Modified DNASE1-LIKE2 for production and therapeutic use

Abstract

The present disclosure provides D1L2 enzymes lacking a proline-rich domain (PRD) and / or lacking a C-terminal Arg residue, which are efficiently produced in microbial expression systems such as Pichia pastoris and have high activity in degrading chromatin at acidic pH. The present disclosure provides methods of making the D1L2 enzymes and methods of using the D1L2 enzymes in therapy.

Description

[Technical field]

[0001] Priority This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 150,433, filed February 17, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Inflammation is an essential host response to control invading microorganisms and heal damaged tissues. Uncontrolled and persistent inflammation causes tissue injury in many inflammatory disorders. Neutrophils are the primary leukocytes in acute inflammation. During infection, neutrophils generate neutrophil extracellular traps (NETs), lattices of DNA filaments decorated with toxic histones and enzymes that immobilize and neutralize bacteria. However, excessive NET formation can harm host cells through cytotoxic, pro-inflammatory, pro-thrombotic, and pro-tumorigenic activities.

[0003] DNASE1-LIKE2 (also referred to herein as DNASE1L2 or D1L2) belongs to the DNASE1 protein family, a homologous group of secreted endonuclease enzymes that can degrade extracellular DNA. D1L2 is the only divalent cation-dependent acid DNase in mammals. D1L2 therefore has unique characteristics that may be physiologically and therapeutically important. Although D1L2 may have desirable enzymatic properties for treating or controlling inflammatory conditions or infections in various tissues, for example by degrading extracellular DNA, D1L2 has not been described as suitable for production using conventional recombinant systems.

[0004] Thus, the present invention provides improved recombinant D1L2 enzymes for production and therapy, as well as methods of making and using recombinant D1L2 enzymes in therapy. This disclosure demonstrates that the recombinant D1L2 enzymes have high chromatin degrading activity, especially at acidic pH. Summary of the Invention

[0005] The present disclosure is based in part on the discovery that D1L2 enzymes lacking the proline-rich domain (PRD) and / or lacking the C-terminal Arg residue are efficiently produced in microbial expression systems and have high activity in degrading chromatin at acidic pH.

[0006] In one aspect, the present disclosure provides a recombinant D1L2 enzyme lacking a PRD domain and / or lacking a C-terminal arginine naturally present in D1L2 isoform 1 and isoform 2. The D1L2 enzyme according to this aspect has high recombinant expression characteristics, including in non-mammalian expression systems such as Pichia pastoris. Thus, in an aspect of the present disclosure, a method of making a recombinant D1L2 enzyme lacking a PRD and / or a C-terminal arginine is provided. The method includes culturing a non-mammalian host cell encoding the D1L2 enzyme and recovering the enzyme from the culture. An exemplary D1L2 enzyme according to the present disclosure is represented by SEQ ID NO:7.

[0007] In various embodiments, the D1L2 gene expressed in the expression system encodes a signal peptide essentially native to wild-type human DNase. In some embodiments, the signal peptide is derived from human D1 (SEQ ID NO: 13), D1L2 (SEQ ID NO: 8), D1L1 (SEQ ID NO: 14) or D1L3 (SEQ ID NO: 15). In some embodiments, the signal peptide is a non-native (to the D1L2 enzyme) signal peptide suitable for secretion from a host cell in a non-mammalian expression system. An exemplary non-native signal peptide is α-mating factor (SEQ ID NO: 9), which may be used with yeast expression systems such as Pichia pastoris.

[0008] In some embodiments, the D1L2 enzyme is fused to a carrier protein, optionally via an amino acid linker. The carrier protein can be an albumin amino acid sequence. Such fusion to a carrier protein can provide benefits not only in recombinant expression, but also in in vivo persistence.

[0009] In various embodiments according to the present disclosure, the recombinant D1L2 enzyme is non-glycosylated. In various embodiments, the D1L2 enzyme produced according to the present disclosure is substantially non-immunogenic.

[0010] In some embodiments, the D1L2 enzyme is modified to have physical, pharmacodynamic and / or enzymatic properties suitable for therapy, including therapy using topical or localized compositions. In various embodiments, the modified D1L2 enzyme may have the same or higher protein-free DNA (naked DNA) degradation activity, the same or higher chromosomal DNA (chromatin) degradation activity, similar or improved protease resistance, and higher production levels in non-mammalian expression systems (such as Pichia pastoris) compared to D1L2 isoform 1 (as defined by SEQ ID NO:1 or SEQ ID NO:4).

[0011] In various embodiments, the D1L2 enzyme comprises one or more polyethylene glycol (PEG) moieties or other conjugates (e.g., fatty acids or acyl groups). In some embodiments, the one or more PEGylated amino acids are selected from lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. In some embodiments, the one or more amino acids suitable for PEGylation are introduced by substitution of one or more amino acids in D1L2 (e.g., SEQ ID NO: 7). In some embodiments, the one or more PEGylated amino acids are cysteine ​​(Cys or C), and PEGylation is performed by thiol conjugation. In some embodiments, primary amines in D1L2 can be PEGylated using NHS-PEG reagents.

[0012] According to the present disclosure, the D1L2 enzyme is encoded by a polynucleotide that can be codon-optimized for expression in a non-mammalian expression system. In some embodiments, the polynucleotide comprises a plasmid vector. In various embodiments, the plasmid vector comprises a nucleic acid encoding a D1L2 enzyme or variant described herein operably linked to an expression control region functional in a host cell. The expression control region is capable of driving expression of the operably linked encoding nucleic acid such that the enzyme is produced in a cell transformed with the expression vector. In another aspect, the present disclosure provides a non-mammalian expression cell ("host cell") comprising an expression vector encoding a D1L2 enzyme described herein. In some embodiments, the cell is a yeast cell, such as Pichia pastoris.

[0013] In yet another aspect, the present disclosure provides a pharmaceutical composition for local or topical administration, comprising a recombinant D1L2 enzyme as described herein or produced according to the present disclosure, and a pharma- ceutically acceptable carrier.

[0014] In some embodiments, the pharmaceutical composition is formulated for topical administration, such as in a cream, lotion, gel, solution, foam, spray, ointment, mouthwash, shampoo, or soap. In some embodiments, the pharmaceutical composition is for localized or topical treatment of tissues having an acidic environment and / or biofilm. For example, the acidic environment may have a pH of about 6.0 or less, about 5.5 or less, or about 5.0 or less, or about 4.5 or less, or about 4.0 or less. In some embodiments, the composition is a skin care composition for maintaining healthy skin and an attractive appearance of the skin. In these embodiments, the composition is useful in methods for treating or preventing skin infection and / or inflammation. In some embodiments using the topical composition, the subject may have a condition that affects the appearance or health of the skin, hair, or nails. In some embodiments, the subject has a condition such as psoriasis, dermatitis, epidermolysis bullosa, or an allergic reaction (including, for example, hives or a rash). In some embodiments, the subject has dry skin, such as xerosis. In some embodiments, the subject has acne vulgaris. In yet other embodiments, the subject's skin may be characterized by an overgrowth of Staphylococcus aureus, which may be associated with atopic dermatitis. In some embodiments, the subject has psoriasis and the composition is applied to psoriatic lesions.

[0015] In yet other embodiments, the composition is formulated for administration to one or more of the eye, ear, nasal or sinus cavity, and oral cavity. The compositions according to these embodiments may be useful in methods of treating or preventing inflammation or infection of the eye of a subject, for example, where the composition is formulated as eye drops or eye washes (including eyelid washes). In some embodiments, the subject may have dry eye disease or may have allergic, bacterial, or viral conjunctivitis. Bacterial conjunctivitis may be exacerbated by biofilm formation. In some embodiments, the subject has inflammation of the ocular canal, which may manifest as blepharitis or related conditions (e.g., chalazions). In still other embodiments, the composition may be useful in treating or preventing inflammation or infection of the ear of a subject, for example, where the composition is formulated as ear drops. In still other embodiments, the composition may be useful in methods of treating or preventing inflammation or infection of the nasal or sinus cavity of a subject, for example, where the composition is formulated as a nasal spray. In some embodiments, the subject has a sinus infection or a recurrent sinus infection, hi yet other embodiments, the subject has an infection or inflammation of the oral cavity, including gingivitis and dental abscesses.

[0016] In some embodiments, the compositions are useful in methods of treating nucleic acid-associated ocular disorders, including dry eye disease, interlaminar keratitis, contact lens-associated keratitis, endophthalmitis, infectious crystalline keratopathy, ocular cicatricial pemphigoid (OCP), keratoconjunctivitis sicca (KCS), Sjogren's syndrome (SS), Sjogren's syndrome-associated keratoconjunctivitis sicca, non-Sjogren's syndrome-associated keratoconjunctivitis sicca, keratitis sicca, sicca syndrome, xerophthalmia, tear film disorders, decreased tear production, aqueous tear deficiency syndrome (ATD), and meibomian gland dysfunction (MGD).

[0017] In yet other embodiments, the composition is formulated for pulmonary delivery, such as a powder or solution aerosol. In some embodiments, the composition is formulated for delivery by a nebulizer. In various embodiments, the composition is useful in a method for treating or preventing pulmonary inflammation or infection in a patient. In some embodiments, the subject has cystic fibrosis, pneumonia, bronchitis, asthma, lower respiratory tract infection, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), transfusion-induced lung injury (TRALI), or chronic obstructive pulmonary disease (COPD). In some embodiments, the subject has pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis, or IPF). In still other embodiments, the subject has a chronic or recurrent pulmonary infection.

[0018] In the case of cystic fibrosis, Pseudomonas aeruginosa infections are difficult to eradicate or control, due in part to the formation of biofilms. By administering a D1L2 enzyme according to the present disclosure, for example by nebulizer, it may be possible to control Pseudomonas infections in cystic fibrosis patients and alleviate some of the overuse of antibiotics. In various embodiments, the D1L2 enzymes provided herein have greater activity in CF lung secretions than PULMOZYME (dornase alfa).

[0019] In yet other embodiments, patients may have indwelling medical devices that tend to favor microbial colonization and biofilm formation, hi some embodiments, the D1L2 enzyme or a composition comprising the D1L2 enzyme is administered to a patient on mechanical ventilation to prevent or reduce biofilm formation.

[0020] Other aspects and embodiments of the present disclosure will become apparent from the following detailed description. [Brief description of the drawings]

[0021] [Figure 1]FIG. 1 shows an amino acid sequence alignment of human DNASE1L2 isoform 1 (DNASE1L2-L, SEQ ID NO: 1, Uniprot identifier: Q92874-1) and human DNASE1L2 isoform 2 (DNASE1L2-S; SEQ ID NO: 2, Uniprot identifier: Q92874-2). The secretory signal peptide (SEQ ID NO: 8) and proline-rich domain (PRD, SEQ ID NO: 3) are highlighted. The sequence rulers indicate the amino acid positions. Included are the mature forms of isoform 1 (SEQ ID NO: 4) and isoform 2 (SEQ ID NO: 6). [Diagram 2] Figure 1: Deletion of the proline-rich domain increases DNASE1L2 expression in Pichia pastoris. Estimated protein concentrations in the supernatants of the three winning strains are shown, calculated by peak area against bovine serum albumin diluted in mock strain matrix of known concentration. The expression constructs include (a) isoform 1 (SEQ ID NO: 4) with native signal peptide (SP, SEQ ID NO: 8), (b) isoform 1 (SEQ ID NO: 4) with alpha mating factor (aMF, SEQ ID NO: 9), and (c) isoform 2 (SEQ ID NO: 6) with alpha mating factor (aMF, SEQ ID NO: 9). [Diagram 3] This figure shows that purified proline-rich domain-deleted DNASE1L2 (PRDD-DNASE1L2) degrades chromatin at acidic pH. Nuclei as a source of high molecular weight chromatin were incubated with purified PRDD-DNASE1L2 at pH levels ranging from 3.5 to 7.5. Degradation of high molecular weight to low molecular weight was observed at acidic pH of 3.5 to 5.5. Basic domain-deleted (BDD)-DNASE1L3, which was used as a control, showed degradation at neutral pH of 6.5 and 7.5. [Figure 4] Figure 1 shows an alignment of DNASE1L2 amino acid sequences from different species extracted from the Uniprot database, which shows that only DNASE1L2 from human, chimpanzee and baboon features a C-terminal arginine residue. [Diagram 5]Figure 3. Deletion of the C-terminal arginine residue increases DNASE1L2 isoform 2 expression in Pichia pastoris. Estimated protein concentrations in the supernatants of the three winning strains are shown, calculated by peak area against bovine serum albumin diluted in mock strain matrix of known concentration. Expression constructs include (a) isoform 1 (SEQ ID NO: 4), (b) isoform 2 (SEQ ID NO: 6), and (c) isoform 2 lacking the C-terminal arginine residue (del278R in SEQ ID NO: 6), generating a new variant (SEQ ID NO: 7). Alpha mating factor (aMF, SEQ ID NO: 9) was used as the secretion signal peptide for all constructs. [Figure 6] FIG. 1 shows D1L2(PRDD) expressed in Pichia pastoris as a fusion with albumin at the N-terminus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present disclosure is based in part on the discovery that D1L2 enzymes lacking the proline-rich domain (PRD) and / or lacking the C-terminal Arg residue are efficiently produced in microbial strains and have high activity in degrading chromatin at acidic pH.

[0023] DNASE1L2 (or D1L2) is expressed and secreted in two isoforms, SEQ ID NO: 1 and SEQ ID NO: 2, which differ depending on the presence or absence of PRD, respectively. Isoform 1 is expressed in terminally differentiated keratinocytes of human epidermis, hair follicles, and nail matrix. Knockdown of D1L2 in a human in vitro skin model prevented nuclear DNA degradation and caused the retention of nuclei in the stratum corneum, a condition known as parakeratosis (Fischer H. et al., DNASE1L2 degrades nuclear DNA during corneocyte formation. J Invest Dermatol. 2007 Jan; 127(1):24-30). Parakeratotic lesions in skin diseases such as psoriasis may be associated with reduced D1L2 expression. Furthermore, D1L2 can suppress biofilm formation, especially in pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus (Eckhart L, et al., DNASE1L2 suppresses biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus. Br J. Dermatol. 2007 156(6):1342-5). Isoform 2 is expressed in leukocytes under inflammatory conditions, but its function is poorly characterized (Shiokawa et al. Characterization of the human DNASE1L2 gene and the molecular mechanism for its transcriptional activation induced by inflammatory cytokines. Genomics. 2004 84:95-105).

[0024] Although D1L2 may have desirable enzymatic properties for treating or controlling inflammatory conditions or infections in various tissues, for example by degrading extracellular DNA, D1L2 has not been described as suitable for production using conventional recombinant systems. Thus, the present disclosure provides in various aspects and embodiments recombinant D1L2 enzymes and methods for making recombinant D1L2 enzymes, including for use in therapy. In various embodiments, the present invention provides high expression of D1L2 enzymes in non-mammalian expression systems, such as yeast expression systems (e.g., Pichia pastoris), which have robust activity for therapeutic applications. In particular, the D1L2 enzymes have robust activity at acidic pH (e.g., less than about 6.0).

[0025] In one aspect, the disclosure provides a method for producing a recombinant D1L2 enzyme. The method includes culturing a non-mammalian host cell encoding a D1L2 enzyme substantially lacking a proline-rich domain (PRD) defined by SEQ ID NO:3. In various embodiments, the D1L2 produced by the host cell includes a signal peptide that directs secretion from the cell, which is generally removed during secretion. The method further includes recovering the D1L2 enzyme from the culture medium.

[0026] Human D1L2-isoform 1 contains a 21 amino acid PRD, as shown in Figure 1. Removal of this PRD (as with isoform 2) substantially improves expression in non-mammalian expression systems. Exemplary expression systems include yeast expression systems and eukaryotic expression systems such as insect cell expression systems or plant cell expression systems. An exemplary non-mammalian expression system is Pichia pastoris. Suitable non-mammalian expression systems include, but are not limited to, insect cell-based expression systems such as Sf9 cells and baculovirus-based vectors, bacterial expression systems such as Escherichia coli, Lactococcus lactis, Pseudomonas fluorescens and Bacillus subtilis, yeast expression systems and filamentous fungal expression systems such as Aspergillus spp., Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica, plant-based expression systems such as Nicotiana benthamiana and Nicotiana tabacum. See, e.g., U.S. Pat. Nos. 10,385,319, 10,899,801, 10,287,555, 9,931,390, 9,879,280, 9,458,487, 8,535,930, 8,067,198, 7,229,792, 6,558,920, and 5,637,477, which are incorporated by reference in their entireties.

[0027] In various embodiments, the D1L2 enzyme lacks at least about 10 amino acids of the PRD (SEQ ID NO:3). In some embodiments, the D1L2 enzyme lacks at least about 15 amino acids or at least about 18 amino acids of the PRD (SEQ ID NO:3), or lacks the PRD entirely. In some embodiments, the PRD is replaced with an alternative sequence that has low or minimal homology to the PRD, such as less than about 8 or less than about 5 identical amino acids aligned at corresponding positions. In some embodiments, the alternative sequence has about 3 or less, or about 2 or less, or 1 or less proline residues. In some embodiments, the D1L2 enzyme with a deleted or modified PRD comprises an amino acid sequence that has at least about 80% sequence identity to the enzyme defined by SEQ ID NO:4 (isoform 1) or SEQ ID NO:6 (isoform 2). In some embodiments, the D1L2 enzyme comprises an amino acid sequence having at least about 85% sequence identity, or at least about 90% sequence identity, to the enzyme defined by SEQ ID NO:4 or SEQ ID NO:6, or at least about 95% sequence identity to the enzyme defined by SEQ ID NO:4 or SEQ ID NO:6, or at least about 97% sequence identity to the enzyme defined by SEQ ID NO:4 or SEQ ID NO:6.

[0028] In some embodiments, the D1L2 enzyme comprises an amino acid sequence having at least about 85% sequence identity or at least about 90% sequence identity to the enzyme defined by SEQ ID NO:6, or at least about 95% sequence identity to the enzyme defined by SEQ ID NO:6, or at least about 97% sequence identity to the enzyme defined by SEQ ID NO:6.

[0029] Alternatively or additionally, the present disclosure provides a recombinant D1L2 enzyme lacking the C-terminal arginine naturally present in D1L2 isoform 1 and isoform 2. The D1L2 enzyme according to this embodiment has high recombinant expression characteristics, including in non-mammalian expression systems such as Pichia pastoris. Thus, in an embodiment of the present disclosure, a method of making a recombinant D1L2 enzyme lacking a C-terminal arginine is provided. The method includes culturing a non-mammalian host cell encoding a D1L2 enzyme lacking a C-terminal Arg residue. Non-limiting examples of D1L2 enzymes lacking a C-terminal Arg residue are SEQ ID NO:5 and SEQ ID NO:7. In some embodiments, the D1L2 enzyme lacking a C-terminal Arg residue comprises an amino acid sequence having at least about 80% sequence identity to the enzyme defined by SEQ ID NO:5 or SEQ ID NO:7. In some embodiments, the D1L2 enzyme lacking a C-terminal Arg residue comprises an amino acid sequence having at least about 85% sequence identity, or at least about 90% sequence identity, to the enzyme defined by SEQ ID NO:5 or SEQ ID NO:7, or at least about 95% sequence identity, to the enzyme defined by SEQ ID NO:5 or SEQ ID NO:7, or at least about 97% sequence identity to the enzyme defined by SEQ ID NO:5 or SEQ ID NO:7. In various embodiments, the D1L2 enzyme produced by the host cell comprises a signal peptide that directs secretion from the cell, which is generally removed during secretion. The method further comprises recovering the D1L2 enzyme from the culture medium.

[0030] In some embodiments, the D1L2 enzyme has a deleted or modified PRD (as described), as well as a truncation of the C-terminal Arg. In embodiments, the D1L2 enzyme substantially lacking a proline-rich domain (PRD) and lacking a C-terminal Arg residue comprises an amino acid sequence having at least about 80% sequence identity to the enzyme defined by SEQ ID NO: 7. In some embodiments, the D1L2 enzyme substantially lacking a proline-rich domain (PRD) and lacking a C-terminal Arg residue comprises an amino acid sequence having at least about 85% sequence identity to the enzyme defined by SEQ ID NO: 7, or at least about 90% sequence identity to the enzyme defined by SEQ ID NO: 7, or at least about 95% sequence identity to the enzyme defined by SEQ ID NO: 7, or at least about 97% sequence identity to the enzyme defined by SEQ ID NO: 7.

[0031] As used herein, when referring to sequence identity between DNase enzymes, unless otherwise specified, the sequence refers to the mature enzyme lacking the signal peptide. Furthermore, unless otherwise specified, amino acid positions are numbered relative to the full translated DNase sequence, including the signal peptide, for clarity.

[0032] The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined by sequence alignment as known in the art. Such alignment can be performed using several art-known algorithms, such as the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ) or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80). Exemplary algorithms are incorporated in the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215: 403-410. When using BLAST programs, the default parameters of the respective programs are used.

[0033] In various embodiments, the encoded signal peptide is essentially native to the wild-type human DNase. In some embodiments, the signal peptide is derived from human D1 (SEQ ID NO: 13), D1L2 (SEQ ID NO: 8), D1L1 (SEQ ID NO: 14) or D1L3 (SEQ ID NO: 15). In some embodiments, one, two or three amino acid substitutions are made to the wild-type signal sequence to enhance production or secretion from a non-mammalian expression system. In some embodiments, the signal peptide is a non-native (to the D1L2 enzyme) signal peptide suitable for secretion from a host cell of a non-mammalian expression system. An exemplary non-native signal peptide is α-mating factor (SEQ ID NO: 9), which may be used with yeast expression systems such as Pichia pastoris. In some embodiments, the D1L2 enzyme is expressed using an albumin secretion signal peptide, exemplified in SEQ ID NO: 10.

[0034] In various embodiments, the non-mammalian expression system (e.g., Pichia pastoris) co-expresses or overexpresses one or more genes that facilitate signal peptide processing. In some embodiments, the genes encode a protease that has the activity to cleave the signal peptide from the secreted protein. An exemplary protease is KEX2.

[0035] In some embodiments, the D1L2 enzyme is fused to a carrier protein, optionally via an amino acid linker. The carrier protein can be a polypeptide, such as albumin, transferrin, an Fc domain, XTEN (see U.S. Patent No. 8,492,530, which is incorporated herein by reference in its entirety), or an elastin-like protein. See, for example, U.S. Patent No. 9,458,218, which is incorporated herein by reference in its entirety. Such fusions can provide benefits not only in recombinant expression, but also in in vivo persistence.

[0036] In some embodiments, the carrier protein is an albumin amino acid sequence, and the albumin amino acid sequence is fused to the N-terminus of D1L2, optionally with an intervening linker. An exemplary albumin amino acid sequence is provided by SEQ ID NO: 11. In some embodiments, the albumin amino acid sequence or domain of the fusion protein has 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% sequence identity to the reference albumin sequence defined by SEQ ID NO: 11. In some embodiments, the albumin amino acid sequence or domain comprises or consists of the reference albumin sequence defined by SEQ ID NO: 11. In various embodiments, the albumin amino acid sequence binds to fetal Fc receptor (FcRn), e.g., human FcRn. The albumin amino acid sequence may be a variant of wild-type HSA (e.g., represented by SEQ ID NO: 11). In various embodiments, the albumin variant may have 1-20 or 1-10 amino acid modifications, independently selected from deletions, substitutions, and insertions, relative to SEQ ID NO: 11. In some embodiments, the albumin amino acid sequence is any mammalian albumin amino acid sequence. A variety of modifications to albumin sequences are known that enhance its ability to function as a carrier, and such modifications can be used with the present invention. Exemplary modifications to albumin amino acid sequences are described in U.S. Pat. Nos. 8,748,380, 10,233,228, 10,208,102, and 10,501,524, each of which is incorporated herein by reference in its entirety. Exemplary modifications include one or more (or all) of E505Q, T527M, and K573P described therein.

[0037] In some embodiments, the albumin amino acid sequence or domain is a fragment of full-length albumin as represented by SEQ ID NO: 11. The term "fragment", when used in the context of albumin, refers to any fragment of full-length albumin or a variant thereof (as described above) that improves the expression of the D1L2 enzyme to which it is fused or conjugated (e.g., in Pichia pastoris) compared to the corresponding unfused D1L2. In some embodiments, a fragment of albumin may refer to an amino acid sequence that includes a fusion of multiple domains of albumin, such as domains I and III, and domains II and III (see, e.g., WO 2011 / 124718). Generally, a fragment of albumin has at least about 100 amino acids of the full-length sequence, or at least about 200 amino acids, or at least about 300 amino acids. In various embodiments, the albumin fragment maintains the ability to bind to human FcRn.

[0038] In some embodiments, the D1L2 enzyme is fused to the carrier polypeptide (such as an albumin amino acid sequence) via a peptide linker. The peptide linker can be a flexible linker, a rigid linker, or in some embodiments, a physiologically cleavable linker (e.g., a protease cleavable linker). In some embodiments, the linker is 5 to 100 amino acids in length, or 5 to 50 amino acids in length. In some embodiments, the linker is about 10 to about 35 amino acids in length, or about 15 to about 35 amino acids in length. In some embodiments, the linker is a flexible linker of 20 to 40 amino acids. The flexible linker can comprise (or consist of) primarily Gly and Ser amino acid residues.

[0039] In some embodiments, the D1L2 enzyme is fused to an Fc domain. See, for example, WO2005047334, WO2004074455, US20070269449, which are incorporated by reference in their entirety. In some embodiments, the human Fc domain is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the human Fc domain is a human IgG Fc domain. In some embodiments, the Fc domain comprises at least two heavy chain constant region domains (CH2 and CH3) and a hinge region. The Fc domain can be joined to D1L2 at the N-terminus and / or C-terminus of the D1L2 enzyme, optionally with an intervening linker. In some embodiments, the D1L2 enzyme comprises an Fc domain sequence fused to the N-terminus of the mature D1L2 enzyme with an intervening amino acid linker. The peptide linker can be a flexible linker, a rigid linker, or in some embodiments, a physiologically cleavable linker. In some embodiments, the D1L2 enzyme comprises an Fc domain sequence fused to the C-terminus of the D1L2 enzyme, optionally via a linker (e.g., a flexible linker).

[0040] Flexible linkers are composed primarily or entirely of small non-polar or polar residues such as Gly, Ser, and Thr. Exemplary flexible linkers include (Gly y Ser) n S z linkers, where y is 1-10 (e.g., 1-5), n is 1 to about 10, and z is 0 or 1. In some embodiments, n is 3 to about 8 or 3 to about 6. In exemplary embodiments, y is 2-4 and n is 3-8. These linkers are unstructured due to their flexibility. More rigid linkers include polyproline or polyPro-Ala motifs and α-helical linkers. An exemplary α-helical linker is A(EAAAK). nA, where n is as defined above (e.g., 1-10 or 3-6). Generally, the linker may be composed primarily of amino acids selected from Gly, Ser, Thr, Ala, and Pro. Exemplary linker sequences include at least 10 amino acids and may range from 10 to about 50 amino acids, or from about 15 to about 40 amino acids, or from about 15 to about 35 amino acids. An exemplary linker design is provided as SEQ ID NO: 12.

[0041] In some embodiments, the D1L2 enzyme includes a linker, and the amino acid sequence of the linker is primarily or essentially composed of glycine and serine residues. In some embodiments, the ratio of Ser to Gly in the linker is about 1:1 to about 1:10, about 1:2 to about 1:6, or about 1:4, respectively. Exemplary linker sequences include or consist of S(GGS)4GSS, S(GGS)9GSS, (GGS)9GSS. In some embodiments, the linker has at least 10 amino acids, or at least 15 amino acids, or at least 20 amino acids, or at least 25 amino acids, or at least 30 amino acids. For example, the linker can have a length of 15 to 40 amino acids. In various embodiments, a longer linker of at least 15 amino acids can provide improved yields upon expression in Pichia pastoris.

[0042] In other embodiments, the linker is a physiologically cleavable linker, such as a protease-cleavable linker. For example, the protease can be a coagulation pathway protease, such as activated factor XII. In some embodiments, the linker comprises an amino acid sequence of factor XI and / or prekallikrein, or a physiologically cleavable fragment thereof. In other embodiments, the linker comprises a peptide sequence targeted for cleavage by neutrophil-specific proteases, such as neutrophil elastase, cathepsin G, and proteinase 3.

[0043] In exemplary embodiments, the production of D1L2 enzyme is carried out by large-scale (e.g., batch) fermentation of at least about 1000 L, or at least about 5000 L, or at least about 10,000 L, or at least about 50,000 L. In various embodiments, the fermentation produces at least about 200 mg / L of D1L2 enzyme. In some embodiments, the fermentation produces at least about 300 mg / L of D1L2 enzyme, at least about 400 mg / L of D1L2 enzyme, at least about 600 mg / L of D1L2 enzyme, or at least about 800 mg / L of D1L2 enzyme, or at least about 1 g / L of D1L2 enzyme, or at least about 1.2 g / L of D1L2 enzyme. In yet other embodiments, the fermentation produces at least about 1.5 g / L of D1L2, or at least about 2 g / L of D1L2 enzyme, or at least about 5 g / L or at least about 10 g / L of D1L2 enzyme.

[0044] In various embodiments according to the present disclosure, the recombinant D1L2 enzyme is non-glycosylated. In various embodiments, the D1L2 enzyme produced according to the present disclosure is substantially non-immunogenic.

[0045] According to another aspect, the present disclosure provides a recombinant D1L2 enzyme produced according to the methods described herein. In some embodiments, the D1L2 enzyme is modified to have physical, pharmacodynamic and / or enzymatic properties suitable for therapy, including therapy with topical compositions. In various embodiments, the modified D1L2 enzyme may have the same or higher protein-free DNA (naked DNA) degradation activity, the same or higher chromosomal DNA (chromatin) degradation activity, similar or improved protease resistance, and higher production levels in non-mammalian expression systems (such as Pichia pastoris) compared to D1L2 isoform 1 (defined by SEQ ID NO:1 or SEQ ID NO:4).

[0046] In some embodiments, the invention uses D1L2 enzymes engineered by transferring a single amino acid or multiple adjacent amino acids, referred to as "building blocks," between two members of the DNase 1 protein family to generate enzymatically active variants of D1L2. A "building block" is defined by amino acids that are variable between two or more members of the DNase 1 protein family. These variable amino acids are adjacent to amino acids ("anchors") that are conserved between two or more members of the DNase 1 protein family. The variable single amino acid or multiple consecutive amino acids ("building blocks") are exchanged between members of the DNase 1 protein family by inserting between the conserved single amino acid or multiple consecutive amino acids ("anchors"). See U.S. Patent No. 10,696,956, which is incorporated herein by reference in its entirety.

[0047] When three or more amino acids are transferred, up to one third of the amino acids may be further replaced. For example, when three or six amino acids are transferred as building blocks, one or up to two residues may be further replaced, respectively. In some embodiments, when four or more amino acids are transferred as building block replacements, up to 25% of the transferred amino acids are further replaced, for example, by conservative or non-conservative amino acid modifications. For example, when four, eight or twelve amino acids are transferred, one, two or three amino acids (respectively) may be further replaced in the building block replacement.

[0048] In some embodiments, the building block substitutions for D1L2 are selected from (e.g., one, two, three, four or five selected from) a non-human D1L2 protein, resulting in a variant of human D1L2 characterized by one or more of the following mutations corresponding to SEQ ID NO:1: L22K, I24V, I29V, S35N, S35H, S35R, S35T, V37A, S38L, A41D, A41V, A41G, G43I, S44G, S44i, I45V, K4 8Q, L55I, L55V, A56T, A56M, P64A, S70D, S70T, A71T, A71L, A71S, A71V, M73L, E74Q, N77H, S78R, E81K, E81R, E83N, S85G, S85N, Q9 0E, Q90K, Q96H, F103Y, V104I, K107D, A109V, A109T, A109K, V110A, V113L, V113M, D114S, D114E, L117Q, P119S, E122G, V124A, V1 24F, S126N, E128D, F134V, A136V, A136T, G138S, G138R, T139S, T139C, S148C, A151P, P154A, A159P, A160G, A161P, A161T, Q162D , Q162K, Q162R, Q162T, N163K, N163E, L164V, L164F, I167V, H174N, Q175H, A178T, A178V, D192N, G195N, T196S, D198V, M199L, M1 99I, S210K, R213K, Q215H, A218P, A219S, E226Q, V227I, S243T, A252V, C253S, A255S, A255V, R256H, L257M, R259K, S260T, L261V, Q264H, T267S, T267A, D270N, G276D, G276S, T280S, T280D, T280A, A284C, I286V, L295F, F297S, F297T, F297P, H298R and R299del.

[0049] Mutants of D1L2 produced by building block technology are disclosed in WO 2020 / 0271953 and WO 2020 / 163264, which are incorporated by reference in their entireties.

[0050] The engineered variant of D1L2 enzyme may include one or more amino acid substitutions, additions (insertions), deletions or truncations in the amino acid sequence of human D1L2 (SEQ ID NO: 7). Amino acid substitutions may include conservative and / or non-conservative substitutions. For example, "conservative substitutions" may be made based on similarity in, for example, polarity, charge, size, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the amino acid residues involved. The 20 naturally occurring amino acids can be classified into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. A "conservative substitution" is defined as the replacement of an amino acid with another amino acid listed in the same group of the six standard amino acid groups shown above. For example, replacement of Asp with Glu retains one negative charge in the modified polypeptide. In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices. As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid listed in a different one of the six standard amino acid groups (1)-(6) shown above.

[0051] DNA fragmentation of D1L2 and variants can be measured using methods known to those skilled in the art, such as gel electrophoresis and DNase activity assays. Methods for quantifying DNA fragmentation activity are described, for example, in U.S. Patent No. 10,696,956, which is incorporated herein by reference in its entirety.

[0052] According to the present disclosure, the D1L2 enzyme is encoded by a polynucleotide that can be codon optimized for expression in a non-mammalian expression system, including, but not limited to, Pichia pastoris. In some embodiments, the polynucleotide comprises a plasmid vector. In various embodiments, the plasmid vector comprises a nucleic acid encoding the D1L2 enzyme or variant described herein operably linked to an expression control region functional in a host cell. The expression control region is capable of driving expression of the operably linked encoding nucleic acid such that the enzyme is produced in a cell transformed with the expression vector.

[0053] Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that affect expression of an operably linked nucleic acid. In one embodiment, an expression control region provides regulatable expression to an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as inhibitory. Typically, the amount of increase or decrease provided by such elements is proportional to the amount of signal present. The greater the amount of signal, the greater the increase or decrease in expression.

[0054] In other aspects, the disclosure provides a non-mammalian expression cell (i.e., a "host cell") as described above, comprising an expression vector encoding the D1L2 enzyme described herein. In some embodiments, the cell is a yeast cell, such as Pichia pastoris.

[0055] In various embodiments, the D1L2 enzyme comprises one or more polyethylene glycol (PEG) moieties or other conjugates, such as fatty acid or acyl conjugates. In some embodiments, the one or more PEGylated amino acids are selected from lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. In some embodiments, one or more amino acids suitable for PEGylation are introduced by substitution of one or more amino acids in D1L2. In some embodiments, the one or more PEGylated amino acids are lysine or arginine, and the PEGylation is performed by amine conjugation. In some embodiments, the one or more PEGylated amino acids are glutamine (Gln or Q), and the PEGylation is performed by transglutaminase (TGase)-mediated enzyme conjugation. In some embodiments, the one or more PEGylated amino acids are cysteine ​​(Cys or C), and the PEGylation is performed by thiol conjugation. For example, a primary amine in D1L2 can be modified using an NHS-PEG reagent. In some embodiments, one or more PEGylated amino acids are conjugated to a PEG moiety that is independently selected from linear or branched PEGs having a molecular weight in the range of about 2 kDa to about 60 kDa. In some embodiments, the PEG moiety has a molecular weight independently selected from the ranges of about 5 kDa to about 60 kDa, or about 5 kDa to about 40 kDa, or about 5 kDa to about 30 kDa, or about 10 kDa to about 60 kDa, or about 10 kDa to about 40 kDa, or about 10 kDa to about 30 kDa.

[0056] In some embodiments, the recombinant expression system has a deletion or inactivation of one or more proteases that cleave at paired basic amino acids. Exemplary enzymes include aspartic proteinase 3 (Ysp1) and kexin (Kex2) expressed by Pichia pastoris or other yeast systems. In some embodiments, these enzymes are not genetically deleted or inactivated, but their activity is inhibited by protease inhibitors during recombinant protein production.

[0057] Additionally, in some embodiments, the growth medium of the non-mammalian expression system is supplemented with polyanions such as dextran sulfate, heparin, ferric citrate, and EDTA. In further embodiments, the growth medium is supplemented with dextran sulfate having an average molecular weight between 5 kDa and 100 kDa. In some embodiments, the dextran sulfate has an average molecular weight of about 10 kDa or less, or about 20 kDa or less, or about 30 kDa or less, or about 40 kDa or less, or about 50 kDa or less, or about 75 kDa or less, or about 100 kDa or less. In various embodiments, the polyanion is added to the culture in an amount sufficient to form a complex with the recombinant protein produced. In some embodiments, the recombinant D1L2 enzyme or variants thereof is purified from the culture medium by a method that includes dissociation of the enzyme from polyanions such as dextran sulfate, heparin, and EDTA. In certain embodiments, the purification method includes a strong anion exchange resin such as triethylaminoethyl.

[0058] In yet another aspect, the present disclosure provides a pharmaceutical composition for local or topical administration, comprising a recombinant D1L2 enzyme as described herein or produced according to the present disclosure, and a pharma- ceutically acceptable carrier.

[0059] In some embodiments, the pharmaceutical composition is formulated for topical administration, such as in a cream, lotion, gel, solution, foam, spray, ointment, mouthwash, shampoo, or soap. In some embodiments, the pharmaceutical composition is for localized or topical treatment of tissues with an acidic environment and / or biofilm. For example, the acidic environment may have a pH of about 6.0 or less, about 5.5 or less, or about 5.0 or less, or about 4.5 or less, or about 4.0 or less. In some embodiments, the composition is a skin care composition for maintaining healthy skin and an attractive appearance of the skin. In these embodiments, the composition is useful in methods for treating or preventing skin infection and / or inflammation. For example, in some embodiments, the composition is applied to skin containing a parakeratotic lesion. Parakeratosis is a mode of keratinization characterized by retention of the nucleus in the stratum corneum. In skin, this process results in the abnormal replacement of annular scales by nucleated cells. Parakeratosis is accompanied by thinning or loss of the granular layer and is commonly seen in diseases of increased cell turnover, whether inflammatory or neoplastic. Parakeratosis is seen in psoriatic plaques and dandruff.

[0060] In some embodiments using the topical composition, the subject may have a condition that affects the appearance or health of the skin, hair, or nails. In some embodiments, the subject has a condition such as psoriasis, dermatitis, epidermolysis bullosa, or an allergic reaction (including, for example, hives or a rash). In some embodiments, the subject has dry skin, such as xerosis. In some embodiments, the subject has acne vulgaris. In still other embodiments, the subject's skin may be characterized by an overgrowth of Staphylococcus aureus, which may be associated with atopic dermatitis. In some embodiments, the subject has psoriasis, and the composition is applied to a psoriatic lesion.

[0061] In yet other embodiments, the topical composition can be used in methods to promote wound healing and / or reduce or prevent fibrosis, for example, as described in U.S. Patent Application Publication No. 2018 / 0271953, which is incorporated herein by reference in its entirety. In some embodiments, the subject has a chronic wound, such as a diabetic ulcer, an amputation wound, or a skin or tissue graft (including a donor site). In some embodiments, the wound is a burn, such as a first or second degree burn. In some embodiments, the subject has a sunburn that can be treated with a topical lotion, gel, or ointment according to the present disclosure.

[0062] In yet other embodiments, the composition is formulated for administration to one or more of the eye, ear, nasal or sinus cavity, and oral cavity. The compositions according to these embodiments may be useful in methods of treating or preventing inflammation or infection of the eye of a subject, for example, where the composition is formulated as eye drops or eye washes (including eyelid washes). In some embodiments, the subject may have dry eye disease or may have allergic, bacterial, or viral conjunctivitis. Bacterial conjunctivitis may be exacerbated by biofilm formation. In some embodiments, the subject has inflammation of the ocular canal, which may manifest as blepharitis or related conditions (e.g., chalazion). In yet other embodiments, the composition may be useful in treating or preventing inflammation or infection of the ear of a subject, for example, where the composition is formulated as ear drops. An exemplary condition is an ear infection, such as otitis media. In yet other embodiments, the composition may be useful in methods of treating or preventing inflammation or infection of the nose or sinus of a subject, for example, where the composition is formulated as a nasal spray. Exemplary conditions include rhinitis, rhinosinusitis, and post-rhinoplasty. In some embodiments, the subject has a sinus infection or recurrent sinus infection. In yet other embodiments, the subject has an infection or inflammation of the oral cavity, including gingivitis and dental abscesses.

[0063] In some embodiments, the compositions are useful in methods of treating nucleic acid-associated ocular diseases, such as those described in U.S. Patent No. 9,867,871, which is incorporated herein by reference in its entirety, including dry eye disease, interlaminar keratitis, contact lens-associated keratitis, endophthalmitis, infectious crystalline keratopathy, ocular cicatricial pemphigoid (OCP), keratoconjunctivitis sicca (KCS), Sjogren's syndrome (SS), Sjogren's syndrome-associated keratoconjunctivitis sicca, non-Sjogren's syndrome-associated keratoconjunctivitis sicca, keratitis sicca, sicca syndrome, xerophthalmia, tear film disorders, decreased tear production, aqueous tear deficiency syndrome (ATD), and meibomian gland dysfunction (MGD).

[0064] In yet other embodiments, the composition is formulated for pulmonary delivery, such as a powder or solution aerosol. In some embodiments, the composition is formulated for delivery by a nebulizer. In various embodiments, the composition is useful in a method for treating or preventing lung inflammation or infection in a patient. In some embodiments, the subject has cystic fibrosis, pneumonia (including ventilator-associated pneumonia, i.e., VAP), bronchitis, asthma, lower respiratory tract infection, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), transfusion-induced lung injury (TRALI), or chronic obstructive pulmonary disease (COPD). In some embodiments, the subject has pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis, i.e., IPF). In yet other embodiments, the subject has chronic or recurrent lung infections.

[0065] In the case of cystic fibrosis, Pseudomonas aeruginosa infections are difficult to eradicate or control, due in part to the formation of biofilms. By administering the D1L2 enzymes according to the present disclosure, for example by nebulizer, it may be possible to control Pseudomonas infections in cystic fibrosis patients and alleviate some of the overuse of antibiotics. In various embodiments, the D1L2 enzymes provided herein have higher activity in CF lung secretions than PULMOZYME (dornase alfa).

[0066] In yet other embodiments, patients may have indwelling medical devices that tend to favor microbial colonization and biofilm formation, hi some embodiments, the D1L2 enzyme or a composition comprising the D1L2 enzyme is administered to a patient under mechanical ventilation to prevent or reduce biofilm formation.

[0067] The compositions described herein are useful for treating or controlling infection and / or colonization by biofilm microorganisms, including on mammalian tissues and on surfaces of indwelling medical devices. Biofilm-forming pathogens and opportunistic bacteria include Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, Haemophilus influenzae, Group A beta-hemolytic streptococci, Enterobacteriaceae, Mycobacteria (including nontuberculous mycobacteria), and Streptococcus pneumoniae. Biofilm formation by these bacteria causes refractoriness to antibiotics. These pathogens cause the following infections (but are not limited to): native valve endocarditis (NVE), otitis media (OM), chronic bacterial prostatitis, cystic fibrosis (CF), bloodstream infections, urinary tract infections (optionally associated with biofilms on urinary catheters), prosthetic valve endocarditis (PVE), eye infections (optionally associated with biofilms on contact lenses), and pelvic inflammatory disease (optionally associated with biofilms on intrauterine devices (IUDs)).

[0068] As used herein, "treatment" or "treat" or "treated" refers to a therapeutic procedure aimed at delaying (alleviating) an undesirable physiological condition, disorder or disease, or achieving a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, reduction in the extent of a condition, disorder or disease, stabilization (i.e., not worsening) of a condition, disorder or disease state, delay in onset or slowing of progression of a condition, disorder or disease, remission of a condition, disorder or disease state, and remission (whether partial or complete), or improvement or reversal of a condition, disorder or disease, whether detectable or undetectable. Treatment includes causing a clinically significant response without excessive levels of side effects. In other embodiments, "treatment" or "treat" or "treated" refers to a preventative measure, the purpose of which is to delay onset or reduce the severity of an undesirable physiological condition, disorder or disease, for example in a person with a predisposition to the disease.

[0069] As used herein, the term "about" means plus or minus 10% of the associated numerical value. EXAMPLES

[0070] Recombinant expression and extracellular chromatin degradation activity of DNASE1L2 Amino acid sequence alignment of DNASE1L2 isoform 1 and isoform 2 identifies a proline-rich domain GERAPPLPSRRALTPPPLPAA (SEQ ID NO: 3) in the middle of DNASE1L2 isoform 1 (Figure 1). DNASE1L2 isoform 1 (SEQ ID NO: 1) was cloned and expressed in Pichia pastoris with the native secretory signal peptide. Analysis of the culture supernatant detected only trace amounts of DNASE1L2 protein, indicating that DNASE1L2 isoform 1 is not suitable for production in Pichia pastoris (Figure 2). The native secretory signal peptide was replaced with α-mating factor prepropeptide (aMF; SEQ ID NO: 9), which enhances protein secretion in Pichia pastoris. However, no significant amount of target protein could be detected in the culture supernatant, and secretion of the new aMF-DNASE1L2 isoform 1 construct by Pichia pastoris remained low.

[0071] In this study, we investigated an expression vector containing aMF and DNASE1L2 isoform 2. Surprisingly, this new construct was robustly expressed and secreted by Pichia pastoris, and significant amounts of the target protein could be detected in the culture supernatant (Figure 2). Thus, removal of the PRD allows for large-scale production of DNASE1L2 in yeast expression systems such as Pichia pastoris.

[0072] Fermentation was performed at a 5L scale, and PRDD-DNASE1L2 (SEQ ID NO: 2) was purified using affinity chromatography. The purified protein was tested for enzymatic activity. Previous reports have measured the degradation of purified DNA by DNASE1L2, but in this example, high molecular weight chromatin derived from intact nuclei of HEK293 cells was used as a substrate. Robust chromatin degradation activity of PRDD-DNASE1L2 was observed at pH levels below 6.5, while basic domain deleted DNASE1L3 showed any chromatin degradation at neutral pH (Figure 3). These data indicate that the deletion of the PRD in DNASE1L2 allows for the large-scale production of an enzyme that efficiently degrades chromatin in an acidic environment.

[0073] Alignment of DNASE1L2 amino acid sequences from different species in Uniprot showed that isoform 1 containing the PRD is expressed in human, chimpanzee and baboon, but not in mouse, rat, rabbit, dog, pig, guinea pig, cow and elephant. Furthermore, only DNASE1L2 from human, chimpanzee and baboon features a C-terminal arginine residue (Figure 4). For this reason, derivatives with deletion of the C-terminal arginine residue were studied in the hope of finding DNASE1L2 mutants with improved characteristics. Surprisingly, as shown in Figure 5, screening of Pichia pastoris clones expressing the DNASE1L2 isoform 2 R278del mutant (SEQ ID NO: 7) identified three clones with substantially increased production levels compared to DNASE1L2 isoform 2 (SEQ ID NO: 6).

[0074] PRDD-DNASE1L2 and its variants are particularly suited to degrade extracellular chromatin, including neutrophil extracellular traps (NETs), not only on the skin, which has a naturally acidic pH, but also under acidic conditions, for example in the acidic sputum of patients with inflammatory lung diseases such as cystic fibrosis and asthma (Kodric M, Shah AN, Fabbri LM, et al. An investigation of airway acidification in asthma using induced sputum: A study of feasibility and correlation. Am J Respir Crit Care Med. 2007;175(9):905-10, Dropping acid: why is cystic fibrosis mucus abnormal? Eur Respir J. 2018 Dec 6;52(6):1802057).

[0075] Recombinant expression of PRDD-DNASE1L2 as a fusion with albumin has also been achieved in Pichia pastoris. Albumin was fused at the N-terminus of PRDD-DNASE1L2 via a flexible linker composed of Gly and Ser. In these embodiments, the PRDD-DNASE1L2 fusion is secreted by the human albumin secretory signal peptide (SEQ ID NO: 10). This system similarly results in good expression of PRDD-DNASE1L2.

[0076] Sequence Listing Wild-type DNASE1L2 enzyme SEQ ID NO:1 TIFF2024508753000002.tif51170

[0077] SEQ ID NO:2 TIFF2024508753000003.tif41170

[0078] Proline-rich domain in DNASE1L2 isoform 1 SEQ ID NO:3 GERAPPLPSRRALTPPPLPAA

[0079] DNASE1L2 mutants SEQ ID NO:4 TIFF2024508753000004.tif41170

[0080] SEQ ID NO:5 TIFF2024508753000005.tif46170

[0081] SEQ ID NO:6 Human D1L2 isoform 2: mature protein LRIGAFNIQSFGDSKVSDPACGSIIAKILAGYDLALVQEVRDPDLSAVSALMEQINSVSEHEYSFVSSQPLGRDQYKEMYLFVYRKDAVSVVDTYLYPDPEDVFSREPFVVKFSAPGTAQNLVLIPLH AAPHQAVAEIDALYDVYLDVIDKWGTDDMLFLGDFNADCSYVRAQDWAAIRLRSSEVFKWLIPDSADTTVGNSDCAYDRIVACGARLRRSLKPQSATVHDFQEEFGLDQTQALAISDHFPVEVTLKFHR

[0082] SEQ ID NO:7 Human D1L2 isoform 2 variant: R278del mutant, mature protein LRIGAFNIQSFGDSKVSDPACGSIIAKILAGYDLALVQEVRDPDLSAVSALMEQINSVSEHEYSFVSSQPLGRDQYKEMYLFVYRKDAVSVVDTYLYPDPEDVFSREPFVVKFSAPGTAQNLVLIPLH AAPHQAVAEIDALYDVYLDVIDKWGTDDMLFLGDFNADCSYVRAQDWAAIRLRSSEVFKWLIPDSADTTVGNSDCAYDRIVACGARLRRSLKPQSATVHDFQEEFGLDQTQALAISDHFPVEVTLKFH

[0083] Secretory signal peptide SEQ ID NO:8 D1L2 signal peptide MGGPRALLAALWALEAAGTAA

[0084] SEQ ID NO:9 Pichia pastoris α-mating factor MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVS

[0085] SEQ ID NO:10 Human albumin secretory signal peptide + propeptide MKWVTFISLLFLFSSAYSRGVFRR

[0086] SEQ ID NO:13 D1 signal peptide MRGMKLLGALLALAALLQGAVS

[0087] SEQ ID NO:14 D1L1 signal peptide MHYPTALLFLILANGAQA

[0088] SEQ ID NO:15 D1L3 signal peptide MSRELAPLLLLLLSIHSALA

[0089] Albumin sequence SEQ ID NO:11 Human serum albumin (mature protein): DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRH PYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAE VENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0090] Linker sequence SEQ ID NO:12 Flexible Peptide Linker: GGGGSGGGGSGGGGS

Claims

1. A pharmaceutical composition comprising a DNASE1L2 (D1L2) enzyme for treating a subject in need of extracellular DNA degradation, extracellular chromatin degradation, extracellular trap (ET) degradation and / or neutrophil extracellular trap (NET) degradation, wherein the D1L2 enzyme substantially lacks a proline-rich domain (PRD) defined by SEQ ID NO:3 and / or lacks a C-terminal arginine residue.

2. 2. The pharmaceutical composition of claim 1, wherein the D1L2 enzyme substantially lacks a PRD.

3. 2. The pharmaceutical composition of claim 1, wherein the D1L2 enzyme lacks a C-terminal arginine residue.

4. 2. The pharmaceutical composition of claim 1, wherein the D1L2 enzyme substantially lacks a PRD and lacks a C-terminal arginine residue.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the D1L2 enzyme is PEGylated.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the D1L2 is fused to a carrier protein at the N-terminus or C-terminus.

7. The pharmaceutical composition of claim 6, wherein the carrier protein is an albumin amino acid sequence.

8. 8. The pharmaceutical composition of claim 7, wherein the albumin amino acid sequence is fused to the N-terminus of D1L2, optionally via a peptide linker.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the subject accumulates extracellular DNA, extracellular chromatin, extracellular traps (ET) and / or neutrophil extracellular traps (NET) in an acidic environment.

10. 10. The pharmaceutical composition of claim 1, wherein the subject has a pulmonary indication, a skin condition, a wound, eye inflammation or infection, ear inflammation or infection, or nasal or sinus inflammation or infection.

11. 11. The pharmaceutical composition of claim 10, wherein the subject has cystic fibrosis, pneumonia, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), transfusion-induced lung injury (TRALI), asthma, chronic obstructive pulmonary disorder (COPD), pulmonary fibrosis or a lung infection.

12. 12. The pharmaceutical composition of any one of claims 1 to 11, wherein the D1L2 enzyme comprises an amino acid sequence having at least about 80% sequence identity, or at least about 85%, at least about 90%, or at least about 95%, or at least about 97% sequence identity to the enzyme defined by SEQ ID NO:5 or SEQ ID NO:

7.

13. A method for producing a recombinant D1L2 enzyme, comprising culturing a non-mammalian expression host cell encoding a D1L2 enzyme that is substantially lacking a proline-rich domain (PRD) defined by SEQ ID NO:3 and / or lacks a C-terminal arginine and includes a signal peptide, and recovering the D1L2 enzyme from the culture medium.

14. A recombinant D1L2 enzyme produced according to the method of claim 13.

15. 15. A polynucleotide encoding the D1L2 enzyme of claim 14, which is codon-optimized for expression in a non-mammalian expression system, and / or the non-mammalian expression system is Pichia pastoris.

16. A non-mammalian host cell comprising a vector comprising the polynucleotide of claim 15 and expressing the D1L2 enzyme.

17. A recombinant D1L2 enzyme according to claim 14, or a D1L2 enzyme substantially lacking the PRD of SEQ ID NO:3 and / or lacking a C-terminal arginine residue. A pharma- ceutically acceptable carrier; Including, Formulated for topical or local administration; Pharmaceutical compositions.

18. 18. A pharmaceutical composition according to claim 17 for treating or preventing infection and / or inflammation of the skin.

19. 18. The pharmaceutical composition according to claim 17 for promoting wound healing.

20. A pharmaceutical composition as described in claim 17 for treating or preventing eye inflammation or infection, wherein the pharmaceutical composition is formulated as an eye drop or eyewash.

21. The pharmaceutical composition of claim 17 for treating or preventing ear inflammation or infection, wherein the pharmaceutical composition is formulated as ear drops.

22. A pharmaceutical composition as described in claim 17 for treating or preventing inflammation or infection of the nasal cavity or paranasal sinuses, wherein the pharmaceutical composition is formulated as a nasal spray.

23. A pharmaceutical composition as described in claim 17 for treating or preventing pulmonary inflammation or infection, wherein the pharmaceutical composition is formulated as a powder aerosol or solution aerosol, or formulated as a nebulizer.

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