in addition, the D1L3 variant contains one or more base pair mutations. In some embodiments, the paired basic residue 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. In some embodiments, variant D1L3 contains one or more substitutions selected from the substitution corresponding to R114T, R114A, R114D, R114Q, K227S, and K227E of SEQ ID NO: 4. In some embodiments, the one or more base pair mutations include an amino acid replacement corresponding to R51K, R81K, R115K and R304K. In some embodiments, a pairwise basic residue is replaced using a corresponding building block replacement. In accordance with these embodiments, the D1L3 variant expressed by a recombinant protein expression system (eg, CHO and Pichia pastoris) will be more resistant to proteases.

In some aspects of the invention, a variant of DNase 1 (D1) is provided that contains an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 1, with one or more cysteine residue substitutions. In some embodiments, the D1 protein variant contains one or more additional modifications that result in increased protein stability, higher levels of production in in vitro expression systems, and/or non-substantially less, equal, or better DNA and/or chromatin degradation activity and /or NET compared to the wild type D1 protein with SEQ ID NO:1. For example, variant D1 may contain at least one additional building block substitution or point mutation described in PCT/US2018/47084, which is incorporated herein by reference in its entirety.

In some embodiments, variant D1 has a substitution of one or both of C123 and C126, which are optionally substituted with Ala, Ser, and Gly. In some embodiments, variant D1 contains the substitution G122_N128delinsYQGDA. In some embodiments, variant D1 contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:1.

- 13 046216

In some embodiments, the invention provides a D1 enzyme containing a PEG moiety conjugated at a position corresponding to C123 and/or C126. In these embodiments, the PEG moiety will provide extended half-life while avoiding disulfide scrambling and/or protein misfolding. In some embodiments, the PEG moiety is conjugated using a maleimide chemical reaction, which can be performed under mild conditions. Other conjugation chemistries are known and can be used, such as the activation chemistries of vinyl sulfone, dithiopyridine and iodoacetamide. The PEG moiety can be linear or branched and can typically be in the range of 10 to 40 kDa or in the range of 20 to 30 kDa.

In other aspects of the invention, a variant of D1L1 is provided that contains an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 2, with one or more cysteine residues substituted. The cysteine residue(s) are optionally non-conserved within the D1 family (eg, C22 and/or C50) and are optionally substituted with Gly, Arg or Ser or substituted as part of a building block replacement. In some embodiments, the D1L1 variant contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO: 2.

In some embodiments, the invention provides a D1L1 enzyme containing a PEG moiety conjugated at a position corresponding to C22 and/or C50. In these embodiments, the PEG moiety will provide extended half-life while avoiding disulfide scrambling and/or protein misfolding. In some embodiments, the PEG moiety is conjugated using a maleimide chemical reaction, which can be performed under mild conditions. Other conjugation chemistries are known and can be used, such as the activation chemistries of vinyl sulfone, dithiopyridine and iodoacetamide. The PEG moiety can be linear or branched and can typically be in the range of 10 to 40 kDa or in the range of 20 to 30 kDa.

In some aspects of the invention, a variant of D1L2 is provided that contains an amino acid sequence that is at least 80% identical to the enzyme defined by SEQ ID NO: 3, with one or more cysteine residues substituted. Cysteine residues may be non-conserved in the D1 family (eg, C43) and are optionally substituted with Gly, Arg or Ser or substituted as part of a building block replacement. In some embodiments, the D1L2 variant contains an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 98% identical to SEQ ID NO:3.

In some embodiments, the invention provides a D1L2 enzyme containing a PEG moiety conjugated at a position corresponding to C43. In these embodiments, the PEG moiety will provide extended half-life while avoiding disulfide scrambling and/or protein misfolding. In some embodiments, the PEG moiety is conjugated using a maleimide chemical reaction, which can be performed under mild conditions. Other conjugation chemistries are known and can be used, such as the activation chemistries of vinyl sulfone, dithiopyridine and iodoacetamide. The PEG moiety can be linear or branched and can typically be in the range of 10 to 40 kDa or in the range of 20 to 30 kDa.

Other aspects of the invention provide isolated polynucleotides encoding the D1, D1L1, D1L2, or D1L3 variants described herein, as well as vectors and host cells. Host cells can be cells of any expression system, including bacterial or eukaryotic, whether non-mammalian, such as Pichia pastoris, or mammalian, such as CHO cells.

In some embodiments, the delivery of polynucleotides is used for therapy. Coding polynucleotides can be delivered as mRNA or as DNA constructs using known procedures, such as electroporation or cell compression, and/or vectors (including viral vectors). The mRNA polynucleotides may include known modifications (mRNA) to avoid activation of the innate immune system. See WO 2014/028429, which is incorporated herein by reference in its entirety. In some embodiments, the polynucleotide is delivered to the body of a subject. In some embodiments, the polynucleotides are delivered to a cell in vitro and the cell is delivered to a subject. The cell may be, for example, a white blood cell (eg, a T cell or macrophage), an endothelial cell, an epithelial cell, a hepatocyte, or a stem cell.

In other aspects of the invention, a method for producing extracellular DNase variants described herein is provided. The method involves culturing cells expressing a polynucleotide encoding extracellular DNase and isolating recombinant DNase protein. Cells can be prokaryotic or eukaryotic. In some embodiments, the DNase is expressed using a non-mammalian expression system, which is optionally Pichia pastoris or Saccharomyces spp. In some variants

- 14 046216 implementation uses a mammalian expression system such as CHO cells.

The invention also provides pharmaceutical compositions comprising an extracellular DNase or variant thereof, as described herein, or, optionally, a polynucleotide or vector, as described, and a pharmaceutically acceptable carrier.

The vector typically 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, ionic or amphiphilic linked polynucleotides, plasmids, and viruses. Typical vectors include an autonomously replicating plasmid or virus. The term should also be taken to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compound, liposomes and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

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

In other aspects of the invention, a method is provided for treating a subject in need of extracellular DNA degradation, extracellular chromatin degradation, extracellular trap (ET) degradation, and/or neutrophil extracellular trap (NET) degradation. The method includes administering a therapeutically effective amount of extracellular DNase or a variant or composition thereof described herein. Examples of indications where a subject requires degradation of extracellular DNA or chromatin (including degradation of ET or NET) are disclosed in PCT/US18/47084, the disclosure of which is incorporated herein by reference. In some embodiments, the invention provides a method of treating a subject in need thereof, comprising administering a therapeutically effective amount of a protein that is represented by any of the sequences of SEQ ID NO: 8 to SEQ ID NO: 30.

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

In various embodiments, the present invention provides a method for treating, preventing, or controlling diseases or conditions characterized by the presence or accumulation of NETs. Such diseases or pathological conditions include, but are not limited to, diseases associated with chronic neutrophilia, neutrophil aggregation and leukostasis, thrombosis and vascular occlusion, ischemic reperfusion injury, surgical and traumatic tissue injury, acute or chronic inflammatory reaction or disease, autoimmune diseases, cardiovascular diseases, metabolic diseases, systemic inflammation, inflammatory diseases of the respiratory tract, inflammatory diseases of the kidneys, inflammatory diseases associated with transplanted tissue (eg, graft-versus-host disease) and cancer (including leukemia).

In some embodiments, the present invention relates to the treatment of diseases or conditions characterized by D1L3 deficiency or D1 deficiency. In some cases, the subject has a mutation (eg, a loss-of-function mutation) in the Dnase1l3 gene or the Dnase1 gene. Such subjects may exhibit an autoimmune disease (eg, 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, the subject has an acquired inhibitor of D1 (eg, anti-DNase 1 and actin) and/or D1L3 (eg, anti-DNase 1l3). Such subjects may also have an autoimmune or inflammatory disease (eg, SLE, systemic sclerosis).

In some embodiments, the subject has or is at risk for NET occlusion in the ductal system. For example, the DNase enzymes described herein can be administered to a subject to treat pancreatitis, cholangitis, conjunctivitis, mastitis, dry eye syndrome, vas deferens obstruction, and kidney disease.

In some embodiments, the subject has, or is at risk for, accumulation of NETs on endothelial surfaces (eg, adhesions caused by surgery), skin (eg, wounds/scars), or in synovial joints (eg, gout and arthritis, eg, rheumatoid arthritis). The DNase enzymes described herein can be administered to a subject to treat a pathological condition characterized by the accumulation of NETs on endothelial surfaces, such as, but not limited to, adhesions caused by surgery.

Other diseases and conditions associated with NETs that may be treated or prevented using the DNase enzymes described herein include

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ANCA-associated vasculitis, asthma, chronic obstructive pulmonary disease, neutrophilic dermatosis, dermatomyositis, burns, cellulitis, meningitis, encephalitis, otitis media, pharyngitis, tonsillitis, pneumonia, endocarditis, cystitis, pyelonephritis, appendicitis, cholecystitis, pancreatitis, uveitis, keratitis, disseminated intravascular coagulation, acute kidney injury, acute respiratory distress syndrome, liver shock, hepatorenal syndrome, myocardial infarction, stroke, intestinal ischemia, limb ischemia, testicular torsion, preeclampsia, eclampsia, and solid organ transplantation (eg, kidney, heart, liver transplantation and/or lungs). In addition, the DNase enzymes described herein can be used to prevent the formation of a scar or contracture, for example, by topical application to the skin of an individual at risk, such as a person with a surgical incision, tear or burn.

In various embodiments, the subject suffers from a disease that is or will be treated with wild-type DNases, including D1 and streptodornase. Similar diseases or conditions include thrombosis, stroke, sepsis, lung injury, atherosclerosis, viral infection, sickle cell disease, myocardial infarction, otitis media, wound healing, liver disease, endocarditis, liver infection, pancreatitis, primary graft dysfunction, ischemia /limb reperfusion, kidney damage, blood coagulation, alum-induced inflammation, hepatorenal injury, pleural exudations, hemothorax, intrahepatic thrombi, post-pneumatic anemia, ulcers, otolaryngological conditions, oral infections, minor injuries, sinusitis, postoperative rhinoplasty, infertility, urinary catheter, wound debridement, skin reaction test, pneumococcal meningitis, gout, leg ulcers, cystic fibrosis, Kartagener's syndrome, asthma, lobar atelectasis, chronic bronchitis, bronchiectasis, lupus, primary ciliary dyskinesia, bronchiolitis, epimyema, pleural infections, cancer, dry eye syndrome, infections lower respiratory tract, chronic hematomas, Alzheimer's disease and obstructive pulmonary disease.

Other aspects and embodiments of the present invention will be apparent from the following examples.

Examples

Almost 70% of all biologics are produced using Chinese hamster ovary (CHO) cells. Indeed, wild-type DNase 1 (D1; dornase alpha) is normally produced in CHO cells. Despite the significant advantages in cell line development and large-scale production using CHO cells, a major challenge still remains in the production of DNase enzymes due to a large degree of variability and the lack of reliable ways to predict or model cell growth characteristics. It is important to note that CHO cells were not able to consistently produce hyperactive D1 variants, preventing their clinical production, and prior to this disclosure, the production properties of other members of the DNase 1 protein family, including DNase 1-like protein 3 (D1L3), were unknown.

Using CHO and microbial expression systems, several problems have been identified in the production of D1L3, including low yield, proteolytic degradation, protein misfolding, and erroneous or unwanted glycosylation. This invention provides technical solutions to these and other manufacturing problems that may also improve the therapeutic properties of D1L3.

Example 1: Expression and characterization of basic domain deletion (BDD) D1L3 in Chinese hamster ovary (CHO) cells and Pichia pastoris.

DNase 1 and DNase 1b3 preferentially cleave DNA without protein and DNA-histone complexes (i.e., chromatin), respectively. Previous studies suggest that the basic domain (BD) at the C terminus of DNase1b3, which is absent in DNase1, is responsible for the different substrate specificities of both enzymes (Sisirak et al., Cell, 2016; Keyel, Developmental Biology, 2017).

A protein engineering technology called protein building block engineering is described in PCT/US18/47084 and US 62/800790, the disclosures of which are incorporated herein by reference in their entirety. This approach can be applied to members of the DNase 1 and DNase 2 protein families. The method is based on the following steps: ensuring protein-protein alignment of donor and recipient DNase enzymes; determining variable amino acid sequences for transfer, wherein the variable amino acids are flanked by one or more conserved amino acids in the donor and recipient DNase enzymes; replacing the variable amino acids of the recipient DNase with the variable amino acids of the donor DNase to create a chimeric DNase and recombinant production of the chimeric DNase.

To characterize the amino acids that are responsible for chromatin degradation activity (chromatinase activity), wild-type D1L3 was replaced with building blocks from D1, as described in PCT/US2018/047084. D1L3 building block substitutions are selected from human D1 and result in human D1L3 variants that have the following mutations: M21_R22delinsLK, C24_S25delinsAA, V28_S30delinsIQT, S34T, Q36_V44delinsMSNATLVSY, K47_K50delinsQILS, C52Y, I55_M58delinsIALVQE, I60_ K61delinsVR, N64_I70delinsHLTAVGK,

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M72_K74delinsLDN, R77_I83delinsQDAPD, N86H, I89V, S91_R92delinsEP, T97S, Q101R, A103L, L105V, K107_L110delinsRPDQ, V113_R115delinsAVD, H118Y, H120D, Y122_A127delinsGCEPCG N, V129T, S131N, F135_V136delinsAI, W138R, Q140_H143delinsFSRF, A145_D148delinsEVRE, V150A, I152V, T156_T157delinsAA, E159_S161delinsGDA, K163A, E167A, V169_E170delinsYD, T173L,

K176_R178delinsQEK, K180_A181delinsGL, N183_F186delinsDVML, P198_A201delinsRPSQ, K203_N204delinsSS, R208W, D210S, R212T, V214Q, G218P, Q220_E221delinsSA, V225_S228delins ATP, N230H, L238_R239delinsVA, Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256N, K259A, K262G, T264_E267delinsSDQL, L269_V271delinsQAI, F275Y, F279_K280delinsVM, Q2 82_S205delinsK regarding SEQ ID NO: 4.

These 63 D1L3 variants were screened for loss or gain of chromatin degradation activity. Briefly, D1L3 variants were transiently expressed in CHO cells using an in vitro expression vector. Culture supernatants were collected and tested for chromatin degradation activity using purified nuclei as a chromatin source. As shown in FIG. 1, replacement with building blocks #17 and #63 from D1 significantly improved the degradation of high molecular weight (HM) chromatin to small fragments compared to wild-type D1L3. The substitution of building block #7 causes a missense mutation Q101R, which replaces glutamine at position 101 with arginine (SEQ ID NO: 8). The substitution of building block #63 causes the mutation Q282_S305delinsK, which removes the entire C-terminal BD of D1L3 at amino acid positions 283 to 305 and replaces glutamine (Q) at position 282 with lysine (SEQ ID NO: 9). Western blot analysis of the supernatants was then performed to determine the expression levels of wild-type D1L3 and both mutants (Fig. 2). To our surprise, two D1L3 variants of different sizes were found in samples with wild-type D1L3 and the Q101R mutant. Samples with Q282_S305delinsK contained only the smaller D1L3 variant. Data suggest that wild-type D1L3 BD is spontaneously cleared (e.g., proteolyzed) during expression or post-secretion in CHO cells. Two D1L3 variants were also detected in the supernatants of CHO cells that stably express WT-D1L3 (Fig. 3). Of note, the basic domain-deleted D1L3 (BDD-D1L3) showed significantly increased chromatinase activity compared to wild-type D1L3.

Pichia pastoris was then tested as an alternative microbial expression system relative to CHO cells. Generally, we observed higher expression levels with BDD-D1L3 compared to wild-type D1L3. At this stage, wild-type D1L3 and BDD-D1L3 were purified and characterized from Pichia pastoris fermentation supernatants (Figure 4). Surprisingly, we discovered that wild-type D1L3 was proteolytically truncated within the BD at amino acid positions K291, K291, or S293, resulting in a heterogeneous mixture of D1L3 variants after purification. In contrast to wild-type D1L3, expression of BDD-D1L3 due to three substitutions of building blocks (F275Y, F279_K280delinsVM, Q282_S205delinsK) generated a pure protein.

The chromatinase activity of D1L3 after both purifications was then compared. We observed that a heterogeneous mixture of D1L3 variants with a BD truncation at positions K291, K291, or S293 had approximately 10-fold lower chromatinase activity compared to a D1L3 variant with a complete BD deletion due to F275Y/F279_K280delinsVM/Q282_S205delinsK. Taken together, the data indicate that proteolytic cleavage of BD can occur naturally in microbial and mammalian expression systems (e.g., CHO and P. pastoris), and removal of BD appears to activate D1L3 chromatin degradation activity.

Example 2: D1L3 expression in CHO cells in bioreactors.

Disclosed herein is the development of stable CHO cell lines producing wild-type D1L3 (SEQ ID NO: 4). Cell lines were cultured in bioreactors using standard CHO culture medium. In particular, in FIG. 5 shows Western blot analysis 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 minor levels of D1L3 and D1L3 fragments were detected. Data show that low yield of D1L3 is a problem in D1L3 production.

As described herein, high levels of wild-type D1L3 production were achieved by adding polyanions to the culture medium. Such polyanions may contain one or more of heparin, dextran sulfate, ferric citrate and ethylenediaminetetraacetic acid and constitute the biologically active ingredient in anti-aggregation reagents. Specifically, we added dextran sulfate to the CHO culture medium and observed a strong increase in D1L3 as well as D1L3 fragments (Figure 5). Data show that polyanions increase the yield of D1L3 production but do not prevent proteolytic degradation.

In fig. 6A shows that polyanions such as dextran sulfate (DS) form a complex with D1L3. The D1L3-DS complex prevents D1L3 from interacting and absorbing negatively charged surfaces during the manufacturing process. Such negatively charged surfaces include, but are not limited to, the cell surface of producing cells (eg, CHO cells, Pichia pastoris, Saccharomyces spp.), DNA exposed by dying cells, and

- 17 046216 surfaces of bioreactors. In fig. 6B and 6C show a two-step process for purifying D1L3 from DS-D1L3 complexes. As shown in FIG. 6, the first step is aimed at dissociating the DS-D1L3 complex. Dissociation can be achieved by incubating the DS-D1L3 complex with strong anion exchange surfaces that bind DS and thus release D1L3. In particular, the purification process may include passing a culture medium containing DS-D1L3 through a chromatography column packed with a strong anion exchange resin, followed by collecting the stream that contains D1L3 without DS. The second stage of the purification process is shown in FIG. 6C and involves affinity purification of D1L3 from a non-DS stream through the use of a strong cation exchange resin. In conclusion, the production yield of D1L3 can be significantly increased by the addition of polyanions such as dextran sulfate.

Example 3. Construction of D1L3 resistant to proteases.

Wild-type D1L3 contains 50 arginine and lysine residues, making the enzyme particularly sensitive to proteases such as trypsin, thrombin and plasmin. In this example, trypsin and plasmin cleavage sites in D1L3 were identified. The sites can be mutated to produce protease-resistant D1L3 variants.

Briefly, purified D1L3 was treated with trypsin. D1L3 fragments were isolated and the amino acid sequence of the fragments was determined using combinations of liquid chromatography (LC) and mass spectrometry (MS). Trypsin was determined to cleave D1L3 at the following arginine and lysine residues: R22, R29, R51, R66, R80, R81, R95, K99, R115, K147, K163, K180, R208, R212, R235, R239, K250, and K262. These arginine and lysine residues can be replaced by small amino acids such as alanine, valine and serine, or amino acids that have similar properties according to the Grantham distance score (eg, histidine, glutamine and glutamate; Fig. 7). Protease-resistant D1 contains arginine and lysine residues corresponding to R51, R95, K99, and R235, suggesting that these residues are not primarily responsible for the proteolytic degradation of D1L3.

Protein building block engineering was used to transfer the following building blocks from D1 to replace D1L3 building blocks that contain trypsin cleavage sites (Figure 7): 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).

Plasmin is a plasma protease that is formed upon activation of its zymogenic plasminogen. Plasminogen activator inhibitor 1 (PAI-1) suppresses plasmin activation. Interestingly, PAI-1 increases D1L3 enzymatic activity in serum, suggesting that plasmin may proteolytically inactivate D1L3. However, plasmin cleavage sites in D1L3 have not been identified.

In silico analysis revealed that the amino acid combination lysine-alanine (KA) or arginine-alanine (RA) is believed to be preferentially cleaved by the protease plasmin or proteases that have plasmin-like activity. D1L3 contains a total of four putative plasmin cleavage sites (Figure 8): (site 1) K180/A181 (K160/A161 without signal peptide), (site 2) K200/A201 (K180/A181 without signal peptide), (site 3) K259/A260 (K239/A240 without signal peptide) and (site 4) R285/A286 (R270/A250 without signal peptide). Using a pairwise alignment of D1 and D1L3, neither plasmin cleavage site was found to be present in D1 (Fig. 8). These data are consistent with the fact that D1 activity is not affected by inactivation by serum proteases such as thrombin and plasmin. Protein building block engineering was used to transfer the following building blocks from D1 to replace D1L3 building blocks that contain plasmin cleavage sites (Figure 9): (site 1) K180_A181delinsGL, (site 2) P198_A201delinsRPSQ, and (site 3) K259A. R285/A286 (site 4) is in a C-terminal extension that is absent in D1. Consequently, we created a D1L3 variant in which all four putative plasmin cleavage sites were mutated: K180_A181delinsGL, P198_A201delinsRPSQ, K259A, and R285A. The chromatin degradation of the D1L3 variant was then analyzed, and strong chromatin degradation activity was observed in the mutated D1L3 (Fig. 10). Taken together, the data indicate that four arginine and lysine residues, K180, K200, K259, and R285, can be mutated to reduce the risk of proteolytic degradation without compromising enzymatic activity.

Purified D1L3 was then treated with purified plasmin. D1L3 fragments were isolated and the amino acid sequence of the fragments was determined using a combination of LC and MS. We determined that plasmin cleaves D1L3 at the following 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 can be replaced by small amino acids such as alanine, valine and serine, or amino acids that have similar properties according to the Grantham distance score (eg, histidine, glutamine and glutamate; Fig. 11). Protease-resistant D1 contains lysine residues corresponding to K45, suggesting that these residues do not carry

- 18 046216 primarily responsible for the proteolytic degradation of D1L3 by plasmin. Protein building block engineering was used to transfer the following building blocks from D1 to replace D1L3 building blocks that contain in silico trypsin cleavage sites (Figure 11): R22 (Mutation: M21_R22delinsLK), R29 (V28_S30delinsIQT), K47 (K47_K50delinsQILS), K74 ( M72_K74delinsLDN), R81 (R77_I83delinsQDAPD), R92 (S91_R92delinsEP), K107 (K107_L110delinsRPDQ), K176 (K176_R178delinsQEK), R212 (R212T), K226 (V225_S228delinsATP), K227 (V225_ S228delinsATP), K250 (K250D), K259 (K259A) and K262 ( K262G).

Finally, recombinantly expressed wild-type D1L3 was isolated and its C-terminus sequenced. Three different amino acid sequences were identified, ending at S290 (SEQ ID NO: 10), K291 (SEQ ID NO: 11) and K292 (SEQ ID NO: 12), respectively (Fig. 4, example 1). These data identify lysine residues 291 and 292 as significant proteolytic cleavage sites of D1L3 during large-scale production.

Example 4: Design of D1L3 to Avoid Degradation.

We observed fragmentation of D1L3 after heterologous expression in Pichia pastoris. Analysis of fragments characterized by paired basic amino acids, arginine (R) and lysine (K) residues as proteolytic cleavage sites. A similar degradation feature was observed after expression of D1L3 in CHO cells. These observations indicate that Pichia pastoris and CHO cells share homologous proteases that cleave D1L3 at paired basic amino acids, although the effect was more significant in CHO cells.

It was determined that paired amino acid cleaving enzyme (PACE) contributed to the fragmentation of DNase1b3. PACE, also known as furin (Uniprot ID: P09958), is expressed in humans and mammals. Pichia pastoris expresses two enzymes targeting paired basic amino acids, namely aspartic proteinase 3 (gene: Ysp1; Uniprot ID: P32329) and kexin (gene: Kex2; Uniprot ID: P13134). Thus, the DNase1b3 and DNaseH3 variants can be expressed in Pichia pastoris and in CHO cells in which furin, aspartic proteinase 3 and kexin are pharmacologically inhibited or genetically depleted.

In addition, mutations of paired basic amino acids in the DNasePb3 and DNasePb3 variants allow their expression in CHO and Pichia pastoris with reduced fragmentation. Analysis of DNAsePb3 fragments revealed features of paired basic amino acids at positions: K50/R51, R80/R81, K114/R115, K199/K200, K226/K227, K291/K292, R297/K298/K299 and K303/R304 in SEQ ID NO: 2

As disclosed in US Provisional Patent Application No. 62/800,790 (which is incorporated herein by reference in its entirety), other species of DNasePb3 contain amino acid substitutions at these cleavage sites, including R114T (mouse), R114A (rat), R114D (guinea pig) , R114Q (cow), K227S (dog) and K227E (elephant). These amino acid substitutions can be applied to human DNase1b3 to render the enzyme resistant to proteolytic degradation, including during expression in CHO and Pichia pastoris cells.

Kexin preferentially cleaves at sites after the KR and RR residues. DNase1P3 has four KEX2 cleavage sites at positions K50/R51, R80/R81, K114/R115 and K303/R304. Amino acid substitutions of these residues make DNasePb3 resistant to the action of KEX2 and enable the expression of DNase1b3 and DNase1b3 variants in Pichia pastoris and CHO cells. These amino acid substitutions may be conservative, such as R51K, R81K, R115K and R304K.

Example 5: Design of D1L3 variants to prevent the formation of high molecular weight aggregates.

During cGMP-compatible expression of D1L3 in CHO cells (Fig. 12A), accumulation of high molecular weight D1L3 aggregates was observed, indicating an additional challenge for the clinical production of D1L3. The formation of high molecular weight aggregates was observed to a much lesser extent in Pichia pastoris.

Application of reducing conditions to the proteins of the bioreactor material led to the dissolution of D1L3 aggregates. Data indicate that the formation of D1L3 aggregates is caused by intra- and/or intermolecular cross-linking via disulfide bridges during protein expression. In particular, as shown in FIG. 12B, the gel was run under non-reducing conditions and showed the accumulation of high molecular weight D1L3 aggregates over time. The gel was run under reducing conditions and no aggregates were detected. Data show that erroneous intra- and intermolecular disulfide bonds cause misfolding of human D1L3 under production conditions.

In fig. 13 shows an alignment of the amino acid sequences of human D1 (SEQ ID NO: 1) and human D1L3 (SEQ ID NO: 4). Signal peptide, conserved amino acids, variable amino acids, non-conserved cysteine residues and conserved cysteine residues are shown. Mutations in non-conserved cysteine residues reduce the potential for intra- and intermolecular disulfide bond formation during protein expression. Analysis of the amino acid sequence of D1L3 (SEQ ID NO: 4) showed the presence of five cysteine (C) residues: C24, C52, C68, C194 and C231 (Fig. 14). Cysteine residues C194 and C231 are conserved among all members of the protein family

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DNase 1 and form disulfide bonds that are essential for the enzymatic activity of DNase 1. The function of the cysteine residues in D1L3 was not known prior to this disclosure. Accordingly, as described herein, mutation of these cysteine residues reduces cross-linking through disulfide bridges and thus increases the yield of protein production.

Cysteine residues can be replaced by other small amino acids, namely alanine (A), serine (S), and glycine (G), among others. These substitutions cause the following amino acid mutations: C24A/S/G, C52A/S/G, C68A/S/G, C194A/S/G and C231A/S/G. In addition, building blocks that contain conserved cysteine residues can be replaced by building blocks from the donor DNase family of DNase 1 proteins (eg, D1 and D1L3). The following building blocks from D1 were used to replace the building blocks of D1L3, which contain non-conserved cysteine residues C24, C52 and C68: C24_S25delinsAA, C52Y and N64_I70delinsHLTAVGK. The chromatin degradation activity of D1L3 variants was quantified as described in PCT/US18/4708. Both common amino acid substitutions (C24A, C52A) and building block substitutions (C24_S25delinsAA, C52Y) caused a complete lack of chromatin degradation, indicating that C24 and C52 are required for D1L3 activity (Fig. 15). It is important to note that mutation of the C68 cysteine, either by a common amino acid substitution [C68A, (SEQ ID NO: 13)] or by a BB mutation (N64_I70delinsHLTAVGK), resulted in the formation of the D1L3 variant with chromatin degradation activity (Fig. 15). Amino acid sequence alignments revealed that the C68 cysteine is not conserved among other members of the DNase 1 protein family, supporting the view that C68 is not required for enzymatic activity. In addition, it was found that the amino acid substitution of the highly conserved cysteine C194 to alanine (C194A), but not the mutation of the highly conserved cysteine C231 to alanine (C231A), resulted in the formation of the enzymatically active variant D1L3 (Fig. 15). Thus, cysteines C68 and C194 can be mutated to reduce the risk of formation of erroneous disulfide bonds during D1L3 production.

A similar approach can be used to mutate non-conserved cysteine residues in other members of the DNase 1 family of proteins: D1, DNase 1-like protein 1 (D1L1), and DNase 1-like protein 2 (D1L2). D1 has two non-conserved cysteines: C123 and C126. D1L1 shows two non-conserved cysteine residues (C22, C50), which correspond to C24 and C52, D1L2 has only one non-conserved cysteine residue: C43. Mutation of non-conserved cysteine residues of members of the DNase1 family of proteins will reduce cross-linking through erroneous disulfide bridges during protein expression and thus allow the production of D1, D1L1, D1L2 and D1L3 for therapeutic use.

Example 6: Construction and expression of D1L3 and albumin-E^3 fusion protein in Pichia pastoris.

The expression of recombinant human extracellular DNases, including D1L3, was tested using Pichia pastoris. As shown in FIG. 16A, first at the N-terminus of D1L3 is the leader sequence for the Saccharomyces cerevisiae α-mating factor (aMF) pre-pro-secretion (SEQ ID NO: 46), a common tool for heterologous protein expression in Pichia pastoris. As disclosed herein, the combination of aMF with D1L3 caused unexpected unprocessing of aMF due to glycosylation (Fig. 16B). Glycosylation of the D1L3 protein limits the use of P. pastoris for clinical production of D1L3. D1L3 is correctly processed when the native D1L3 secretory signal peptide is found first at the N terminus [Fig. 16B, (SEQ ID NO: 48)]. Importantly, aMF increased D1L3 expression by 3- to 5-fold compared to native D1L3 signal peptide. Therefore, the processing of D1L3 fusion proteins was analyzed. In pilot studies, an N-terminal fusion of aMF and human serum albumin [HSA, (SEQ ID NO: 39)] with D1L3 was obtained (Fig. 17A). Some variants contained a linker peptide [eg, (GSSSS) ₃ ] between HSA and D1L3. As shown in FIG. 17B and 17C, expression of the fusion protein in P. pastoris generated non-glycosylated and enzymatically active D1L3. In addition, expression levels were increased 5- to 10-fold compared to native secretory signal peptide-driven D1L3 expression. Taken together, the data indicate that fusion of D1L3 to albumin enables production in Pichia pastoris.

Based on these pilot studies, various HSA fusion constructs with wild-type D1L3 and BDD-D113 were constructed and screened for target protein expression levels (SEQ ID NO: 17-28). As shown in FIG. 18, N-terminal fusion of human serum albumin (SEQ ID: NO: 17) with variant BDD-D113 (SEQ ID NO: 16) did not significantly increase expression levels. However, it should be noted that the inventors did find a strong increase in expression levels after insertion of a flexible linker consisting of glycine (G) and serine (S) residues between HSA and BDD-D1L3. In addition, the length of the linker sequence was correlated with increased expression. For example, although 12 ± 1.9 relative units of expression were obtained with a 5 amino acid linker (SEQ ID NO: 18), and with a 15 amino acid linker (SEQ ID NO: 19) the expression level was 32 ± 3.2 relative units, An approximately 7.5-fold improvement was observed when fused to HSA without a linker. In addition, the N-terminal location was critical for improved expression levels, as C-terminal fusion of linker-HSA constructs resulted in low levels of expression (SEQ ID NO: 20, SEQ ID NO: 21). It should be noted that the N-terminal HSA fusion via

- 20046216 flexible linker also significantly increased the expression of wild-type D1L3 (SEQ ID NO: 22), approximately 20-fold compared to native D1L3 (SEQ ID NO: 4). In conclusion, fusion of HSA via an N-terminal linker allows the production of D1L3 variants as well as BDD-D1L3.

We then tested whether the nature of the linker sequence was critical for improving D1L3 expression. Two additional sequences were tested: APAPAPAPAPAPAP (SEQ ID NO: 33, 14 amino acids, rigid linker) and AEAAAKEAAAKA (SEQ ID NO: 34, 12 amino acids, rigid, helical linker). As shown in FIG. 19, both test constructs (SEQ ID NO: 23, SEQ ID NO: 24) showed a strong increase in expression, but the rigid coiled-coil linker did not achieve the same strong increase in expression levels as the GGGGSGGGGSGGGGS linker. Thus, the length and acidic composition of the linker influence D1L3 expression levels.

The relationship between linker length, expression level, and enzymatic activity was then analyzed. For these tests, expression vectors containing an N-terminal HSA GS linker fusion with BDD-D1L3 variants (SEQ ID NO: 25-27) were constructed. Three linkers of different lengths were tested:

SGGSGSS [7 amino acids, (SEQ ID NO: 35)],

SGGGSGGSGGSGGSGSS [16 amino acids, (SEQ ID NO: 36)] and SGGSGGSGGSGGSGGSGGSGGSGGSGGSGSS [31 amino acids: (SEQ ID NO: 37)].

As shown in FIG. 20, it was observed that extension of the linker sequence from 7 to 16 amino acids leads to an increase in the expression level. Further extension from 16 to 31 amino acids did not increase protein expression, but did increase enzymatic activity, as determined by the decomposition of HMW chromatin into LMW chromatin. Biologics fused to albumin often exhibit reduced activity because albumin sterically interferes with the interaction with substrates and ligands. Thus, peptide linkers can be used to increase the distance between albumin and a fusion protein or peptide. However, the observation that insertion of a linker sequence between HSA and D1L3 simultaneously improved enzymatic activity and expression levels was unexpected.

The chromatin degradation activity of BDD-D1L3 (SEQ ID NO: 14) was compared with its hybrid counterpart (SEQ ID NO: 19). Briefly, Dnase1 ^-/- Dnase113 ^-/- mice were injected with SEQ ID NO: 4 or SEQ ID NO: 19. Serum was collected 15 min after injection. As shown in FIG. 21A, similar serum chromatin degradation activity was observed in both animals. Importantly, fusion of albumin to the N terminus of D1L3 and other human cell-free DNAs provides therapeutics with extended DNase half-lives. As described herein, the half-life of SED 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 is characterized by transgenic expression of human FcRn, which is responsible for the long half-life of albumin in the bloodstream. While unconjugated D1L3 (e.g., SEQ ID NO: 4) has a very short half-life in the circulation (<30 min), fusion with albumin extended the half-life to 3.3 days, thereby significantly improving systemic exposure as well as providing rapid absorption with t _max 5 min (Fig. 21B). Taken together, the data demonstrate that N-terminal fusion of HSA to D1L3 via a linker sequence not only facilitates production but also improves the pharmacokinetic properties of D1L3 in vivo.

Finally, a double fusion of HSA to the N- and C-terminus of D1L3 was tested. The C terminus of D1L3 was first analyzed for potential attachment sites. We identified two serine residues at positions 283 and 284 that provide a flexible connection of BD (RAFTNSKKSVTLRKKTKSKRS) to the central part of D1L3. Thus, we removed the BD and decided to attach HSA via a flexible GS linker (SEQ ID NO: 38) to S284. As shown in FIG. 22, the HSA fusion to the N- and C-terminus of BDD-D1L3 (SEQ ID NO: 28) maintained the high expression levels that were observed with the HSA fusion to the N-terminus (SEQ ID NO: 27).

Example 7: Development of cleavable linker sequences.

The results disclosed in this document have implications beyond manufacturing. For example, D1L3 variants with C-terminal amino acid deletions that retain their chromatin and/or NET degrading enzymatic activity, as indicated by SEQ ID NO: 9 to SEQ ID NO: 12, can be used for D1L3 therapy. In addition, site-specific alkylation of unpaired cysteine thiol is commonly used to create biologics with extended half-life for therapeutic applications. In particular, the nonessential cysteines C68 and C194 of D1L3 can be used for site-specific PEGylation (PEG, polyethylene glycol). Additionally, D1L3 variants resistant to plasmin inactivation due to mutations such as K180_A181delinsGL, P198_A201delinsRPSQ, K259A and R285A are expected to have improved half-life and hence efficacy in therapeutic applications.

Importantly, fusion of albumin to the N terminus of D1L3 and other human cell-free DNA provides therapeutics with extended DNase half-lives

- 21 046216 (Fig. 23A). Several linker sequences were used to reduce the steric inhibition of D1L3 by albumin. In addition, a physiologically cleavable peptide linker was developed. The peptide linker was designed to cleave when the fusion protein is in close proximity to neutrophil extracellular traps (NETs). Peptide sequences targeted by neutrophil-specific proteases, such as neutrophil elastase, cathepsin G, and proteinase 3, are candidates for a cleavable linker sequence.

A cleavable linker sequence has been developed that is cleaved intravascularly and is thus optimal for intravenous and intra-arterial application of DNase-based therapeutics. When developing the peptide, it was taken into account that NETs have the ability to activate blood coagulation factors, in particular 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 revealed that the FXIIa cleavage site is conserved between FXI and PK (Fig. 23B). In FXI, the cleavage site is between arginine 387 and isoleucine 388. In PK, the cleavage site is between arginine 390 and isoleucine 391. Indeed, FXI and PK are homologous proteins. As described herein, the inventors have developed several peptide linkers that contain all or parts of the FXI sequence from position 380 to position 403 (SEQ ID NO: 42, SEQ ID NO: 43) or the PK sequence from position 383 to position 406 (SEQ ID NO: 44).

Ultimately, the FXIIa cleavable linker can be used to produce extended half-life variants of other biologics (Figure 24), including, but not limited to, variants of other extracellular DNase, human coagulation factors (e.g., factor VII, factor VIII, and factor IX), as well as complement factors (for example, factor H).

All patents and patent publications cited herein are incorporated herein by reference in their entirety.

Wild-type human DNase.

SEQ ID NO: 1

DNase1 (NP_005212.2): signal peptide, mature protein:

MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDS HLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFN REPAIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVR P SQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQ AISDHYPVEVMLK

SEQ ID NO: 2

DNase1-like protein 1 (NP_006721.1): signal peptide; mature protein:

MHYPTALLFLILANGAQAFRICAFNAQRLTLAKVAREQVMDTLVRILARCDIMVLQEVVDSSGSA IPLLLRELNRFDGSGPYSTLSSPQLGRSTYMETYVYFYRSHKTQVLSSYVYNDEDDVFAREPFVAQFSLP SNVLPSLVLVPLHTTPKAVEKELNALYDVFLEVSQHWQSKDVILLGDFNADCASLTKKRLDKLELRTEP GFHWVIADGEDTTVRASTHCTYDRVVLHGERCRSLLHTAAAFDFPTSFQLTEEEALNISDHYPVEVELK LSQAHSVQPLSLTVLLLLLSLLSPQLCPAA

SEQ ID NO: 3

DNase1-like protein 2 (NP_001365.1): signal peptide, mature protein:

MGGPRALLAALWALEAAGTAALRIGAFNIQSFGDSKVSDPACGSIIAKILAGYDLALVQEVRDPDL SAVSALMEQINSVSEHEYSFVSSQPLGRDQYKEMYLFVYRKDAVSVVDTYLYPDPEDVFSREPFVVKFS APGTGERAPPLPSRRALTPPPLPAAAQNLVLIPLHAAPHQAVAEIDALYDVYLDVIDKWGTDDMLFL GD FNADCSYVRAQDWAAIRLRSSEVFKWLIPDSADTTVGNSDCAYDRIVACGARLRRSLKPQSATVHDFQ EEFGLDQTQALAISDHFPVEVTLKFHR

SEQ ID NO: 4

DNase1-like protein 3, isoform 1 (NP_004935.1): signal peptide, mature protein:

MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRI CPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFV VWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKK AWKNI RLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDH FPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS

SEQ ID NO: 5

DNase1-like protein 3, isoform 2 (NP_001243489.1): signal peptide; mature protein:

MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRI CPILMEKLNREKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDE LVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVK KSTNCAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKR S

SEQ ID NO: 6

DNase 2A (O00115): signal peptide; mature protein:

- 22 046216

MIPLLLAALLCVPAGALTCYGDSGQPVDWFVVYKLPALRGSGEAAQRGLQYKYLDESSGGWRD GRALINSPEGAVGRSLQPLYRSNTSQLAFLLYNDQPPQPSKAQDSSMRGHTKGVLLLDHDGGFWLVHS VPNFPPPASSAAYSWPHSACTYGQTLLCVSFPFAQFSKMGKQLTYTYPWVYNYQLEGIFAQEFPDLENV V KGHHVSQEPWNSSITLTSQAGAVFQSFAKFSKFGDDLYSGWLAAALGTNLQVQFWHKTVGILPSNCS DIWQVLNVNQIAFPGPAGPSFNSTEDHSKWCVSPKGPWTCVGDMNRNQGEEQRGGGTLCAQLPALWK AFQPLVKNYQPCNGMARKPSRAYKI

SEQ ID NO: 7

DNase 2B (Q8WZ79): signal peptide; mature protein:

MKQKMMARLLRTSFALLFLGLFGVLGAATISCRNEEGKAVDWFTFYKLPKRQNKESGETGLEYL YLDSTTRSWRKSEQLMNDTKSVLGRTLQQLYEAYASKSNNTAYLIYNDGVPKPVNYSRKYGHTKGLL LWNRVQGFWLIHSIPQFPPIPEEGYDYPPTGRRNGQSGICITFKYNQYEAIDSQLLVCNPNVY SCSIPATF HQELIHMPQLCTRASSSEIPGRLLTTLQSAQGQKFLHFAKSDSFLDDIFAAWMAQRLKTHLLTETWQRK RQELPSNCSLPYHVYNIKAIKLSRHSYFSSYQDHAKWCISQKGTKNRWTCIGDLNRSPHQAFRSGGFICT QNWQIYQAFQGLVLYYESCK

Human DNAse3 variant

SEQ ID NO: 8

DNase1-like protein 3, Q101R (signal peptide; mature protein)

MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRI CPILMEKLNRNSRRGITYNYVISSRLGRNTYKERYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFV VWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAW KNI RLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDH FPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS

SEQ ID NO: 9

DNase33, Q282_S305delinksK (signal peptide; mature protein):

MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRI CPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFV VWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKK AWKNI RLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDH FPVEFKLK

SEQ ID NO: 10

DNase33, S305delinsK (signal peptide; mature protein):

MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRI CPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFV VWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWGLENFIFMGDFNAGCSYVRPSQ WKNI RLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQAAYKLTEEEALDVSDH FPVEFKLQSSRAFTNS

SEQ ID NO: 11

DNase1b3, K292_S305del (signal peptide; mature protein):

MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRI CPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFV VWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWGLENFIFMGDFNAGCSYVRPSQ WKNI RLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQAAYKLTEEEALDVSDH FPVEFKLQSSRAFTNSK

SEQ ID NO: 12

DNase1b3, S293_S305del (signal peptide; mature protein):

MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRI CPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFV VWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWGLENFIFMGDFNAGCSYVRPSQ WKNI RLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQAAYKLTEEEALDVSDH FPVEFKLQSSRAFTNSKK

SEQ ID NO: 13

DNase1b3, C68A (signal peptide; mature protein):

MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRI APILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPF VVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAW K NIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVS DHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS

SEQ ID NO: 14

DNase1b3, F275Y/F279_K280delinsVM/Q282_S305delinsK (signal peptide; mature protein):

MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRI CPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFV VWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKK AWKNI

- 23 046216

RLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSSNSVFDFQKAYKLTEEEALDVSDH YPVEVMLK

SEQ ID NO: 15

DNase33, S283_S305del (signal peptide; mature protein):

MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRI CPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFV VWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKK AWKNI RLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDH FPVEFKLQ

SEQ ID NO: 16

DNase33, R285_S305del (signal peptide; mature protein):

MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRI CPILMEKLNREKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDE LVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVK KSTNCAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSS

DNase1b3 fused to albumin and variants

SEQ ID NO: 17

Albumin is a variant of DNAse1b3 fusion protein. (Albumin, variant DNase1b3):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLMRICSFNVRSFGESK QEDKNAMD VIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVK RSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAE NFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAY DRIVLRGQEIVSSVVPKS NSVFDFQKAYKLTEEEALDVSDHYPVEVMLK

SEQ ID NO: 18

Albumin is a variant of DNAse1b3 fusion protein. (Albumin, variant DNase1b3):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSMRICSFNVRSF GESKQEDK NAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKEK LVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHR WKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKK STNCAYDRIVLRGQEIVSS VVPKSNSVFDFQKAYKLTEEEALDVSDHYPVEVMLK

SEQ ID NO: 19

Albumin is a variant of DNAse1b3 fusion protein. (Albumin, variant DNase1b3):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSMR ICSFNV RSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYK EQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDEL VEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQ EDTTVKKSTNCAYDR IVLRGQEIVSSVVPKSSNSVFDFQKAYKLTEEEALDVSDHYPVEVMLK

SEQ ID NO: 20

- 24 046216

DNase33 variant - Albumin - fusion protein. (Albumin, variant of DNaseIII):

MRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNY VISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLI GDQEDTT VKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHYPVEVMLKGGGGSDAHKS EVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDK LCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLK KYLY EIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGE RAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSV V LLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTK KVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL VNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVK HKPKATKEQLKAVMDD FAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

SEQ ID NO: 21

DNaseIII3 variant - Albumin - fusion protein. (Albumin, DNaseIII variant):

MRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNY VISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLI GDQEDTT VKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHYPVEVMLKGGGGSGGGGS GGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCD KSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTA FHDN EETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEY AR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKK QTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

SEQ ID NO: 22

Albumin - DNaseIII - fusion protein. (Albumin, DNaseIII3):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGGSGGGGSGGGGSMR ICSFNV RSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYK EQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDEL VEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQ EDTTVKKSTNCAYDR IVLRGQEIVSSVVPKSSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS

SEQ ID NO: 23

Albumin - DNaseIII - fusion protein. (Albumin, DNaseIII3):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAPAPAPAPMRICS FNVRSF GESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQ YAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVE VYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQED TTVKKSTNCAYDRIV LRGQEIVSSVVPKSSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS

SEQ ID NO: 24

Albumin - DNaseIII - fusion protein. (Albumin, DNaseIII3):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK

- 25 046216

SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN Q DSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEK TPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAEAAKEAAAKAMRICSFNVRSF GESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISS RLGRNTYKEQ YAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVE VYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIV LRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRA FTNSKKSVTLRKKTKSKRS

SEQ ID NO: 25

Albumin is a variant of DNaseIII fusion protein. (Albumin, DNase1b3 variants):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLSGGSGSSMRICSFNVRS FGESKQED KNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQYAFLYKE KLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKH RWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVK KSTNCAYDRIVLRGQEIVS SVVPKSSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQ

SEQ ID NO: 26

Albumin is a variant of DNAse1b3 fusion protein. (Albumin, variant DNase1b3):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLSGGSGGSGGSGGSGSSMR ICSFNV RSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYK EQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDEL VEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQ EDTTVKKSTNCAYDR IVLRGQEIVSSVVPKSSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQ

SEQ ID NO: 27

Albumin is a variant of DNAse1b3 fusion protein. (Albumin, variant DNase1b3):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLSGGSGGSGGSGGSGGSG GSGGSG GSGGSGSSMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGI TYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVI IPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRL RTDPRFVWLIGDQ EDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQ

SEQ ID NO: 28

Albumin is a variant of DNAse1b3 - Albumin is a fusion protein. (Albumin, DNaseIII variant):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK

- 26 046216

CASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKF QN ALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLSGGSGGG SGGSGGSGGSGGSG GSGGSGSSMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGI TYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVI IPLHTTPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKK AWKNIRLRTDPRFVWLIGDQ EDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSGGSGG SGGSGGSGGSGGSGGSGGSGGSGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVN EVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQ EPERNECFLQHKDDNPNL PRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCH GDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLA ADFVESKDVC KNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEP QNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDY LSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVP KEFNAETFTFHADICTLSEKERQ IKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

SEQ ID NO: 29

Albumin isoform 2 DNAse1b3 fusion protein. (Albumin, DNase1b3 isoform 2):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLSGGSGGSGGSGGSGGSG GSGGSG GSGGSGSSMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNREQYAF LYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYT DVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDT TVKKSTNCAYDRIVLRG QEIVSSVVPKSSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS

SEQ ID NO: 30

Albumin is a variant of DNAse1b3 isoform 2 - fusion protein. (Albumin, DNaseIII isoform 2):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLSGGSGGSGGSGGSGGSG GSGGSG GSGGSGSSMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNREQYAF LYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYT DVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDT TVKKSTNCAYDRIVLRG QEIVSSVVPKSSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQ

LINKER SEQUENCE

SEQ ID NO: 31

GGGGS

SEQ ID NO: 32

GGGGSGGGGSGGGGS

SEQ ID NO: 33

APAPAPAPAPAPAP

SEQ ID NO: 34

AEAAAAKEAAAKA

SEQ ID NO: 35

SGGSGSS

SEQ ID NO: 36

SGGSGGSGGSGGSGSS

- 27 046216

SEQ ID NO: 37

SGGSGGSGGSGGSGGSGGSGGSGGSGGSGS

SEQ ID NO: 38

GGSGGSGGSGGSGGSGGSGGSGGSGGSGS

OTHER SEQUENCES

SEQ ID NO: 39

Human serum albumin (mature protein):

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDK SLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNE ETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQ KFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR RHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQN ALLV RYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE QLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

SEQ ID NO: 40

Human factor XI:

MIFLYQVVHFILFTSVSGECVTQLLKDTCFEGGDITTVFTPSAKYCQVVCTYHPRCLLFTFTAESPS EDPTRWFTCVLKDSVTETLPRVNRTAAISGYSFKQCSHQISACNKDIYVDLDMKGINYNSSVAKSAQEC QERCTDDVHCHFFTYATRQFPSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLACI RDIFPN TVFADSNIDSVMAPDAFVCGRICTHHPGCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGF SLQSCRHSIPVFCHSSFYHDTDFLGEELDIVAAKSHEACQKLCTNAVRCQFFTYTPAQASCNEGKGKCY LKLSSNGSPTKILHGRGGISGYTLRLCKMDNECTTKIKPRIVGGTASVRGEWPWQVTLHTTSPT QRHLC GGSIIGNQWILTAAHCFYGVESPKILRVYSGILNQSEIKEDTSFFGVQEIIIHDQYKMAESGYDIALLKLET TVNYTDSQRPICLPSKGDRNVIYTDCWVTGWGYRKLRDKIQNTLQKAKIPLVTNEECQKRYRGHKITH KMICAGYREGGKDACKGDSGGPLSCKHNEVWHLVGITSWGEGCA QRERPGVYTNVVEYVDWILEKT QAV

SEQ ID NO: 41

Human prekallikrein:

I FQHLAFSDVDVARVLTPDAFVCRTICTYHPNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENT ISGYSLLTCKRTLPEPCHSKIYPGVDFGGEELNVTFVKGVNVCQETCTKMIRCQFFTYSLLPEDCKEEKC KCFLRLSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTTKTSTRIVGGTNSSWGEWPWQVSLQVK LT AQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFSQIKEIIIHQNYKVSEGNHDI ALIKLQAPLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPLVTNEECQKRYQD YKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGC ARREQPGVYTKVAEYMD WILEKTQSSDGKAQMQSPA

ACTIVATE LINKER SEQUENCES

SEQ ID NO: 42

FXIIa-sensitive linker (factor XI peptide):

CTTKIKPRIVGGTASVRGEWPWQVT

SEQ ID NO: 43

FXIIa-sensitive linker

GGGGSPRIGGGGS

SEQ ID NO: 44

FXIIa-sensitive linker (prekallikrein peptide):

VCTTKTSTRIVGGTNSSWGEWPWQVS

EQ ID NO: 45

FXIIa-sensitive linker (prekallikrein peptide):

STRIVGG

SIGNAL PEPTIDES

SEQ ID NO: 46 α-Mating Factor (P01149):

MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFIN TTIASIAAKEEGVS

SEQ ID NO: 47

Human albumin secretory signal peptide+propeptide (P02768):

MKWVTFISLLFLFSSAYSRGVFRR

SEQ ID NO: 48

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Claims (11)

Human DNase3 signal peptide (Q13609): MSRELAPLLLLLLSIHSALA CLAIM 1. DNase1-like protein 3 (D1L3) variant, where the D1L3 variant is a fusion protein that contains: an amino acid sequence that is at least 95% identical to the mature enzyme defined by amino acids 21-282 of SEQ ID NO: 4; the amino acid sequence of albumin at the N-terminal side of the mature enzyme, wherein said albumin has an amino acid sequence at least 95% identical to SEQ ID NO: 39; and a linker amino acid sequence between the amino acid sequence of albumin and the amino acid sequence of the mature enzyme, wherein said linker amino acid sequence has at least 15 amino acids and consists of serine and glycine residues, wherein the D1L3 variant has a deletion of at least 5 amino acids of the C-terminal basic domain, wherein The C-terminal basic domain is defined by the 23 C-terminal amino acids of SEQ ID NO: 4. 2. The D1L3 variant of claim 1, wherein the D1L3 variant contains an amino acid sequence that is at least 98% identical to the mature enzyme defined by SEQ ID NO: 4 or SEQ ID NO: 5. 3. The D1L3 variant of claim 1, wherein D1L3 has a deletion of at least 10 amino acids or at least 15 amino acids of the C-terminal basic domain or a deletion of the entire C-terminal basic domain. 4. The D1L3 variant according to any one of claims 1 to 3, wherein D1L3 has an amino acid substitution at a position corresponding to position 101 of SEQ ID NO: 4, optionally wherein D1L3 has an amino acid substitution that is Q101R. 5. The D1L3 variant of claim 1, wherein the linker is a flexible linker, a rigid linker, or contains a protease cleavage site, optionally wherein the linker has at least 10 amino acids or at least 15 amino acids, optionally wherein the linker has from 15 to 35 amino acids. 6. The D1L3 variant of claim 1, wherein the variant comprises an amino acid sequence of any of SEQ ID NO: 8-30, in each case optionally having from one to twenty amino acid modifications independently selected from insertions, deletions or substitutions, optionally wherein the variant has 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. 7. Variant D1L3 according to any one of claims 1 to 6, containing one or more substitutions of building blocks from D1 and/or one or more deletions or substitutions of cysteine residues compared to the enzyme SEQ ID NO: 4, optionally, where the amino acid corresponding to C68 in SEQ ID NO: 4, optionally, wherein the amino acid corresponding to C68 in SEQ ID NO: 4 is replaced with an amino acid selected from Ala, Ser and Gly, optionally, wherein the variant contains the substitution N64_I70delinsHLTAVGK, which is optionally modified with one, two or three amino acids substitutions, deletions or insertions, provided that the substitution of a building block is not modified to include a cysteine residue. 8. Option D1L3 according to claim 1, containing PEG conjugation with an amino acid corresponding to C68 and/or C194. 9. The D1L3 variant according to claim 1, having one or more substitutions of the arginine and lysine residues that are present in SEQ ID NO: 4 and/or SEQ ID NO: 5, where the substitution makes the D1L3 variant less sensitive to proteolysis by plasmin, thrombin and/ or trypsin compared to protein SEQ ID NO: 4 or protein SEQ ID NO: 5, optionally, wherein one or more arginine and lysine substitutions correspond to the following position(s) of SEQ ID NO: 4: K180, K200, K259 and R285; optionally, wherein the arginine or lysine substitution is selected from one or more of K18OA, K2OOA, K259A and R285A with respect to SEQ ID NO: 4; optionally, wherein the variant contains a substitution selected from K180_A181delinsGL, P198_A201delinsRPSQ and K259A with respect to SEQ ID NO: 4, wherein the variant contains one or more mutations of a paired basic residue, wherein the paired basic residue corresponds to a position selected from K50/R51, R80/R81, K114/R115, K199/K200, K226/K227, K291/K292, R297/K298/K299 and K303/R304 from SEQ ID NO: 4; optionally wherein one or more paired base residue mutations include an amino acid substitution selected from R114T, R114A, R114D, R114Q, K227S and K227E with respect to SEQ ID NO: 4, optionally wherein one or more paired base residue mutations include an amino acid substitution selected of R51K, R81K, R115K and R304K with respect to SEQ ID NO: 4. 10. Option D1L3 according to claim 6, wherein the linker is cleaved by a coagulation system protease, optionally wherein the linker is cleaved by factor XII or a neutrophil protease. 11. An isolated polynucleotide encoding the D1L3 variant according to any one of claims 1 to 10. -