WO2016069889A1

WO2016069889A1 - Therapeutic nuclease-transferrin fusions and methods

Info

Publication number: WO2016069889A1
Application number: PCT/US2015/058039
Authority: WO
Inventors: James Arthur Posada; Chris GABEL
Original assignee: Resolve Therapeutics, Llc
Priority date: 2014-10-31
Filing date: 2015-10-29
Publication date: 2016-05-06

Abstract

The invention provides for hybrid nuclease-transferrin molecules with increased pharmacokinetic properties. The hybrid nuclease-transferrin molecules of the invention have one or more nuclease domains (e.g., an RNase and/or DNase domain) operably coupled to a transferrin, or a variant or fragment thereof. The invention also provides methods of treating or preventing a condition associated with an abnormal immune response.

Description

THERAPEUTIC NUCLEASE-TRANSFERRIN FUSIONS AND METHODS RLATED APPLICATIONS

This application claims the benefit of the priority date of U.S. Provisional Application No.62/073,857, which was filed on October 31, 2014. The content of this provisional application is hereby incorporated by reference in its entirety. BACKGROUND

Excessive release of (ribo)nucleoprotein particles from dead and dying cells can cause lupus pathology by two mechanisms: (i) Deposition or in situ formation of chromatin / anti-chromatin complexes causes nephritis and leads to loss of renal function; and (ii) nucleoproteins activate innate immunity through toll-like receptor (TLR) 7, 8, and 9 as well as TLR-independent pathway(s). Release of nucleoproteins can serve as a potent antigen for autoantibodies in SLE, providing amplification of B cell and DC activation through co-engagement of antigen receptors and TLRs. Thus, there exists a need for a means to remove inciting antigens and/or attenuate immune stimulation, immune amplification, and immune complex mediated disease in subjects in need thereof, for example, with long-acting nuclease molecules that attack circulating immune complexes by digesting nucleic acids contained therein. SUMMARY OF THE INVENTION

The invention relates, in part, to a hybrid nuclease-transferrin molecule comprising a first nuclease domain and a transferrin, or a variant or fragment thereof, wherein the first nuclease domain is operably coupled to the N- or C-terminus of transferrin, or variant or fragment thereof (i.e., hybrid nuclease-transferrin molecules), wherein the hybrid nuclease-transferrin molecule exhibits enhanced pharmacokinetic activity relative to the first nuclease domain alone. Such hybrid nuclease-transferrin molecules exhibit altered, e.g., improved, serum half-life relative to the first nuclease domain alone. In some embodiments, the hybrid nuclease-transferrin molecule further includes a first linker domain, and the first nuclease domain is operably coupled to the transferrin, or a variant or fragment thereof, via the first linker domain. In some embodiments, the first nuclease domain is an RNase or DNase, for example, RNase1 and DNase1, respectively. In some embodiments, the hybrid nuclease-transferrin molecule further includes a second nuclease domain (e.g., an RNase or DNase domain), which is operably coupled to the first nuclease domain or the N- or C-terminus of transferrin, or a variant or fragment thereof, optionally via a linker. In some embodiments, the first and second nuclease domains are the same, e.g., RNase and RNase, or DNase and DNase. In other embodiments, the first and second nuclease domains are different, e.g., RNase and DNase. In some embodiments, the RNase domain is a wild-type RNase, such as wild-type human RNase1. In other embodiments, the RNase domain is a mutant RNase, such as an aglycosylated, underglycosylated, or deglycosylated RNase 1, such as human RNase1 N34S/N76S/N88S (SEQ ID NO: 61). In some embodiments, the RNase containing hybrid nuclease-transferrin molecule degrades circulating RNA and RNA in immune complexes, or inhibits interferon-alpha production, or both. In yet other embodiments, the activity of the RNase is not less than about 10-fold less, such as 9-fold less, 8-fold less, 7-fold less, 6-fold less, 5-fold less, 4-fold less, 3-fold less, or 2-fold less than the activity of a control RNase molecule. In yet other embodiments, the activity of the RNase is about equal to the activity of a control RNase molecule. In some embodiments, the DNase domain is wild type DNase, such as wild type, human DNase1. In other embodiments, the DNase domain is a mutant DNase domain, such as mutant, human DNase1 A114F (SEQ ID NO: 52) or an aglycosylated, underglycosylated, or deglycosylated human DNase, such as mutant, human DNase1 N18S/N106S/A114F (SEQ ID NO: 72). In some embodiments, the DNase containing hybrid nuclease- transferrin molecule degrades circulating DNA and DNA in immune complexes, or inhibits interferon-alpha production, or both. In yet other embodiments, the activity of the DNase is not less than about 10-fold less, such as 9-fold less, 8-fold less, 7-fold less, 6-fold less, 5-fold less, 4-fold less, 3-fold less, or 2-fold less than the activity of a control DNase molecule. In yet other embodiments, the activity of the DNase is about equal to the activity of a control DNase molecule. In some embodiments, the hybrid nuclease-transferrin molecule has a gly-ser linker separating the first and second nuclease domains, and/or the nuclease domains from the transferrin, or a variant or fragment thereof. In some embodiments, the hybrid nuclease-transferrin molecule has an increased serum half-life and/or activity relative to a molecule that does not contain the transferrin, or variant or fragment thereof. In some embodiments, the transferrin, a variant or fragment thereof, is from human, cow, pig, sheep, dog, rabbit, rat, mouse, hamster, echnida, platypus, chicken, frog, hornworm, monkey, horse, or bovine. Preferably, the transferrin is human serum transferrin (HST; SEQ ID NO: 1). In some embodiments, the transferrin variant is more than 80%, such greater than 85%, greater than 90%, or greater than 95% identical to the amino acid sequence of HST (SEQ ID NO: 1). In some embodiments, the transferrin is a fragment of transferrin or a variant thereof. In some embodiments, the fragment is at least 20 amino acids, such as at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, or at least 500 amino acids in length. In some aspects, the hybrid nuclease-transferrin molecule may include the mutant, human DNase1 A114F domain set forth in SEQ ID NO: 52. In another embodiment, the hybrid nuclease-transferrin molecule may include the mutant, human DNase1

N18S/N106S/A114F domain set forth in SEQ ID NO: 72. In some embodiments, the DNase domain is mutant human DNase1 E13R/N74K/A114F/T205K (SEQ ID NO: 86). In other embodiments, the DNase domain is mutant human DNase1

E13R/N74K/A114F/T205K/N18S/N106S (SEQ ID NO:87). In another embodiment, the hybrid nuclease-transferrin molecule may include the human, wild-type RNase1 domain set forth in SEQ ID NO: 60. In another embodiment, the hybrid nuclease-transferrin molecule may include the human, mutant RNase1

N34S/N76S/N88S domain set forth in SEQ ID NO: 61. In another embodiment, the hybrid nuclease-transferrin molecule may include HST set forth in SEQ ID NO: 1. In another embodiment, the hybrid nuclease-transferrin molecule may include the

(Gly₄Ser)3 linker domain set forth in SEQ ID NO: 73. In another embodiment, the hybrid nuclease-transferrin molecule may include a VK3LP leader (SEQ ID NO: 83). These individual domains can be operably coupled to each other in any order to form a hybrid nuclease-transferrin molecule that is enzymatically active. In some aspects, the invention provides hybrid nuclease-transferrin molecules having the amino acid sequences set forth in SEQ ID NOs: 3-50. In other aspects, the hybrid nuclease-transferrin molecules have amino acid sequences at least 90% identical to the amino acid sequences set forth in SEQ ID NOs: 3-50. In some aspects, the invention provides compositions including the hybrid nuclease- transferrin molecules and a carrier, such as a pharmaceutically acceptable carrier or diluent. In some aspects, the invention provides nucleic acid molecules that encode the hybrid nuclease-transferrin molecules disclosed herein. In some embodiments, the invention provides a recombinant expression vector having a nucleic acid molecule that encodes the hybrid nuclease-transferrin molecules disclosed herein. In some embodiments, the invention provides host cells transformed with the recombinant expression vectors containing the nucleic acid sequences encoding the hybrid nuclease-transferrin molecules disclosed herein. Also disclosed herein is a method of making a hybrid nuclease- transferrin molecule disclosed herein involving providing a host cell comprising a nucleic acid sequence that encodes the hybrid nuclease-transferrin molecule; and maintaining the host cell under conditions in which the hybrid nuclease-transferrin molecule is expressed. Also disclosed herein is a method for treating or preventing a condition associated with an abnormal immune response by administering to a patient in need thereof an effective amount of an isolated hybrid nuclease-transferrin molecule disclosed herein. In some embodiments, the condition is an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, an experimental form of uveoretinitis, Hashimoto’s thyroiditis, primary myxoedema, thyrotoxicosis, pernicious anaemia, autoimmune atrophic gastritis, Addison’s disease, premature menopause, male infertility, juvenile diabetes, Goodpasture’s syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune haemolytic anaemia, idiopathic leucopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerative colitis, Sjogren’s syndrome, scleroderma, Wegener’s granulomatosis, polymyositis, dermatomyositis, discoid LE, systemic lupus erythematosus (SLE), and connective tissue disease. In some

embodiments, the autoimmune disease is SLE or Sjogren’s syndrome. Also disclosed herein is a method of treating SLE or Sjogren’s syndrome comprising administering to a subject a nuclease-containing composition in an amount effective to degrade immune complexes containing RNA, DNA or both RNA and DNA. In some aspects, the composition includes a pharmaceutically acceptable carrier and a hybrid nuclease-transferrin molecule as described herein. In other aspects, the composition includes a hybrid nuclease-transferrin molecule with the amino acid sequences set forth in SEQ ID NO: 3-50. In another aspect, the invention relates to hybrid nuclease-transferrin molecules for use in treating diseases characterized by defective clearance or processing of apoptotic cells and cell debris, such as SLE. In some embodiments, the hybrid nuclease-transferrin molecules are those set forth in SEQ ID NOs: 3-50. In another aspect, the invention relates to the use of the hybrid nuclease-transferrin molecules for manufacturing a medicament for treating diseases characterized by defective clearance or processing of apoptotic cells and cell debris, such as SLE. In some embodiments, the hybrid nuclease-transferrin molecules are those set forth in SEQ ID NOs: 3-50. BRIEF DESCRIPTION OF THE DRAWINGS These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawing, where: FIG.1 is a schematic depicting the various configurations of the hybrid nuclease- transferrin fusions described herein. DETAILED DESCRIPTION

Overview Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by the presence of high titer autoantibodies directed against self nucleoproteins. There is strong evidence that defective clearance or processing of dead and dying cells in SLE leads to disease, predominantly through accumulation of ribo- and deoxy- ribonucleoproteins (abbreviated nucleoproteins). The nucleoproteins cause damage through three mechanisms: i) activation of the innate immune system to produce inflammatory cytokines; ii) serve as antigens to generate circulating immune complexes; and iii) serve as antigens to generate in situ complex formation at local sites such as the kidney. The present invention provides methods for treating diseases characterized by defective clearance or processing of apoptotic cells and cell debris, such as SLE and Sjogren’s syndrome, by administering an effective amount of a long-acting nuclease activity to degrade extracellular RNA and DNA containing immune complexes. Such treatment can inhibit production of Type I interferons (IFNs) which are prominent cytokines in SLE and are strongly correlated with disease activity and nephritis. The present invention relates, in part, to the provision of such long-acting nucleases. In particular, the invention relates to nucleases that are operatively coupled to transferrin, or a variant or fragment thereof. Transferrin is an iron-binding blood plasma glycoprotein that controls free iron levels. Wild-type serum transferrin has a serum half-life of about 7-10 days. When fused to nucleases, the resulting hybrid nuclease-transferrin molecules exhibit altered serum half-life. Another advantage conferred by transferrin is that it does not activate effector Fc receptors, and thus the hybrid nuclease-transferrin molecules may avoid toxicity associated with activating these receptors. Accordingly, in one embodiment, a subject with a disease characterized by defective clearance or processing of apoptotic cells and cell debris is treated by administering a hybrid nuclease-transferrin molecule, which includes one or more nuclease domains (e.g., a DNase, RNase or combination) coupled to a transferrin, or a variant or fragment thereof, such that the hybrid nuclease-transferrin molecule has increased bioavailability and/or serum half-life relative to the non-conjugated nuclease domain. In one aspect, a hybrid nuclease-transferrin molecule includes first and second nuclease domains. In another aspect, a method of treating SLE or Sjogren’s syndrome is provided in which a sufficient or effective amount of a nuclease-transferrin molecule-containing composition is administered to a subject. In one aspect, treatment results in degradation of immune complexes containing RNA, DNA or both RNA and DNA. In another aspect, treatment results in inhibition of Type I interferons, such as interferon-α, in a subject. In one aspect, a method of treating a subject comprises administering an effective amount of a composition of a hybrid nuclease-transferrin molecule having an amino acid sequence set forth in SEQ ID NOs: 3-50. In another aspect, the invention relates to hybrid nuclease-transferrin molecules for use in treating diseases characterized by defective clearance or processing of apoptotic cells and cell debris, such as SLE. In some embodiments, the hybrid nuclease-transferrin molecules are those set forth in SEQ ID NOs: 3-50. In another aspect, the invention relates to the use of the hybrid nuclease-transferrin molecules for manufacturing a medicament for treating diseases characterized by defective clearance or processing of apoptotic cells and cell debris, such as SLE. In some embodiments, the hybrid nuclease-transferrin molecules are those set forth in SEQ ID NOs: 3-50. Definitions

Terms used in the claims and specification are defined as set forth below unless otherwise specified. "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes. An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different "replacement" amino acid residue. An "amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, larger "peptide insertions" can be made, e.g. insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) may be naturally occurring or non-naturally occurring as disclosed above. An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence. "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer. "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res 1991;19:5081; Ohtsuka et al., JBC 1985;260:2605-8); Rossolini et al., Μοl Cell Probes 1994;8:91-8). For arginine and leucine, modifications at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. Polynucleotides of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms. As used herein, the term“operably linked” or“operably coupled” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. As used herein, the term "hybrid nuclease-transferrin molecule" refers to polynucleotides or polypeptides that comprise at least one nuclease domain and a transferrin, or a variant or fragment thereof. Hybrid nuclease-transferrin molecules are also referred to as fusion protein(s) and fusion gene(s). For example, in one embodiment, a hybrid nuclease- transferrin molecule can be a polypeptide comprising a transferrin, or a variant or fragment thereof, operably coupled to a nuclease domain such as DNase and/or RNase. As another example, a hybrid nuclease-transferrin molecule can include an RNase nuclease domain, a linker domain, and a transferrin, or a variant or fragment thereof. SEQ ID NOs: 3-50 are examples of hybrid nuclease-transferrin molecules. Other examples are described in more detail below. In one embodiment a hybrid nuclease- transferrin molecule of the invention can have altered glycosylation or include additional modifications. In another embodiment, a hybrid nuclease-transferrin molecule may be modified to add a functional moiety (e.g., a drug or label). As used herein, the term“transferrin” refers to a vertebrate glycoprotein that functions to bind and transport iron. Human transferrin is a glycosylated 698 amino acid protein (SEQ ID NO: 2). In one embodiment, the transferrin is human serum transferrin (“HST”; SEQ ID NO: 1), which lacks the 19 amino acid leader found in human transferrin. The term“transferrin activity” refers to the ability of transferrin, or a variant or fragment thereof, to prolong the half-life of a hybrid nuclease-transferrin molecule compared to a nuclease not fused to transferrin. As used herein, the term "wild-type" (WT) transferrin means transferrin having the same amino acid sequence as naturally found in an animal or in a human being. As used herein, the term“variant” refers to a polypeptide derived from a wild-type transferrin and differs from the wild-type transferrin by one or more alteration(s), i.e., a substitution, insertion, and/or deletion, at one or more positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid. A deletion means removal of an amino acid occupying a position. An insertion means adding 1 or more, such as 1-3 amino acids, immediately adjacent to an amino acid occupying a position. Variant transferrins necessarily have less than 100% sequence identity or similarity with the wild-type transferrin. In a preferred embodiment, the variant transferrin will have an amino acid sequence from about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of wild-type transferrin, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o, 99%) and most preferably from about 95% to less than 100%, e.g., over the length of the variant molecule. For purposes of the present invention, the polypeptide of human serum transferrin (HST) set forth in SEQ ID NO: 1 is used to determine the corresponding amino acid residue in another transferrin, e.g., a transferrin variant or natural transferrin variant. The amino acid sequence of another transferrin is aligned with the mature polypeptide set forth in SEQ ID NO: 1, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the mature polypeptide set forth in SEQ ID NO: 1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, JMB 1970;48:443-53) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., Trends Genet 2000;16:276-7), preferably version 3.0.0 or later. Identification of the

corresponding amino acid residue in another transferrin can be confirmed by an alignment of multiple polypeptide sequences using "ClustalW" (Larkin et al.,

Bioinformatics 2007;23:2947-8). When the other polypeptide (or protein) has diverged from the polypeptide set forth in SEQ ID NO: 1 such that traditional sequence-based comparison fails to detect their relationship (Lindahl and Elofsson, JMB 2000;295:613- 15), other pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searching can be attained using search programs that utilize probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologs (Atschul et al., Nucleic Acids Res 1997;25:3389- 402). Even greater sensitivity can be achieved if the family or superfamily for the polypeptide has one or more representatives in the protein structure databases. Programs such as GenTHREADER (Jones, JMB 1999;287:797-815; McGuffin and Jones,

Bioinformatics 2003;19:874-81) utilize information from a variety of sources (PSI- BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as inputs to a neural network that predicts the structural fold for a query sequence. Similarly, the method of Gough et al., JMB 2000;313:903-19, can be used to align a sequence of unknown structure within the superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose. The term "fragment," when used in the context of transferrin, refers to any fragment of HST or a variant thereof that extends the half-life of a nuclease domain to which it is fused or conjugated to relative to the corresponding non-fused nuclease domain. A fragment of a transferrin may be referred to as a“portion,”“region,” or“moiety.” In some embodiments, a fragment of a transferrin can refer to a polypeptide comprising a fusion of multiple domains of transferrin (see, e.g., WO 2011/124718), such as the C and N domains, as described in more detail infra. As used herein, the term“serum half-life” refers to the time required for the in vivo serum hybrid nuclease-transferrin molecule concentration to decline by 50%. The shorter the serum half-life of the hybrid nuclease-transferrin molecule, the shorter time it will have to exert a therapeutic effect, although in some embodiments as discussed infra, a shorter serum half-life of the hybrid nuclease-transferrin molecule is desirable. A "longer serum half-life" and similar expressions are understood to be in relationship to the

corresponding wild-type transferrin molecule (e.g., HST with the amino acid sequence of SEQ ID NO: 1). Thus, a variant with longer serum half-life means that the variant has a longer serum half-life than the corresponding wild-type transferrin. As used herein, the term“glycosylation” or“glycosylated” refers to a process or result of adding sugar moieties to a molecule (e.g., a hybrid nuclease-transferrin molecule). As used herein, the term“altered glycosylation” refers to a molecule that is

aglycosylated, deglycosylated, or underglycosylated. As used herein,“glycosylation site(s)” refers to both sites that potentially could accept a carbohydrate moiety, as well as sites within the protein on which a carbohydrate moiety has actually been attached and includes any amino acid sequence that could act as an acceptor for an oligosaccharide and/or carbohydrate. As used herein, the term“aglycosylation” or“aglycosylated” refers to the production of a molecule (e.g., a hybrid nuclease-transferrin molecule) in an unglycosylated form (e.g., by engineering a hybrid nuclease-transferrin molecule to lack amino acid residues that serve as acceptors of glycosylation). Alternatively, the hybrid nuclease-transferrin molecule can be expressed in, e.g., E. coli, to produce an aglycosylated hybrid nuclease- transferrin molecule. As used herein, the term“deglycosylation” or“deglycosylated” refers to the process or result of enzymatic removal of sugar moieties on a molecule. As used herein, the term“underglycosylation” or“underglycosylated” refers to a molecule in which one or more carbohydrate structures that would normally be present if produced in a mammalian cell has been omitted, removed, modified, or masked. In certain aspects, the hybrid nuclease-transferrin molecules of the invention can employ one or more "linker domains," such as polypeptide linkers. As used herein, the term "linker domain" refers to a sequence which connects two or more domains in a linear sequence. As used herein, the term "polypeptide linker" refers to a peptide or

polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) which connects two or more domains in a linear amino acid sequence of a polypeptide chain. For example, polypeptide linkers may be used to connect a nuclease domain to a transferrin, or a variant or fragment thereof. Preferably, such polypeptide linkers can provide flexibility to the polypeptide molecule. In certain embodiments the polypeptide linker is used to connect (e.g., genetically fuse) a transferrin, or a variant or fragment thereof, with one or more nuclease domains. A hybrid nuclease-transferrin molecule of the invention may comprise more than one linker domain or peptide linker. Various peptide linkers are known in the art. As used herein, the term "gly-ser polypeptide linker" refers to a peptide that consists of glycine and serine residues. An exemplary gly/ser polypeptide linker comprises the amino acid sequence (Gly₄Ser)n. In some embodiments, n is 1 or more, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (e.g., (Gly₄Ser)10). Another exemplary gly/ser polypeptide linker comprises the amino acid sequence Ser(Gly₄Ser)n. In some embodiments, n is 1 or more, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (e.g., Ser(Gly₄Ser)10). As used herein, the terms“coupled,”“linked,”“fused,” or“fusion,” are used

interchangeably. These terms refer to the joining together of two more elements or components or domains, by whatever means including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art. A polypeptide or amino acid sequence "derived from" a designated polypeptide or protein refers to the origin of the polypeptide. Preferably, the polypeptide or amino acid sequence which is derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the sequence. Polypeptides derived from another peptide may have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions. In one embodiment, there is one amino acid difference between a starting polypeptide sequence and the sequence derived therefrom. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. In one embodiment, a polypeptide of the invention consists of, consists essentially of, or comprises an amino acid sequence selected from Table 1 and functionally active variants thereof. In an embodiment, a polypeptide includes an amino acid sequence at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence set forth in Table 1. In some embodiments, a polypeptide includes a contiguous amino acid sequence at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a contiguous amino acid sequence set forth in Table 1. In some embodiments, a

polypeptide includes an amino acid sequence having at least 10, such as at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 200, at least 300, at least 400, or at least 500 (or any integer within these numbers) contiguous amino acids of an amino acid sequence set forth in Table 1. In some embodiments, the peptides of the invention are encoded by a nucleotide sequence. Nucleotide sequences of the invention can be useful for a number of

applications, including: cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of a host in need thereof, antibody generation for, e.g., passive immunization, PCR, primer and probe generation, siRNA design and generation (see, e.g., the Dharmacon siDesign website), and the like. In some

embodiments, the nucleotide sequence of the invention comprises, consists of, or consists essentially of, a nucleotide sequence that encodes the amino acid sequence of the hybrid nuclease-transferrin molecules selected from Table 1. In some embodiments, a nucleotide sequence includes a nucleotide sequence at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence encoding the amino acid sequence of the hybrid nuclease-transferrin molecules in Table 1. In some embodiments, a nucleotide sequence includes a

contiguous nucleotide sequence at least 80%, such as at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a contiguous nucleotide sequence encoding the amino acid sequences set forth in Table 1. In some embodiments, a nucleotide sequence includes a nucleotide sequence having at least 10, such as at least 15, such as at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 200, at least 300, at least 400, or at least 500 (or any integer within these numbers) contiguous nucleotides of a nucleotide sequence encoding the amino acid sequences set forth in Table 1. In some embodiments, polypeptide sequences of the invention are not immunogenic and/or have reduced immunogenicity. It will also be understood by one of ordinary skill in the art that the hybrid nuclease- transferrin molecules of the invention may be altered such that they vary in sequence from the naturally occurring or native sequences from which their components (e.g., nuclease domains, linker domains, and transferrin domains) are derived, while retaining the desirable activity of the native sequences. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at "non-essential" amino acid residues may be made. An isolated nucleic acid molecule encoding a non-natural variant of a hybrid nuclease-transferrin molecule derived from a transferrin (e.g., HST) can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the transferrin such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR- mediated mutagenesis. The hybrid nuclease-transferrin molecules of the invention may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non- essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in a hybrid nuclease-transferrin molecule is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members. Alternatively, in another embodiment, mutations may be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into hybrid nuclease- transferrin molecules of the invention and screened for their ability to bind to the desired target. The term "ameliorating" refers to any therapeutically beneficial result in the treatment of a disease state, e.g., an autoimmune disease state (e.g., SLE, Sjogren’s syndrome), including prophylaxis, lessening in the severity or progression, remission, or cure thereof. The term "in situ" refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture. The term "in vivo" refers to processes that occur in a living organism. The term "mammal" or "subject" or "patient" as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines. The term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence

comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test

sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv Appl Math 1981;2:482, by the homology alignment algorithm of Needleman & Wunsch, J Mol Biol 1970;48:443, by the search for similarity method of Pearson & Lipman, PNAS 1988;85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al, infra). One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J Mol Biol 1990;215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. The term "sufficient amount" means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell. The term "therapeutically effective amount" is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a "prophylactically effective amount" as prophylaxis can be considered therapy. The term“about” will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used,“about” will mean up to plus or minus 10% of the particular value. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Hybrid nuclease-transferrin molecules

The hybrid nuclease-transferrin molecules of the invention include a transferrin, or a variant or fragment thereof, that alters the serum half-life of the nuclease molecules to which it is fused compared to nuclease molecules that are not fused to the transferrin, or a variant or fragment thereof. Such hybrid nuclease-transferrin molecules are referred to herein as “hybrid nuclease-transferrin molecules” or “hybrid transferrin-nuclease molecules,” which are used interchangeably. In some embodiments, a composition of the invention includes a hybrid nuclease- transferrin molecule. In some embodiments, a hybrid nuclease-transferrin molecule includes a nuclease domain operably coupled to a transferrin, or a variant or fragment thereof. In some embodiments the hybrid nuclease-transferrin molecule is a nuclease protein. In some embodiments, the hybrid nuclease-transferrin molecule is a nuclease polynucleotide. In some embodiments, the nuclease domain is operably coupled to the transferrin, or a variant or fragment thereof, via a linker domain. In some embodiments, the linker domain is a linker peptide. In some embodiments, the linker domain is a linker nucleotide. In some embodiments, the hybrid nuclease-transferrin molecule includes a leader molecule, e.g., a leader peptide. In some embodiments, the leader molecule is a leader peptide positioned at the N-terminus of the nuclease domain. In embodiments, a hybrid nuclease-transferrin molecule of the invention comprises a leader peptide at the N- terminus of the molecule, wherein the leader peptide is later cleaved from the hybrid nuclease-transferrin molecule. Methods for generating nucleic acid sequences encoding a leader peptide fused to a recombinant protein are well known in the art. In some embodiments, any of the hybrid nuclease-transferrin molecule of the present invention can be expressed either with or without a leader fused to their N-terminus. The protein sequence of a hybrid nuclease-transferrin molecule of the present invention following cleavage of a fused leader peptide can be predicted and/or deduced by one of skill in the art. Examples of hybrid nuclease-transferrin molecules of the present invention additionally including a VK3 leader peptide (VK3LP), wherein the leader peptide is fused to the N- terminus of the hybrid nuclease-transferrin molecule, are set forth in SEQ ID NOs: 3-26. Such leader sequences can improve the level of synthesis and secretion of the hybrid nuclease-transferrin molecules in mammalian cells. In some embodiments, the leader is cleaved, yielding hybrid nuclease-transferrin molecules having the sequences set forth in SEQ ID NOs: 27-50. In some embodiments, a hybrid nuclease-transferrin molecule of the present invention is expressed without a leader peptide fused to its N-terminus, and the resulting hybrid nuclease-transferrin molecule has an N-terminal methionine. In some embodiments, the hybrid nuclease-transferrin molecule will include a stop codon. In some embodiments, the stop codon will be at the C-terminus of the transferrin, or a variant or fragment thereof. In other embodiments, the stop codon will be at the C- terminus of the nuclease domain (e.g., RNase and/or DNase domain). Appropriate positioning of a stop codon will differ depending on the configuration of components within the hybrid nuclease-transferrin molecule, and will be evident to the skilled artisan. In some embodiments, the hybrid nuclease-transferrin molecule further includes a second nuclease domain. In some embodiments, the second nuclease domain is operably coupled to the transferrin, or variant or fragment thereof, via a second linker domain. In some embodiments, the second linker domain will be at the C-terminus of the transferrin, or a variant or fragment thereof. In some embodiments, the hybrid nuclease-transferrin molecule includes two nuclease domains operably coupled to each other in tandem and further operably coupled to the N- or C-terminus of the transferrin, or a variant or fragment thereof. Figure 1 displays exemplary configurations of the hybrid nuclease-transferrin molecules, and Table 1 provides the sequences of exemplary hybrid nuclease-transferrin molecules of various configurations. In some embodiments, a hybrid nuclease-transferrin molecule is an RNase domain or DNase domain or a multi-nuclease domain (e.g., both RNase and DNase or two RNA or DNA nucleases with different specificity for substrate) fused to a transferrin, or a variant or fragment thereof, that specifically binds to extracellular immune complexes. In other embodiments, the hybrid nuclease-transferrin molecule has activity against single and/or double-stranded RNA substrates. In one embodiment, the nuclease domain is operably coupled (e.g., chemically conjugated or genetically fused (e.g., either directly or via a polypeptide linker)) to the N- terminus of a transferrin, or a variant or fragment thereof. In another embodiment, the nuclease domain is operably coupled (e.g., chemically conjugated or genetically fused (e.g., either directly or via a polypeptide linker)) to the C-terminus of a transferrin, or a variant or fragment thereof. In other embodiments, a nuclease domain is operably coupled (e.g., chemically conjugated or genetically fused (e.g., either directly or via a polypeptide linker)) via an amino acid side chain of a transferrin, or a variant or fragment thereof. In certain embodiments, the hybrid nuclease-transferrin molecules of the invention comprise two or more nuclease domains and at least one transferrin, or a variant or fragment thereof. For example, nuclease domains may be operably coupled to both the N-terminus and C-terminus of a transferrin, or a variant or fragment thereof, with optional linkers between the nuclease domains and the transferrin, or variant or fragment thereof. In some embodiments, the nuclease domains are identical, e.g., RNase and RNase, or DNase1 and DNase1. In other embodiments, the nuclease domains are different, e.g., DNase and RNase. In other embodiments, two or more nuclease domains are operably coupled to each other (e.g., via a polypeptide linker) in series, and the tandem array of nuclease domains is operably coupled (e.g., chemically conjugated or genetically fused (e.g., either directly or via a polypeptide linker)) to either the C-terminus or the N-terminus of a transferrin, or a variant or fragment thereof. In other embodiments, the tandem array of nuclease domains is operably coupled to both the C-terminus and the N-terminus of a transferrin, or a variant or fragment thereof. In other embodiments, one or more nuclease domains may be inserted between two transferrins, or variants or fragments thereof. For example, one or more nuclease domains may form all or part of a polypeptide linker of a hybrid nuclease-transferrin molecule of the invention. Preferred hybrid nuclease-transferrin molecules of the invention comprise at least one nuclease domain (e.g., RNase or DNase), at least one linker domain, and at least one transferrin, or a variant or fragment thereof. Accordingly, in some embodiments, the hybrid nuclease-transferrin molecules of the invention comprise transferrin, or a variant or fragment thereof, as described supra, thereby increasing serum half-life and bioavailability of the hybrid nuclease-transferrin molecules. In some embodiments, a hybrid nuclease-transferrin molecule is as shown in any of SEQ ID NOs: 3-50. It will be understood by the skilled artisan that other configurations of the nuclease domains and transferrin are possible, with the inclusion of optional linkers between the nuclease domains and/or between the nuclease domains and transferrin. It will also be understood that domain orientation can be altered, so long as the nuclease domains are active in the particular configuration tested. In certain embodiments, the hybrid nuclease-transferrin molecules of the invention have at least one nuclease domain specific for a target molecule which mediates a biological effect. In another embodiment, binding of the hybrid nuclease-transferrin molecules of the invention to a target molecule (e.g. DNA or RNA) results in the reduction or elimination of the target molecule, e.g., from a cell, a tissue, or from circulation. In other embodiments, the hybrid nuclease-transferrin molecules of the invention may be assembled together or with other polypeptides to form binding proteins having two or more polypeptides ("multimers"), wherein at least one polypeptide of the multimer is a hybrid nuclease-transferrin molecule of the invention. Exemplary multimeric forms include dimeric, trimeric, tetrameric, and hexameric altered binding proteins and the like. In one embodiment, the polypeptides of the multimer are the same (i.e., homomeric altered binding proteins, e.g., homodimers, homotetramers). In another embodiment, the polypeptides of the multimer are different (e.g., heteromeric). In some embodiments, a nuclease-transferrin hybrid molecule has a serum half-life that is increased at least about 1.5-fold, such as at least 3-fold, at least 5-fold, at least 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600- fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, or 1000-fold or greater relative to the corresponding nuclease molecules not fused to the transferrin, or a variant or fragment thereof. In other embodiments, a nuclease-transferrin hybrid molecule has a serum half-life that is decreased at least about 1.5-fold, such as at least 3-fold, at least 5-fold, at least 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or 500-fold or lower relative to the corresponding nuclease molecules not fused to the transferrin, or a variant or fragment thereof. Routine art-recognized methods can be used to determine the serum half-life of nuclease-transferrin hybrid molecules of the invention. In some embodiments, the activity of the RNase in the hybrid nuclease-transferrin molecule is not less than about 10-fold less, such as 9-fold less, 8-fold less, 7-fold less, 6- fold less, 5-fold less, 4-fold less, 3-fold less, or 2-fold less than the activity of a control RNase molecule. In some embodiments, the activity of the RNase in the hybrid nuclease- transferrin molecule is about equal to the activity of a control RNase molecule. In some embodiments, the activity of the DNase in the hybrid nuclease-transferrin molecule is not less than about 10-fold less, such as 9-fold less, 8-fold less, 7-fold less, 6- fold less, 5-fold less, 4-fold less, 3-fold less, or 2-fold less than the activity of a control DNase molecule. In some embodiments, the activity of the DNase in the hybrid nuclease-transferrin molecule is about equal to the activity of a control DNase molecule. In some embodiments, the hybrid nuclease-transferrin molecules can be active towards extracellular immune complexes containing DNA and/or RNA, e.g., either in soluble form or deposited as insoluble complexes. In some embodiments, the activity of the hybrid nuclease-transferrin molecule is detectable in vitro and/or in vivo. In some embodiments, the hybrid nuclease-transferrin molecule binds to a cell, a malignant cell, or a cancer cell and interferes with its biologic activity. In another aspect, a multifunctional RNase or DNase molecule is provided that is attached to another enzyme or antibody having binding specificity, such as an scFv targeted to RNA or DNA or a second nuclease domain with the same or different specificities as the first domain. In some embodiments, linker domains include (gly4ser) 3, 4 or 5 variants that alter the length of the linker by 5 amino acid progressions. In another embodiment, a linker domain is approximately 18 amino acids in length and includes an N-linked glycosylation site, which can be sensitive to protease cleavage in vivo. In some embodiments, an N- linked glycosylation site can protect the hybrid nuclease-transferrin molecules from cleavage in the linker domain. In some embodiments, an N-linked glycosylation site can assist in separating the folding of independent functional domains separated by the linker domain. In some embodiments, the hybrid nuclease-transferrin molecule includes substantially all or at least an enzymatically active fragment of a DNase. In some embodiments, the DNase is a Type I secreted DNase, preferably a human DNase such as mature human pancreatic DNase 1 (UniProtKB entry P24855, SEQ ID NO: 51). In some embodiments, a naturally occurring variant allele, A114F (SEQ ID NO: 52), which shows reduced sensitivity to actin is included in a DNase1 hybrid nuclease-transferrin molecule (see Pan et al., JBC 1998;273:18374-81; Zhen et al., BBRC 1997;231:499-504; Rodriguez et al., Genomics 1997;42:507-13). In other embodiments, a naturally occurring variant allele, G105R (SEQ ID NO: 53), which exhibits high DNase activity relative to wild type DNase1, is included in a DNase1 hybrid nuclease-transferrin molecule (see Yasuda et al., Int J Biochem Cell Biol 2010;42:1216-25). In some embodiments, this mutation is introduced into a hybrid nuclease-transferrin molecule to generate a more stable derivative of human DNase1. In some embodiments, the DNase is human, wild type DNase1 or human, DNase1 A114F mutated to remove all potential N-linked

glycosylation sites, i.e., asparagine residues at positions 18 and 106 of the DNase1 domain set forth in SEQ ID NO: 51 (i.e., human DNase1 N18S/N106S/A114F, SEQ ID NO: 72), which correspond to asparagine residues at positions 40 and 128, respectively, of full length pancreatic DNase1 with the native leader (SEQ ID NO: 54). In some embodiments, the DNase is a human DNase1 comprising one or more basic (i.e., positively charged) amino acid substitutions to increase DNase functionality and chromatin cleavage. In some embodiments, basic amino acids are introduced into human DNase1 at the DNA binding interface to enhance binding with negatively charged phosphates on DNA substrates (see US 7407785; US 6391607). This hyperactive DNase1 may be referred to as "chromatin cutter." In some embodiments, 1, 2, 3, 4, 5 or 6 basic amino acid substitutions are introduced into DNase1. For example, one or more of the following residues is mutated to enhance DNA binding: Gln9, Glu13, Thr14, His44, Asn74, Asn110, Thr205. In some embodiments one or more of the foregoing amino acids are substituted with basic amino acids such as, arginine, lysine and/or histidine. For example, a mutant human DNase can include one or more of the following substitutions : Q9R, E13R, T14K, H44K, N74K, N110R, T205K. In some embodiments, the mutant human DNase1 also includes an A114F substitution, which reduces sensitivity to actin (see US 6348343). In one embodiment, the mutant human DNase1 includes the following substitutions: E13R, N74K, A114F and T205K. In some embodiments, the mutant human DNase1 further includes mutations to remove potential glycosylation sites, e.g., asparagine residues at positions 18 and 106 of the DNase1 domain set forth in SEQ ID NO:66, which correspond to asparagines residues at positions 40 and 128, respectively of full length pancreatic DNase1 with the native leader (SEQ ID NO:67). In one embodiment, the mutant human DNase1 includes the following substitutions: E13R/N74K/A114F/T205K/N18S/N106S. In some embodiments, the DNase is DNase 1-like (DNaseL) enzyme, 1-3 (UniProtKB entry Q13609; SEQ ID NO: 55). In some embodiments, the DNase is three prime repair exonuclease 1 (TREX1; UniProtKB entry Q9NSU2; SEQ ID NO: 56). In some embodiments, the DNase is DNase2. In some embodiments, the DNase2 is DNAse2 alpha (i.e., DNase2; UnitProtKB entry O00115SEQ ID NO: 57) or DNase2 beta (i.e., DNase2-like acid DNase; UnitProtKB entry Q8WZ79; SEQ ID NO: 58). In some embodiments, the N-linked glycosylation sites of DNase 1L3, TREX1, DNase2 alpha, or DNase2 beta are mutated such as to remove potential N-linked glycosylation sites. In some embodiments, a DNase-linker-transferrin containing a 20 or 25 aa linker domain is made. In some embodiments, hybrid nuclease-transferrin molecules include RNase- transferrin-linker-DNase1, wherein the DNase1 domain is located at the COOH side of the transferrin. In other embodiments, hybrid nuclease-transferrin molecules include DNase1-transferrin-linker-RNase, wherein the DNase1 domain is located at the NH2 side of the transferrin. In some embodiments, hybrid nuclease-transferrin molecules are made that incorporate DNase1 and include: DNase1-transferrin, DNase1-linker-transferrin, transferrin-DNase1, transferrin-linker-DNase1, DNase1-transferrin-RNase, RNase- transferrin-DNase1, DNase1-linker-transferrin-linker-RNase, RNase-linker-transferrin- linker-DNase1, DNase1-linker-RNase-transferrin, RNase-linker-DNase1-transferrin, transferrin-DNase1-linker-RNase, and transferrin-RNase-linker-DNase1. Exemplary configurations of the hybrid nuclease-transferrin molecules comprising DNase1 are shown in Figure 1. In these embodiments, RNase can be, for example, human RNase1. In some embodiments, a hybrid nuclease-transferrin molecule includes TREX1 (SEQ ID NO: 56). In some embodiments, a TREX1 hybrid nuclease-transferrin molecule can digest chromatin. In some embodiments, a TREX1 hybrid nuclease-transferrin molecule is expressed by a cell. In some embodiments, the expressed hybrid nuclease-transferrin molecule includes murine TREX-1 and a transferrin, or a variant or fragment thereof. In some embodiments, a hydrophobic region of approximately 72 aa can be removed from the COOH end of TREX-1 prior to fusion to transferrin, or a variant or fragment thereof, via the linker domain. In some embodiments, a 20 amino acid linker domain version of the hybrid nuclease-transferrin molecule exhibits high expression levels compared to controls and/or other hybrid nuclease-transferrin molecules. In some embodiments, kinetic enzyme assays are used to compare the enzyme activity of hybrid nuclease- transferrin molecules and controls in a quantitative manner. In some embodiments, further optimization of the fusion junction chosen for truncation of a TREX1 enzyme can be used to improve expression of the hybrid nuclease-transferrin molecules. In some embodiments, the hybrid nuclease-transferrin molecule includes a human TREX1-linker-transferrin domain hybrid nuclease-transferrin molecule with 20 and/or 25 aa linker domains. In some embodiments, the linker domain(s) are variants of a

(gly4ser)4 or (gly4ser)5 cassette with one or more restriction sites attached for incorporation into the hybrid nuclease-transferrin molecules construct. In some embodiments, because of the head-to-tail dimerization useful for TREX1 enzyme activity; a flexible, longer linker domain can be used to facilitate proper folding. In some embodiments, the hybrid nuclease-transferrin molecule is a TREX1-tandem hybrid nuclease-transferrin molecule. In some embodiments, an alternative method for facilitating head-to-tail folding of TREX1 is to generate a TREX1-TREX1-transferrin hybrid nuclease-transferrin molecule that incorporates two TREX1 domains in tandem, followed by a linker domain and a transferrin domain. In some embodiments, positioning of TREX1 cassettes in a head-to-tail manner can be corrected for head-to tail folding on either arm of the immunoenzyme and introduce a single TREX1 functional domain into each arm of the molecule. In some embodiments, each immunoenzyme of a hybrid nuclease-transferrin molecule has two functional TREX1 enzymes attached to a single HST, or a variant or fragment thereof. In some embodiments, the hybrid nuclease-transferrin molecule includes TREX1-linker1- transferrin-linker2-RNase. In some embodiments, the hybrid nuclease-transferrin molecule includes RNase-transferrin-linker-TREX1. In some embodiments, cassettes are derived for both amino and carboxyl fusion of each enzyme for incorporation into hybrid nuclease-transferrin molecules where the enzyme configuration is reversed. In some embodiments, the RNase enzyme exhibits comparable functional activity regardless of its position in the hybrid nuclease-transferrin molecules. In some embodiments, alternative hybrid nuclease-transferrin molecules can be designed to test whether a particular configuration demonstrates improved expression and/or function of the hybrid nuclease- transferrin molecule components. In some embodiments, the hybrid nuclease-transferrin molecule includes 1L3-transferrin. In some embodiments, the 1L3 DNase is constructed from a human (SEQ ID NO: 55) and murine (SEQ ID NO: 59) sequence and expressed. In some embodiments, a human 1L3 DNase-transferrin-RNase hybrid nuclease-transferrin molecule is constructed and expressed. In some embodiments, the molecule includes human 1L3-transferrin, human 1L3-transferrin-RNase, and/or human RNase-transferrin-1L3. In some embodiments, the hybrid nuclease-transferrin molecule includes DNase2 alpha (SEQ ID NO: 57) or DNase2 beta (SEQ ID NO: 58). In some embodiments, a human DNase2 alpha-transferrin-RNase or human DNase2 beta-transferrin-RNase hybrid nuclease-transferrin molecule is constructed and expressed. In some embodiments, the molecule includes human DNase2 alpha-transferrin, human DNase2 alpha-transferrin- RNase, and/or human RNase-transferrin-DNase2 alpha. In other embodiments, the molecule includes human DNase2 beta-transferrin, human DNase2 beta-transferrin- RNase, and/or human RNase-transferrin-DNase2 beta. In some embodiments, the hybrid nuclease-transferrin molecule includes a RNase1, preferably human pancreatic RNase1 (UniProtKB entry P07998; SEQ ID NO: 60) of the RNase A family. In some embodiments, the human RNase1 is mutated to remove all potential N-linked glycosylation sites, i.e., asparagine residues at positions 34, 76, and 88 of the RNase1 domain set forth in SEQ ID NO: 60 (human RNase1 N34S/N76S/N88S, SEQ ID NO: 61), which correspond to asparagine residues at positions 62, 104, and 116, respectively, of full length pancreatic RNase1 with the native leader (SEQ ID NO: 62). In some embodiments, a RNase1-linker-transferrin containing a 20 or 25 aa linker domain is made. In some embodiments, hybrid nuclease-transferrin molecules include DNase-transferrin-linker-RNase1, wherein the RNase1 domain is located at the COOH side of the transferrin. In other embodiments, hybrid nuclease-transferrin molecules include RNase1-transferrin-linker-DNase, wherein the RNase1 domain is located at the NH2 side of the transferrin. In some embodiments, hybrid nuclease-transferrin molecules are made that incorporate RNase1 and include: RNase1-transferrin, RNase1-linker- transferrin, transferrin-RNase1, transferrin-linker-RNase1, RNase1-transferrin-DNase, DNase-transferrin-RNase1, RNase1-linker-transferrin-linker-DNase, DNase-linker- transferrin-linker-RNase1, RNase1-linker-DNase-transferrin, DNase-linker-RNase1- transferrin, transferrin-RNase1-linker-DNase, and transferrin-DNase-linker-RNase1. Exemplary configurations of the hybrid nuclease-transferrin molecules comprising RNase1 are shown in Figure 1. In these embodiments, DNase can be, for example, human DNase1. In some embodiments, fusion junctions between enzyme domains and the other domains of the hybrid nuclease-transferrin molecule is optimized. In some embodiments, the targets of the RNase enzyme activity of RNase hybrid nuclease-transferrin molecules are primarily extracellular, consisting of, e.g., RNA contained in immune complexes with anti-RNP autoantibody and RNA expressed on the surface of cells undergoing apoptosis. In some embodiments, the RNase hybrid nuclease- transferrin molecule is active in the acidic environment of the endocytic vesicles. In some embodiments, an RNase hybrid nuclease-transferrin molecule including a transferrin, or a variant or fragment thereof, is adapted to be active both extracellularly and in the endocytic environment. In some aspects, this allows an RNase hybrid nuclease-transferrin molecule including a wild-type HST, or a variant or fragment thereof, to stop TLR7 signaling through previously engulfed immune complexes or by RNAs that activate TLR7 after viral infection. In some embodiments, the wild type RNase of an RNase hybrid nuclease-transferrin molecule is not resistant to inhibition by an RNase cytoplasmic inhibitor. In some embodiments, the wild type RNase of an RNase hybrid nuclease-transferrin molecule is not active in the cytoplasm of a cell. In some embodiments, hybrid nuclease-transferrin molecules include both DNase and RNase. In some embodiments, these hybrid nuclease-transferrin molecules can improve the therapy of SLE because they can, e.g., digest immune complexes containing RNA, DNA, or a combination of both RNA and DNA; and when they further include a transferrin, or a variant or fragment thereof, they are active both extracellularly and in the endocytic compartment where TLR7 and TLR9 can be located. Transferrin, or a variant or fragment thereof The transferrin for use in the hybrid nuclease-transferrin molecules can be a transferrin from, e.g., human, cow, pig, sheep, dog, rabbit, rat, mouse, hamster, echnida, platypus, chicken, frog, hornworm, monkey, horse, and bovine. These transferrin sequences are readily available in Genbank and other public databases. In one embodiment, the transferrin is a human transferrin (SEQ ID NO: 2). In a preferred embodiment, the transferrin is HST (SEQ ID NO: 1). The hybrid nuclease-transferrin molecules of the invention can include any transferrin protein, variant, fragment, domain, or engineered domain. In some embodiments, hybrid nuclease-transferrin molecules include transferrin without the 19 amino acid native leader sequence (SEQ ID NO: 1). In some embodiments, a nuclease domain or nuclease domains can be operably coupled to the N-terminus of transferrin, or a variant or fragment thereof. In other embodiments, a nuclease domain can be operably coupled to the C-terminus of transferrin, or a variant or fragment thereof. In yet other embodiments, nuclease domains can be operably coupled to both the N-terminus and C-terminus of transferrin, or a variant or fragment thereof. In other embodiments, a nuclease domain or nuclease domains can be inserted between the N and C domains of transferrin, or a variant or fragment thereof. In yet other embodiments, a nuclease or nuclease domains can be inserted into one or more of the loops of transferrin, or a variant or fragment thereof (see, e.g., Allet al., JBC 1999;274:24066-73). For example, nucleases may be inserted into all five loops of transferrin, or a variant or fragment thereof, to form a pentavalent hybrid nuclease- transferrin molecule. In some embodiments, the transferrin is a variant transferrin or a fragment thereof. The transferrin variant or fragment thereof generally has a sequence identity to the sequence of HST set forth in SEQ ID NO: 1 of at least 50%, such as at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the number of alterations, e.g., substitutions, insertions, or deletions, in the transferrin variants of the present invention is 1 -20, e.g., 1-10 and 1-5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations compared to the corresponding wild-type transferrin. Variants of transferrin include those that remove potential N-glycosylation sites. HST has two such sites at amino acids corresponding to positions N413 (N432 of SEQ ID NO: 2) and N611 (N630 of SEQ ID NO: 2). These sites can be mutated to an amino acid that does not serve as a N-glycosylation acceptor to yield an aglycoyslated transferrin. In some embodiments, transferrin is O-linked glycosylated (e.g., when produced in Pichia pastoris) at S32 of the transferrin precursor set forth in SEQ ID NO: 2 (i.e., corresponding to S12 of SEQ ID NO: 1). Accordingly, in one embodiment, S12 is deleted or substituted with an amino acid that does not serve as an acceptor of O-linked glycosylation (i.e., any amino acid other than threonine). Transferrin binds to iron and the transferrin receptor, allowing it to cross the blood-brain barrier and be internalized into cells. Accordingly, in some embodiments, transferrin variants are engineered so as to alter binding to iron and/or the transferrin receptor. Exemplary positions of HST that can be alter the interaction with transferrin receptor and/or metal are disclosed in, e.g., US Pat. No.7,176,278 and US Pat. No.5,986,067, and include, but are not limited to, Asp63 (Asp82 of SEQ ID NO: 2), Gly65 (Gly84 of SEQ ID NO: 2), Tyr95 (Tyr114 of SEQ ID NO: 2), Tyr188 (Tyr207 of SEQ ID NO: 2), Lys206 (Lys225 of SEQ ID NO: 2), His207 (His226 of SEQ ID NO: 2), His249 (His278 of SEQ ID NO: 2), Asp392 (Asp411 of SEQ ID NO: 2), Tyr426 (Tyr445 of SEQ ID NO: 2), Tyr514 (Tyr533 of SEQ ID NO: 2), Tyr517 (Tyr536 of SEQ ID NO: 2), and His585 (His604 of SEQ ID NO: 2). In some embodiments, the transferrin variant has a mutation (i.e., substitution, deletion, or insertion) in at least one of the amino acid residues listed above. It is within the abilities of the skilled artisan to determine and test which one of the sites or combination of sites to mutate, and if the mutation is a substitution or insertion, what amino acid to substitute or insert, at these positions to increase, reduce, or eliminate binding to metal and/or the transferrin receptor. In yet other embodiments, at least one amino acid at positions Thr120, Arg124, Ala126, Gly127, Thr452, Arg456, Ala458, and Gly459 of SEQ ID NO: 1 are mutated to alter binding to carbonate, which may affect metal and/or transferrin receptor binding. In one embodiment, the hybrid nuclease-transferrin molecules can include a transferrin splice variant, such as a human transferrin splice variant. One exemplary human transferrin splice variant is set forth in Genbank Acc. No. AAA61140. It will be understood by the skilled artisan that any transferrin variant or natural variant with increased serum half-life compared to the corresponding wild-type transferrin, or that increases the serum half-life of the nuclease domain it is fused or conjugated to, is suitable for use in hybrid nuclease-transferrin molecules. In certain embodiments, it may be desirable for the variant transferrin, or fragment thereof, to decrease the serum half-life of a hybrid nuclease-transferrin molecule. Hybrid nuclease-transferrin molecules with decreased serum half-lives are useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g., for in vivo diagnostic imaging or in situations where the starting polypeptide has toxic side effects when present in the circulation for prolonged periods. Full length human transferrin has two main domains, an N domain (amino acids 1-330; SEQ ID NO: 63) and a C domain (amino acids 340-679; SEQ ID NO: 64), which correspond to an N domain (amino acids 20-339; SEQ ID NO: 65) and a C domain (amino acids 340-679; SEQ ID NO: 64) of HST (SEQ ID NO: 1). These two domains are further divided into two subdomains each (i.e., N1, N2, C1, and C2 domains).

Transferrin carries two iron molecules, which are complexed in the space between N1 and N2, and C1 and C2. Accordingly, in some embodiments, the hybrid nuclease- transferrin molecules include a single transferrin domain, such as the N domain or C domain. In other embodiments, the hybrid nuclease-transferrin molecules include at least two N or at least two C domains. Other exemplary fragments, domains, or engineered domains suitable for use in a hybrid nuclease-transferrin molecule can be found in, e.g., US Patent No.7,176,278. In some embodiments, when the N domain or a portion of the N domain is part of the transferrin of a hybrid nuclease-transferrin molecule, the domain has mutations at positions corresponding to S12 of SEQ ID NO: 1, to form a domain that is not O- glycosylated, and/or at positions corresponding to Asp63, Gly65, Tyr95, Thr120, Arg124, Ala126, Gly127, Tyr188, Lys206, His207, and/or His249 of SEQ ID NO: 1 to alter binding to carbonate, metal, and/or the transferrin receptor. In some embodiments, when the C domain or a portion of the C domain is part of the transferrin of a hybrid nuclease-transferrin molecule, the domain has mutations at positions corresponding to N413 and/or N611 of SEQ ID NO: 1, to form an

aglycosylated domain, or at positions corresponding to Asp392, Tyr 426, Thr452, Arg456, Ala458, Gly459, Tyr514, Tyr617, and/or His585 of SEQ ID NO: 1, to alter binding to carbonate, metal, and/or the transferrin receptor. A fragment of transferrin or a variant thereof will typically be at least 20 amino acids in length, such as at least 40 amino acids, at least 60 amino acids, at least 80 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 550 amino acids, at least 600 amino acids, at least 650 amino acids, or at least 670 amino acids in length, and will be sufficient to alter (e.g., increase) the serum half-life or bioavailability of the nuclease domain it is fused to (e.g., RNase and/or DNase domain) relative to the corresponding non-fused nuclease domain. A fragment of transferrin or a variant thereof may comprise multiple fragments of transferrin, as single or multiple heterologous fusions of different transferrin fragments. A fragment may comprise or consist of at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 % of a transferrin or variant thereof, or a domain of transferrin or a variant thereof. In some embodiments, fragments of transferrin, or fragments of transferrin variants, are suitable for use in the hybrid nuclease-transferrin molecules of the invention provided that they alter (e.g., increase) the serum half-life or bioavailability of the hybrid nuclease- transferrin molecules relative to the corresponding nuclease domain that is not fused to the transferrin fragment, or a variant thereof. In some embodiments, transferrin can be engineered to not bind iron and/or not bind the transferrin receptor. In other embodiments, transferrin can be engineered to not bind iron but bind the transferrin receptor. In yet other embodiments, transferrin can be engineered to bind iron but not the transferrin receptor. In some embodiments, binding to the transferrin receptor can be controlled via strategic use of transferrin domains that retain particular functions. For example, the N domain alone will not bind to transferrin receptor when loaded with iron, and the iron-bound C domain will bind transferrin receptor, but not with the same affinity as compared to full length transferrin. In some embodiments, mutations may be made to transferrin, or a variant or fragment thereof, to alter its three dimensional structure. For example, mutations made in or around amino acid residues 94-96, 245-247, and/or 316-318 of the N domain, and/or amino acid residues 425-427, 581-582, and/or 652-658 of the C domain, or to or around the flanking regions of these sites can alter transferrin structure and function. Nuclease molecules may also be operably coupled to transferrin-related molecules, such as lactoferrin (SEQ ID NO: 66; GenBank Acc: NM_002343) or melanotransferrin (SEQ ID NO: 67; Genbank Acc. NM_013900), or their variants, such as splice variants, e.g., the lactoferrin splice variant with an amino acid sequence set forth in SEQ ID NO: 68 (neutrophil lactorerrin; Genbank Acc: AAA59479). The neutrophil lactoferrin splice variant can comprise the following amino acid sequence: EDCIALKGEADA (SEQ ID NO: 69), which includes a novel region of splice-variance. In other embodiments, nuclease molecules may be operably coupled to fragments of transferrin-related molecules or variants thereof. One or more positions of transferrin, or a variant or fragment thereof, can be altered to provide reactive surface residues for, e.g., conjugation with a DNase and/or RNase domain. Alternatively or additionally, a cysteine residue may be added to the N or C terminus of transferrin. In one embodiment, the conjugates may conveniently be linked via a free thio group present on the surface of transferrin using art-recognized methods. Linker Domains

In some embodiments, a hybrid nuclease-transferrin molecule includes a linker domain. In some embodiments, a hybrid nuclease-transferrin molecule includes a plurality of linker domains. In some embodiments, the linker domain is a polypeptide linker. In certain aspects, it is desirable to employ a polypeptide linker to fuse transferrin, or a variant or fragment thereof, with one or more nuclease domains to form a hybrid nuclease-transferrin molecule. In one embodiment, the polypeptide linker is synthetic. As used herein, the term

"synthetic" with respect to a polypeptide linker includes peptides (or polypeptides) which comprise an amino acid sequence (which may or may not be naturally occurring) that is linked in a linear sequence of amino acids to a sequence (which may or may not be naturally occurring) (e.g., a transferrin sequence) to which it is not naturally linked in nature. For example, the polypeptide linker may comprise non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides (e.g., comprising a mutation such as an addition, substitution or deletion) or which comprise a first amino acid sequence (which may or may not be naturally occurring). The polypeptide linkers of the invention may be employed, for instance, to ensure that transferrin, or a variant or fragment thereof, is juxtaposed to ensure proper folding and formation of a functional transferrin, or a variant or fragment thereof. Preferably, a polypeptide linker compatible with the instant invention will be relatively non- immunogenic and not inhibit any non-covalent association among monomer subunits of a binding protein. In certain embodiments, the hybrid nuclease-transferrin molecules of the invention employ a polypeptide linker to join any two or more domains in frame in a single polypeptide chain. In one embodiment, the two or more domains may be independently selected from any of the transferrins, or variants or fragments thereof, or nuclease domains discussed herein. For example, in certain embodiments, a polypeptide linker can be used to fuse identical transferrin fragments, thereby forming a homomeric transferrin region. In other embodiments, a polypeptide linker can be used to fuse different transferrin fragments (e.g., N and C domains of transferrin), thereby forming a heteromeric transferrin region. In other embodiments, a polypeptide linker of the invention can be used to genetically fuse the C-terminus of a first transferrin fragment to the N-terminus of a second transferrin fragment to form a complete transferrin domain. In one embodiment, a polypeptide linker comprises a portion of a transferrin, or a variant or fragment thereof. For example, in one embodiment, a polypeptide linker can comprise a transferrin fragment (e.g., C or N domain), or a different portion of a transferrin or variant thereof. In another embodiment, a polypeptide linker comprises or consists of a gly-ser linker. As used herein, the term“gly-ser linker” refers to a peptide that consists of glycine and serine residues. An exemplary gly/ser linker comprises an amino acid sequence of the formula (Gly₄Ser)n, wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5). A preferred gly/ser linker is (Gly₄Ser)4. Another preferred gly/ser linker is (Gly₄Ser)3. Another preferred gly/ser linker is (Gly₄Ser)5. In certain embodiments, the gly-ser linker may be inserted between two other sequences of the polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In other embodiments, a gly-ser linker is attached at one or both ends of another sequence of the polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In yet other embodiments, two or more gly-ser linker are incorporated in series in a polypeptide linker. In other embodiments, a polypeptide linker of the invention comprises a biologically relevant peptide sequence or a sequence portion thereof. For example, a biologically relevant peptide sequence may include, but is not limited to, sequences derived from an anti-rejection or anti-inflammatory peptide. Said anti-rejection or anti-inflammatory peptides may be selected from the group consisting of a cytokine inhibitory peptide, a cell adhesion inhibitory peptide, a thrombin inhibitory peptide, and a platelet inhibitory peptide. In a preferred embodiment, a polypeptide linker comprises a peptide sequence selected from the group consisting of an IL-1 inhibitory or antagonist peptide sequence, an erythropoietin (EPO)-mimetic peptide sequence, a thrombopoietin (TPO)-mimetic peptide sequence, G-CSF mimetic peptide sequence, a TNF-antagonist peptide sequence, an integrin-binding peptide sequence, a selectin antagonist peptide sequence, an anti- pathogenic peptide sequence, a vasoactive intestinal peptide (VIP) mimetic peptide sequence, a calmodulin antagonist peptide sequence, a mast cell antagonist, a SH3 antagonist peptide sequence, an urokinase receptor (UKR) antagonist peptide sequence, a somatostatin or cortistatin mimetic peptide sequence, and a macrophage and/or T-cell inhibiting peptide sequence. Exemplary peptide sequences, any one of which may be employed as a polypeptide linker, are disclosed in U.S. Pat. No.6,660,843, which is incorporated by reference herein. Other linkers that are suitable for use in the hybrid nuclease-transferrin molecules are known in the art, for example, the serine-rich linkers disclosed in US 5,525,491, the helix forming peptide linkers (e.g., A(EAAAK)nA (n=2-5)) disclosed in Arai et al., Protein Eng 2001;14:529-32, and the stable linkers disclosed in Chen et al., Mol Pharm

2011;8:457-65, i.e., the dipeptide linker LE, a thrombin-sensitive disulfide cyclopeptide linker, and the alpha-helix forming linker LEA(EAAAK)₄ALEA(EAAAK)₄ALE (SEQ ID NO: 85). Other exemplary linkers include GS linkers (i.e., (GS)n), GGSG (SEQ ID NO: 70) linkers (i.e., (GGSG)n), GSAT linkers (SEQ ID NO: 71), SEG linkers, and GGS linkers (i.e., (GGSGGS)n), wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5). Other suitable linkers for use in the hybrid nuclease- transferrin molecules can be found using publicly available databases, such as the Linker Database (ibi.vu.nl/programs/linkerdbwww). The Linker Database is a database of inter-domain linkers in multi-functional enzymes which serve as potential linkers in novel fusion proteins (see, e.g., George et al., Protein Engineering 2002;15:871-9). It will be understood that variant forms of these exemplary polypeptide linkers can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding a polypeptide linker such that one or more amino acid substitutions, additions or deletions are introduced into the polypeptide linker. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR- mediated mutagenesis. Polypeptide linkers of the invention are at least one amino acid in length and can be of varying lengths. In one embodiment, a polypeptide linker of the invention is from about 1 to about 50 amino acids in length. As used in this context, the term“about” indicates +/- two amino acid residues. Since linker length must be a positive integer, the length of from about 1 to about 50 amino acids in length, means a length of from 1 to 48-52 amino acids in length. In another embodiment, a polypeptide linker of the invention is from about 10-20 amino acids in length. In another embodiment, a polypeptide linker of the invention is from about 15 to about 50 amino acids in length. In another embodiment, a polypeptide linker of the invention is from about 20 to about 45 amino acids in length. In another embodiment, a polypeptide linker of the invention is from about 15 to about 25 amino acids in length. In another embodiment, a polypeptide linker of the invention is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 ,57, 58, 59, 60, or 61 or more amino acids in length. Polypeptide linkers can be introduced into polypeptide sequences using techniques known in the art. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transform host cells for stable production of the polypeptides produced. Exemplary hybrid nuclease-transferrin molecules

The hybrid nuclease-transferrin molecules of the invention are modular, and can be configured to incorporate various individual domains. For example, in one embodiment, the hybrid nuclease-transferrin molecule may include the mutant, human DNase1 A114F domain set forth in (SEQ ID NO: 52). In another embodiment, the hybrid nuclease- transferrin molecule may include the mutant, human DNase1 N18S/N106S/A114F domain set forth in SEQ ID NO: 72. In another embodiment, the hybrid nuclease- transferrin molecule may include the human, wild-type RNase1 domain set forth in SEQ ID NO: 60. In another embodiment, the hybrid nuclease-transferrin molecule may include the human, mutant RNase1 N34S/N76S/N88S domain set forth in SEQ ID NO: 61. In another embodiment, the hybrid nuclease-transferrin molecule may include HST set forth in SEQ ID NO: 1. In another embodiment, the hybrid nuclease-transferrin molecule may include the (Gly₄Ser)3 linker domain set forth in SEQ ID NO: 73. In another embodiment, the hybrid nuclease-transferrin molecule may include a VK3LP leader (SEQ ID NO: 83). It will be understood to the skilled artisan that these individual domains can be operably coupled to each other in any order to form a hybrid nuclease- transferrin molecule that is enzymatically active. For example, as detailed in the specific examples below, RNase1 can be operably coupled to HST. In another example, RNase1 can be operatively coupled to HST via a (Gly₄Ser)3 linker domain. In yet another example, DNase1 A114F can be operatively coupled to HST. In yet another example, DNase1 A114F can be operatively coupled to HST via a (Gly₄Ser)3 linker domain. Various other configurations are possible, with non-limiting exemplary configurations detailed below and in Figure 1. In some embodiments, a hybrid nuclease-transferrin molecule comprises a wild-type, human RNase1 domain operably coupled to a wild-type transferrin, or a variant or fragment thereof. In one embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader (SEQ ID NO: 83), followed by a wild-type, human RNase1 domain operably coupled to the C-terminus of wild-type HST, or a variant or fragment thereof (e.g., an HST-RNase molecule; SEQ ID NO: 3)). In one embodiment, the transferrin-RNase molecule lacks the VK3LP leader (SEQ ID NO: 27). In another embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a wild-type, human RNase1 domain operably coupled to the N- terminus of wild-type HST, or a variant or fragment thereof (e.g., an RNase-HST molecule; SEQ ID NO: 4). In one embodiment, the RNase-HST molecule lacks the VK3LP leader (SEQ ID NO: 28). In some embodiments, a hybrid nuclease-transferrin molecule comprises a wild-type, human RNase1 domain operably coupled via a (Gly₄Ser)3 linker domain to a wild-type transferrin, or mutant or fragment thereof. In one embodiment, the hybrid nuclease- transferrin molecule comprises a VK3LP leader, followed by a wild-type, human RNase1 domain operably coupled via a (Gly₄Ser)3 linker domain to the C-terminus of wild-type HST, or a variant or fragment thereof (e.g., an HST-linker-RNase molecule; SEQ ID NO: 5). In one embodiment, the HST-linker-RNase molecule lacks the VK3LP leader (SEQ ID NO: 29). In another embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a wild-type, human RNase1 domain operably coupled via a

(Gly₄Ser)3 linker domain to the N-terminus of wild-type HST, or a variant or fragment thereof (e.g., an RNase-linker-HST molecule; SEQ ID NO: 6). In one embodiment, the RNase-linker-HST molecule lacks the VK3LP leader (SEQ ID NO: 30). In some embodiments, a hybrid nuclease-transferrin molecule comprises a wild-type, human RNase1 domain operably coupled to a wild-type transferrin, or mutant or fragment thereof, which is operably coupled to a second wild-type, human RNase1 domain. In one embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a first wild-type, human RNase1 domain operably coupled to the N-terminus of wild-type HST, or a variant or fragment thereof, and a second wild- type, human RNase1 domain operably coupled to the C-terminus of the wild-type HST, or a variant or fragment thereof (e.g., an RNase-HST-RNase molecule; SEQ ID NO: 7). In one embodiment, the RNase-HST-RNase molecule lacks the VK3LP leader (SEQ ID NO: 31). In some embodiments, a hybrid nuclease-transferrin molecule comprises a wild-type, human RNase1 domain operably coupled via a (Gly₄Ser)3 linker to a wild-type transferrin, or mutant or fragment thereof, which is operably coupled via a (Gly₄Ser)4 linker to a second wild-type, human RNase1 domain. In one embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a first wild-type, human RNase1 domain operably coupled via a (Gly₄Ser)3 linker domain to the N- terminus of wild-type HST, or mutant or fragment thereof, and a second wild-type, human RNase1 domain operably coupled via a (Gly₄Ser)3 linker domain to the C- terminus of the wild-type HST, or a variant or fragment thereof (e.g., an RNase-linker- HST-linker-RNase molecule; SEQ ID NO: 8). In one embodiment, the RNase-linker- HST-linker-RNase molecule lacks the VK3LP leader (SEQ ID NO: 32). In some embodiments, a hybrid nuclease-transferrin molecule comprises a wild-type, human RNase1 domain operably coupled to a wild-type transferrin, or mutant or fragment thereof, which is operably coupled to a second, mutant, human DNase1 domain. In one embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a wild-type, human RNase1 domain operably coupled to the N-terminus of wild-type HST, or a variant or fragment thereof, and the mutant, human DNase1 A114F domain is operably coupled to the C-terminus of the wild-type HST, or a variant or fragment thereof (e.g., an RNase-HST-DNase A114F molecule; SEQ ID NO: 9). In one embodiment, the RNase-HST-DNase A114F molecule lacks the VK3LP leader (SEQ ID NO: 33). In another embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a mutant, human DNase1 A114F domain operably coupled to the N- terminus of wild-type HST, or a variant or fragment thereof, and the wild-type, human RNase domain is operably coupled to the C-terminus of the wild-type HST, or a variant or fragment thereof (e.g., a DNase A114F-HST-RNase molecule; SEQ ID NO: 10). In one embodiment, the DNase A114F-HST-RNase molecule lacks the VK3LP leader (SEQ ID NO: 34). In some embodiments, a hybrid nuclease-transferrin molecule comprises a wild-type, human RNase1 domain operably coupled via a (Gly₄Ser)4 linker domain to a wild-type transferrin, or mutant or fragment thereof, which is operably coupled via a (Gly₄Ser)4 linker domain to a mutant, human DNase1 domain. In one embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a wild-type, human RNase1 domain is operably coupled via a (Gly₄Ser)3 linker domain to the N- terminus of wild-type HST, or a variant or fragment thereof, and the mutant, DNase A114F domain is operably coupled via a (Gly₄Ser)3 linker domain to the C-terminus of the wild-type HST, or a variant or fragment thereof (e.g., an RNase-linker-HST-linker- DNase A114F molecule; SEQ ID NO: 11). In one embodiment, the RNase-linker-HST- linker-DNase A114F molecule lacks the VK3LP leader (SEQ ID NO: 35). In another embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a mutant, DNase1 A114F domain is operably coupled via a

(Gly₄Ser)3 linker domain to the N-terminus of wild-type HST, or a variant or fragment thereof, and a wild-type, human RNase1 domain operably coupled via a (Gly₄Ser)3 linker domain to the C-terminus to the wild-type HST, or a variant or fragment thereof (e.g., a DNase1 A114F-linker-HST-linker-RNase1 molecule; SEQ ID NO: 12). In one embodiment, the DNase1 A114F-linker-HST-linker-RNase1 molecule lacks the VK3LP leader (SEQ ID NO: 36). In some embodiments, a hybrid nuclease-transferrin molecule comprises a mutant, human DNase1 domain operably coupled to a wild-type transferrin, or a variant or fragment thereof. In one embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a mutant, human DNase1 A114F domain operably coupled to the N-terminus of wild-type HST, or a variant or fragment thereof (e.g., a DNase A114F- HST molecule; SEQ ID NO: 13). In one embodiment, the DNase A114F-HST molecule lacks the VK3LP leader (SEQ ID NO: 37). In another embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a mutant, human DNase1 A114F domain operably coupled to the C- terminus of wild-type HST, or a variant or fragment thereof (e.g., an HST-DNase A114F molecule; SEQ ID NO: 14). In one embodiment, the HST-DNase A114F molecule lacks the VK3LP leader (SEQ ID NO: 38). In some embodiments, a hybrid nuclease-transferrin molecule comprises a mutant, human DNase1 domain operably coupled via a (Gly₄Ser)3 linker domain to a wild-type transferrin, or a variant or fragment thereof. In one embodiment, the hybrid nuclease- transferrin molecule comprises a VK3LP leader, followed by a mutant, human DNase1 A114F domain operably coupled via a (Gly₄Ser)3 linker domain to the N-terminus of wild-type HST, or a variant or fragment thereof (e.g., a DNase A114F-linker-HST molecule; SEQ ID NO: 15)). In one embodiment, the DNase A114F-linker-HST molecule lacks the VK3LP leader (SEQ ID NO: 39). In another embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a mutant, human DNase1 A114F domain operably coupled via a (Gly₄Ser)3 linker domain to the C-terminus of wild-type HST, or a variant or fragment thereof (e.g., an HST-linker-DNase A114F molecule; SEQ ID NO: 16). In one embodiment, the HST-linker-DNase A114F molecule lacks the VK3LP leader (SEQ ID NO: 40). In some embodiments, the hybrid nuclease-transferrin molecule has altered glycosylation and comprises a mutant, human RNase1 domain operably coupled via a (Gly₄Ser)3 linker to a wild-type transferrin, or mutant or fragment thereof. In one embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a mutant, human RNase1 N34S/N76S/N88S domain operably coupled via a (Gly₄Ser)3 linker to the N- terminus of a wild-type HST, or variant or fragment thereof (e.g., an RNase1

N34S/N76S/N88S-linker-HST molecule; SEQ ID NO: 17). In one embodiment, the RNase1 N34S/N76S/N88S-linker-HST molecule lacks the VK3LP leader (SEQ ID NO: 41). In another embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a mutant, human RNase1 N34S/N76S/N88S domain operably coupled via a (Gly₄Ser)3 linker to the C-terminus of a wild-type HST, or variant or fragment thereof (e.g., an HST-linker-RNase1 N34S/N76S/N88S molecule; SEQ ID NO: 18). In one embodiment, the HST-linker-RNase1 N34S/N76S/N88S molecule lacks the VK3LP leader (SEQ ID NO: 42). In some embodiments, a hybrid nuclease-transferrin molecule comprises a mutant, human DNase1 domain operably coupled via a (Gly₄Ser)3 linker to a wild-type transferrin, or mutant or fragment thereof. In one embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by mutant, human DNase1 N18S/N106S/A114F domain operably coupled via a (Gly₄Ser)3 linker to the N-terminus of a wild-type HST, or variant or fragment thereof (e.g., an DNase1 N18S/N106S/A114F- linker-HST molecule; SEQ ID NO: 19). In one embodiment, the DNase1

N18S/N106S/A114F-linker-HST molecule lacks the VK3LP leader (SEQ ID NO: 43). In another embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by mutant, human DNase1 N18S/N106S/A114F domain operably coupled via a (Gly₄Ser)3 linker to the C-terminus of a wild-type HST, or variant or fragment thereof (e.g., an HST-linker-DNase1 N18S/N106S/A114F molecule; SEQ ID NO: 20). In one embodiment, the HST-linker-DNase1 N18S/N106S/A114F molecule lacks the VK3LP leader (SEQ ID NO: 44). In some embodiments, a hybrid nuclease-transferrin molecule comprises a mutant, human RNase1 domain operably coupled via a (Gly₄Ser)3 linker to a wild-type transferrin, or mutant or fragment thereof, which is operably coupled via a (Gly₄Ser)3 linker to a mutant, human DNase1 domain. In one embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a mutant, human RNase1 N34S/N76S/N88S domain operably coupled via a (Gly₄Ser)3 linker to the N-terminus of a wild-type HST, or variant or fragment thereof, and a mutant, DNase A114F domain operably coupled via a (Gly₄Ser)3 linker to the C-terminus of the wild-type HST, or variant or fragment thereof (e.g., an RNase1 N34S/N76S/N88S-linker-HST-linker-DNase1 A114F molecule; SEQ ID NO: 21). In some embodiments, the RNase1 N34S/N76S/N88S-linker-HST-linker- DNase1 A114F molecule lacks the VK3LP leader (SEQ ID NO: 45). In another embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a mutant, DNase A114F domain operably coupled via a (Gly₄Ser)3 linker to the N-terminus of a wild-type HST, or variant or fragment thereof, and a mutant, human RNase1 N34S/N76S/N88S domain operably coupled via a (Gly₄Ser)3 linker to the C-terminus of the wild-type HST, or variant or fragment thereof (e.g., a DNase1 A114F-linker-HST-linker-RNase1 N34S/N76S/N88S molecule; SEQ ID NO: 22). In some embodiments, the DNase1 A114F-linker-HST-linker-RNase1 N34S/N76S/N88S molecule lacks the VK3LP leader (SEQ ID NO: 46). In another embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by mutant, human RNase1 N34S/N76S/N88S domain operably coupled via a (Gly₄Ser)3 linker to the N-terminus of a wild-type HST, or variant or fragment thereof, and a mutant, DNase N18S/N106S/A114F domain operably coupled via a (Gly₄Ser)3 linker to the C-terminus of the wild-type HST, or variant or fragment thereof (e.g., an RNase1 N34S/N76S/N88S-linker-HST-linker-DNase1 N18S/N106S/A114F molecule; SEQ ID NO: 23). In some embodiments, the RNase1 N34S/N76S/N88S- linker-HST-linker-DNase1 N18S/N106S/A114F molecule lacks the VK3LP leader (SEQ ID NO: 47). In another embodiment, the hybrid nuclease-transferrin molecule comprises a VK3LP leader, followed by a mutant, DNase N18S/N106S/A114F domain operably coupled via a (Gly₄Ser)3 linker to the N-terminus of a wild-type HST, or variant or fragment thereof, and a mutant, human RNase1 N34S/N76S/N88S domain operably coupled via a

(Gly₄Ser)3 linker to the C-terminus of the wild-type HST, or variant or fragment thereof (e.g., a DNase1 N18S/N106S/A114F-linker-HST-linker-RNase1 N34S/N76S/N88S molecule; SEQ ID NO: 24). In some embodiments, the DNase1 N18S/N106S/A114F- linker-HST-linker-RNase1 N34S/N76S/N88S molecule lacks the VK3LP leader (SEQ ID NO: 48). In some embodiments, a hybrid nuclease-transferrin molecule comprises a mutant, DNase1 A114F domain operably coupled via a (Gly₄Ser)3 linker domain to a wild-type, human RNase1 domain, which is operably coupled to a wild-type transferrin, or mutant or fragment thereof. In one embodiment, a mutant, human DNase1 A114F domain is operably coupled via a (Gly₄Ser)3 linker domain to a wild-type, human RNase1 domain, and the wild-type, human RNase1 domain is operatively coupled to the N-terminus of a wild-type HST, or mutant or fragment thereof (e.g., a DNase A114F-linker-RNase-HST molecule; SEQ ID NO: 25)). In some embodiments, the DNase A114F-linker-RNase- HST molecule lacks the VK3LP leader (SEQ ID NO: 49). In another embodiment, a wild-type, human RNase1 domain is operably coupled to the C-terminus of a wild-type HST, or mutant or fragment thereof, and a mutant, human DNase1 A114F domain further operably coupled via a (Gly₄Ser)3 linker domain to the wild-type, human RNase1 domain (e.g., an HST-RNase-linker-DNase A114F molecule; SEQ ID NO: 26)). In some embodiments, the HST-RNase-linker-DNase A114F molecule lacks the VK3LP leader (SEQ ID NO: 50). In some embodiments, a hybrid nuclease-transferrin molecule has an amino acid sequence at least 80% identical, such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 99.5% identical to an amino acid sequence of any one of SEQ ID NOs: 3-50. It will be understood by one of ordinary skill that the leader and linker sequences are optional and are not limited to those described in the embodiments above. For example, the RNase and/or DNase domains can be directly fused to the N- and/or C-terminus of HST, or variant or fragment thereof; the leader domain can be any of those known in the art to be useful for its intended purpose, e.g., to increase protein expression and/or secretion (e.g., a Gaussia luciferase signal peptide (MGVKVLFALICIAVAEA; SEQ ID NO: 74)); the linker can be any linker known in the art, e.g., (Gly₄Ser)n, NLG (VDGAAASPVNVSSPSVQDI; SEQ ID NO: 84), LE, thrombin-sensitive disulphide cyclopeptide linker, LEA(EAAAK)₄ALEA(EAAAK)₄ (SEQ ID NO: 75), or an in vivo cleavable disulphide linker, as described herein. It will also be understood that it is within the abilities of a skilled artisan to make the corresponding changes to the amino acid sequences of the hybrid nuclease molecule using routine cloning and recombination methods. It will also be understood that the asparagine residues in the nuclease domains (i.e., N34, N76, and N88 in RNase1, and N18 and N106 in DNase1) can be substituted with an amino acid other than serine (e.g., glutamine), as long as the amino acid does not serve as an acceptor for N-linked glycosylation. It will also be understood that the potential N-linked glycosylation sites in transferrin can also be mutated to eliminate N- linked glycosylation. Methods of Making Hybrid Nuclease-transferrin Molecules The hybrid nuclease-transferrin molecules of this invention largely may be made in transformed or transfected host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used. The invention also includes a vector capable of expressing the peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides operably coupled to appropriate expression control sequences. Methods of affecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal nuclease domains, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation. The resulting vector having the DNA molecule thereon is used to transform or transfect an appropriate host. This transformation or transfection may be performed using methods well known in the art. Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation or transfection, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli), yeast (such as Saccharomyces) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art. In a preferred embodiment, the hybrid nuclease-transferrin molecules are produced in CHO cells. Next, the transformed or transfected host is cultured and purified. Host cells may be cultured under conventional fermentation or culture conditions so that the desired compounds are expressed. Such fermentation and culture conditions are well known in the art. Finally, the peptides are purified from culture by methods well known in the art. The compounds may also be made by synthetic methods. For example, solid phase synthesis techniques may be used. Suitable techniques are well known in the art, and include those described in Merrifield (1973), Chem. Polypeptides, pp.335-61

(Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc.85: 2149; Davis et al., Biochem Intl 1985;10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No.3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides. Compounds that contain derivatized peptides or which contain non-peptide groups may be synthesized by well-known organic chemistry techniques. Other methods are of molecule expression/synthesis are generally known in the art to one of ordinary skill. Hybrid nuclease-transferrin molecules with altered glycosylation

Glycosylation (e.g., O-lined or N-linked glycosylation) can impact the serum half-life of the hybrid nuclease-transferrin molecules of the invention by, e.g., minimizing their removal from circulation by mannose and asialoglycoprotein receptors and other lectin- like receptors. Accordingly, in some embodiments, the hybrid nuclease-transferrin molecules of the invention are prepared in aglycosylated, deglycosylated, or

underglycosylated form. Preferably, N-linked glycosylation is altered and the hybrid nuclease-transferrin molecule is aglycosyated. In some embodiments, all asparagine residues in a hybrid nuclease-transferrin molecule that conform to the Asn-X-Ser/Thr (X can be any other naturally occurring amino acid except Pro) consensus are mutated to residues that do not serve as acceptors of N-linked glycosylation (e.g., serine, glutamine), thereby eliminating glycosylation of the hybrid nuclease-transferrin molecule when synthesized in a cell that glycosylates proteins. In some embodiments, hybrid nuclease-transferrin molecules lacking N-linked glycosylation sites are produced in mammalian cells. In one embodiment, the mammalian cell is a CHO cell. Accordingly, in a specific embodiment, an aglycosylated hybrid nuclease-transferrin molecule is produced in a CHO cell. In other embodiments, a reduction or lack of N-glycosylation is achieved by, e.g., producing hybrid nuclease-transferrin molecules in a host (e.g., bacteria such as E. coli), mammalian cells engineered to lack one or more enzymes important for glycosylation, or mammalian cells treated with agents that prevent glycosylation, such as tunicamycin (an inhibitor of Dol-PP-GlcNAc formation). In some embodiments, the hybrid nuclease-transferrin molecules are produced in lower eukaryotes engineered to produce glycoproteins with complex N-glycans, rather than high mannose type sugars (see, e.g., US2007/0105127). In some embodiments, glycosylated hybrid nuclease-transferrin molecules (e.g., those produced in mammalian cells such as CHO cells) are treated chemically or enzymatically to remove one or more carbohydrate residues (e.g., one or more mannose, fucose, and/or N-acetylglucosamine residues) or to modify or mask one or more carbohydrate residues. Such modifications or masking may reduce binding of the hybrid nuclease-transferrin molecules to mannose receptors, and/or asialoglycoprotein receptors, and/or other lectin- like receptors. Chemical deglycosylation can be achieved by treating a hybrid nuclease- transferrin molecule with trifluoromethane sulfonic acid (TFMS), as disclosed in, e.g., Sojar et al., JBC 1989;264:2552-9 and Sojar et al., Methods Enzymol 1987;138:341-50, or by treating with hydrogen fluoride, as disclosed in Sojar et al. (1987, supra).

Enzymatic removal of N-linked carbohydrates from hybrid nuclease-transferrin molecules can be achieved by treating a hybrid nuclease-transferrin molecule with protein N-glycosidase (PNGase) A or F, as disclosed in Thotakura et al. (Methods Enzymol 1987;138:350-9). Other art-recognized commercially available deglycosylating enzymes that are suitable for use include endo-alpha-N-acetyl-galactosaminidase, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3, and endoglycosidase H. In some embodiments, one or more of these enzymes can be used to deglycosylate the hybrid nuclease-transferrin molecules of the invention. Alternative methods for deglycosylation are disclosed in, e.g., US 8,198,063. In some embodiments, the hybrid nuclease-transferrin molecules are partially

deglycosylated. Partial deglycosylation can be achieved by treating the hybrid nuclease- transferrin molecules with an endoglycosidase (e.g., endoglycosidase H), which cleaves N-linked high mannose carbohydrate but not complex type carbohydrates, leaving a single GlcNAc residue linked to the asparagine. Hybrid nuclease-transferrin molecules treated with endoglycosidase H will lack high mannose carbohydrates, resulting in a reduced interaction with the hepatic mannose receptor. Although this receptor recognizes terminal GlcNAc, the probability of a productive interaction with the single GlcNAc on the protein surface is not as great as with an intact high mannose structure. In other embodiments, glycosylation of a hybrid nuclease-transferrin molecule is modified, e.g., by oxidation, reduction, dehydration, substitution, esterification, alkylation, sialylation, carbon-carbon bond cleavage, or the like, to reduce clearance of the hybrid nuclease-transferrin molecules from blood. In some embodiments, the hybrid nuclease-transferrin molecules are treated with periodate and sodium borohydride to modify the carbohydrate structure. Periodate treatment oxidizes vicinal diols, cleaving the carbon-carbon bond and replacing the hydroxyl groups with aldehyde groups;

borohydride reduces the aldehydes to hydroxyls. Many sugar residues include vicinal diols and, therefore, are cleaved by this treatment. Prolonged serum half-life with periodate and sodium borohydride is exemplified by the sequential treatment of the lysosomal enzyme β-glucuronidase with these agents (see, e.g., Houba et al. (1996) Bioconjug Chem 1996:7:606-11; Stahl et al. PNAS 1976;73:4045-9; Achord et al. Pediat. Res 1977;11:816-22; Achord et al. Cell 1978;15:269-78). A method for treatment with periodate and sodium borohydride is disclosed in Hickman et al., BBRC 1974;57:55-61. A method for treatment with periodate and cyanoborohydride, which increases the serum half-life and tissue distribution of ricin, is disclosed in Thorpe et al. Eur J Biochem 1985;147:197-206. In one embodiment, the carbohydrate structures of a hybrid nuclease-transferrin molecule can be masked by addition of one or more additional moieties (e.g., carbohydrate groups, phosphate groups, alkyl groups, etc.) that interfere with recognition of the structure by a mannose or asialoglycoprotein receptor or other lectin-like receptors. In some embodiments, one or more potential glycosylation sites are removed by mutation of the nucleic acid encoding the hybrid nuclease-transferrin molecule, thereby reducing glycosylation (underglycosylation) of the hybrid nuclease-transferrin molecule when synthesized in a cell that glycosylates proteins, e.g., a mammalian cell such as a CHO cell. In some embodiments, it may be desirable to selectively underglycosylate the nuclease domain of the hybrid nuclease-transferrin molecules by mutating the potential N-linked glycosylation sites therein if, e.g., the underglycosylated hybrid nuclease-transferrin molecule exhibits increased activity or contributes to increased serum half-life. In other embodiments, it may be desirable to underglycosylate portions of the hybrid nuclease- transferrin molecule such that regions other than the nuclease domain lack N- glycosylation if, for example, such a modification improves the serum half-life of the hybrid nuclease-transferrin molecule. Alternatively, other amino acids in the vicinity of glycosylation acceptors can be modified, disrupting a recognition motif for glycosylation enzymes without necessarily changing the amino acid that would normally be

glycosylated. In some embodiments, glycosylation of a hybrid nuclease-transferrin molecule can be altered by introducing glycosylation sites. For example, the amino acid sequence of the hybrid nuclease-transferrin molecule can be modified to introduce the consensus sequence for N-linked glycosylation of Asp-X-Ser/Thr (X is any amino acid other than proline). Additional N-linked glycosylation sites can be added anywhere throughout the amino acid sequence of the hybrid nuclease-transferrin molecule. Preferably, the glycosylation sites are introduced in position in the amino acid sequence that does not substantially reduce the nuclease (e.g., RNase and/or DNase) activity of the hybrid nuclease-transferrin molecule. The addition of O-linked glycosylation sites has been reported to alter serum half-life of proteins, such as growth hormone, follicle-stimulating hormone, IGFBP-6, Factor IX, and many others (e.g., as disclosed in Okada et al., Endocr Rev 2011;32:2-342; Weenen et al., J Clin Endocrinol Metab 2004;89:5204-12; Marinaro et al., European Journal of Endocrinology 2000;142:512-6; US 2011/0154516). Accordingly, in some embodiments, O-linked glycosylation (on serine/threonine residues) of the hybrid nuclease-transferrin molecules is altered. Methods for altering O-linked glycosylation are routine in the art and can be achieved, e.g., by beta-elimination (see, e.g., Huang et al., Rapid

Communications in Mass Spectrometry 2002;16:1199-204; Conrad, Curr Protoc Mol Biol 2001; Chapter 17:Unit17.15A; Fukuda, Curr Protoc Mol Biol 2001;Chapter 17;Unit 17.15B; Zachara et al., Curr Protoc Mol Biol 2011; Unit 17.6; ); by using commercially available kits (e.g., GlycoProfile^TM Beta-Elimination Kit, Sigma); or by subjecting the hybrid nuclease-transferrin molecule to treatment with a series of exoglycosidases such as, but not limited to, β1-4 galactosidase and β–N-acetylglucosaminidase, until only Gal β1-3GalNAc and/or GlcNAc β1-3GalNAc remains, followed by treatment with, e.g., endo-α-N-acetylgalactosaminidase (i.e., O-glycosidase). Such enzymes are commercially available from, e.g., New England Biolabs. In yet other embodiments, the hybrid nuclease-transferrin molecules are altered to introduce O-linked glycosylation in the hybrid nuclease-transferrin molecule as disclosed in, e.g., Okada et al. (supra), Weenen et al. (supra), US2008/0274958; and US2011/0171218. In some embodiments, one or more O-linked glycosylation consensus sites are introduced into the hybrid nuclease-transferrin molecule, such as CXXGGT/S-C (SEQ ID NO: 76) (van den Steen et al., In Critical Reviews in Biochemistry and Molecular Biology, Michael Cox, ed., 1998;33:151-208), NST-E/D-A (SEQ ID NO: 77), NITQS (SEQ ID NO: 78), QSTQS (SEQ ID NO: 79), D/E-FT-R/K-V (SEQ ID NO: 80), C-E/D-SN (SEQ ID NO: 81), and GGSC-K/R (SEQ ID NO: 82). Additional O-linked glycosylation sites can be added anywhere throughout the amino acid sequence of the hybrid nuclease-transferrin molecule. Preferably, the glycosylation sites are introduced in position in the amino acid sequence that does not substantially reduce the nuclease (e.g., RNase and/or DNase) activity of the hybrid nuclease-transferrin molecule. Alternatively, O-linked sugar moieties are introduced by chemically modifying an amino acid in the hybrid nuclease-transferrin molecule as described in, e.g., WO 87/05330 and Aplin et al., CRC Crit Rev Biochem 1981;259-306). In some embodiments, both N-linked and O-linked glycosylation sites are introduced into the hybrid nuclease-transferrin molecules, preferably in positions in the amino acid sequence that do not substantially reduce the nuclease (e.g., RNase and/or DNase) activity of the hybrid nuclease-transferrin molecule. It is well within the abilities of the skilled artisan to introduce, reduce, or eliminate glycosylation (e.g., N-linked or O-linked glycosylation) in a hybrid nuclease-transferrin molecule and determine using routine methods in the art whether such modifications in glycosylation status increases or decreases the nuclease activity or serum half-life of the hybrid nuclease-transferrin molecule. In some embodiments, the hybrid nuclease-transferrin molecule may comprise an altered glycoform (e.g., an underfucosylated or fucose-free glycan). In some embodiments, a hybrid nuclease-transferrin molecule with altered glycosylation has a serum half-life that is increased at least about 1.5-fold, such as at least 3-fold, at least 5-fold, at least 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, or 1000-fold or greater relative to the corresponding glycosylated hybrid nuclease-transferrin molecules (e.g., a hybrid nuclease-transferrin molecule in which potential N-linked glycosylation sites are not mutated). Routine art-recognized methods can be used to determine the serum half-life of hybrid nuclease-transferrin molecules with altered glycosylation status. In some embodiments, a hybrid nuclease-transferrin molecule with altered glycosylation (e.g., a aglycosylated, deglycosylated, or underglycosylated hybrid nuclease-transferrin molecules) retains at least 50%, such as at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the activity of the corresponding glycosylated hybrid nuclease-transferrin molecule (e.g., a hybrid nuclease-transferrin molecule in which potential N-linked glycosylation sites are not mutated). In some embodiments, altering the glycosylation status of the hybrid nuclease-transferrin molecules may increase nuclease activity, either by directly increasing enzymatic activity, or by increasing bioavailability (e.g., serum half-life). Accordingly, in some

embodiments, the nuclease activity of a hybrid nuclease-transferrin molecule with altered glycosylation is increased by at least 1.3-fold, such as at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5- fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5 fold, or 10-fold or greater, relative to the corresponding glycosylated hybrid nuclease-transferrin molecule (e.g., a hybrid nuclease-transferrin molecule in which potential N-linked glycosylation sites are not mutated). The skilled artisan can readily determine the glycosylation status of hybrid nuclease- transferrin molecules using art-recognized methods. In a preferred embodiment, the glycosylation status is determined using mass spectrometry. In other embodiments, interactions with Concanavalin A (Con A) can be assessed to determine whether a hybrid nuclease-transferrin molecule is underglycosylated. An underglycosylated hybrid nuclease-transferrin molecule is expected to exhibit reduced binding to Con A-Sepharose when compared to the corresponding glycosylated hybrid nuclease-transferrin molecule. SDS-PAGE analysis can also be used to compare the mobility of an underglycosylated protein and corresponding glycosylated protein. The underglycosylated protein is expected to have a greater mobility in SDS-PAGE compared to the glycosylated protein. Other suitable art-recognized methods for analyzing protein glycosylation status are disclosed in, e.g., Roth et al., International Journal of Carbohydrate Chemistry 2012;1- 10. Pharmacokinetics, such as serum half-life, of hybrid nuclease-transferrin molecules with different glycosylation status can be assayed using routine methods, e.g., by introducing the hybrid nuclease-transferrin molecules in mice, e.g., intravenously, taking blood samples at pre-determined time points, and assaying and comparing levels and/or enzymatic activity of the hybrid nuclease-transferrin molecules in the samples. Pharmaceutical Compositions In certain embodiments, a hybrid nuclease-transferrin molecule is administered alone. In certain embodiments, a hybrid nuclease-transferrin molecule is administered prior to the administration of at least one other therapeutic agent. In certain embodiments, a hybrid nuclease-transferrin molecule is administered concurrent with the administration of at least one other therapeutic agent. In certain embodiments, a hybrid nuclease-transferrin molecule is administered subsequent to the administration of at least one other therapeutic agent. In other embodiments, a hybrid nuclease-transferrin molecule is administered prior to the administration of at least one other therapeutic agent. As will be appreciated by one of skill in the art, in some embodiments, the hybrid nuclease- transferrin molecule is combined with the other agent/compound. In some embodiments, the hybrid nuclease-transferrin molecule and other agent are administered concurrently. In some embodiments, the hybrid nuclease-transferrin molecule and other agent are not administered simultaneously, with the hybrid nuclease-transferrin molecule being administered before or after the agent is administered. In some embodiments, the subject receives both the hybrid nuclease-transferrin molecule and the other agent during a same period of prevention, occurrence of a disorder, and/or period of treatment. Pharmaceutical compositions of the invention can be administered in combination therapy, i.e., combined with other agents. In certain embodiments, the combination therapy comprises the hybrid nuclease-transferrin molecule, in combination with at least one other agent. Agents include, but are not limited to, in vitro synthetically prepared chemical compositions, antibodies, antigen binding regions, and combinations and conjugates thereof. In certain embodiments, an agent can act as an agonist, antagonist, allosteric modulator, or toxin. In certain embodiments, the invention provides for pharmaceutical compositions comprising a hybrid nuclease-transferrin molecule together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. In certain embodiments, the invention provides for pharmaceutical compositions comprising a hybrid nuclease-transferrin molecule and a therapeutically effective amount of at least one additional therapeutic agent, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant. In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In some embodiments, the formulation material(s) are for s.c. and/or I.V. administration. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as gelatin); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide);

solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company (1995). In some embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH 5.2, 9% Sucrose. In certain embodiments, a hybrid nuclease-transferrin molecule and/or a therapeutic molecule is linked to a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, polyethylene glycol, glycogen (e.g., glycosylation of the hybrid nuclease-transferrin molecule), and dextran. Such vehicles are described, e.g., in U.S. application Ser. No.09/428,082, now U.S. Pat. No.6,660,843 and published PCT

Application No. WO 99/25044. In certain embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antibodies of the invention. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In some embodiments, the saline comprises isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum transferrin are further exemplary vehicles. In certain

embodiments, pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about H 4.0-5.5, which can further include sorbitol or a suitable substitute therefore. In certain embodiments, a composition comprising a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agents, can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agent, can be formulated as a lyophilizate using appropriate excipients such as sucrose. In certain embodiments, the pharmaceutical composition can be selected for parenteral delivery. In certain embodiments, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. The preparation of such

pharmaceutically acceptable compositions is within the ability of one skilled in the art. In certain embodiments, the formulation components are present in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. In certain embodiments, when parenteral administration is contemplated, a therapeutic composition can be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising a desired hybrid nuclease-transferrin molecule, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In certain embodiments, a vehicle for parenteral injection is sterile distilled water in which a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution, properly preserved. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that can provide for the controlled or sustained release of the product which can then be delivered via a depot injection. In certain embodiments, hyaluronic acid can also be used, and can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired molecule. In certain embodiments, a pharmaceutical composition can be formulated for inhalation. In certain embodiments, a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agent, can be formulated as a dry powder for inhalation. In certain embodiments, an inhalation solution comprising a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agent, can be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration is further described in PCT application no.

PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins. In certain embodiments, it is contemplated that formulations can be administered orally. In certain embodiments, a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agents, that is administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. In certain embodiments, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. In certain embodiments, at least one additional agent can be included to facilitate absorption of a hybrid nuclease-transferrin molecule and/or any additional therapeutic agents. In certain embodiments, diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed. In certain embodiments, a pharmaceutical composition can involve an effective quantity of a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agents, in a mixture with non-toxic excipients which are suitable for the manufacture of tablets. In certain embodiments, by dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. In certain embodiments, suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc. Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agent(s), in sustained- or controlled-delivery formulations. In certain embodiments, techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See for example, PCT Application No. PCT/US93/00829 which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. In certain embodiments, sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g. films, or

microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No.3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers, 22:547-556 (1983)), poly (2- hydroxyethyl-methacrylate) (Langer et al., J Biomed Mater Res, 15: 167-277 (1981) and Langer, Chem Tech, 12:98-105 (1982)), ethylene vinyl acetate (Langer et al, supra) or poly-D(-)-3-hydroxybutyric acid (EP 133,988). In certain embodiments, sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al, PNAS, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949. The pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, this can be accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration can be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. In certain embodiments, once the pharmaceutical composition has been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to

administration. In certain embodiments, kits are provided for producing a single-dose administration unit. In certain embodiments, the kit can contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) are included. In certain embodiments, the effective amount of a pharmaceutical composition

comprising a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agent, to be employed therapeutically will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment, according to certain embodiments, will thus vary depending, in part, upon the molecule delivered, the indication for which a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agent, is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. In certain embodiments, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. In certain embodiments, a typical dosage can range from about 0.1 µg/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In certain embodiments, the dosage can range from 0.1 µg/kg up to about 100 mg/kg; or 1 µg/kg up to about 100 mg/kg; or 5 µg/kg up to about 100 mg/kg. In certain embodiments, the frequency of dosing will take into account the pharmacokinetic parameters of a hybrid nuclease-transferrin molecule and/or any additional therapeutic agents in the formulation used. In certain embodiments, a clinician will administer the composition until a dosage is reached that achieves the desired effect. In certain embodiments, the composition can therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose- response data. In certain embodiments, the route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous,

intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device. In certain embodiments, the composition can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration. In certain embodiments, it can be desirable to use a pharmaceutical composition comprising a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agent, in an ex vivo manner. In such instances, cells, tissues and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising a hybrid nuclease-transferrin molecule, with or without at least one additional therapeutic agent, after which the cells, tissues and/or organs are subsequently implanted back into the patient. In certain embodiments, a hybrid nuclease-transferrin molecule and/or any additional therapeutic agents can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, in order to decrease the chance of an

immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In certain embodiments, the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues. In vitro assays Various in vitro assays known in the art can be used to assess the efficacy of the hybrid nuclease-transferrin molecules of the invention. For example, cultured human PBMCs from normal or lupus patient PBMCs are isolated, cultured, and treated with various stimuli (e.g., TLR ligands, costimulatory antibodies, immune complexes, and normal or autoimmune sera), in the presence or absence of the hybrid nuclease-transferrin molecules. Cytokine production by the stimulated cells can be measured using commercially available reagents, such as the antibody pair kits from Biolegend (San Diego, CA) for various cytokines (e.g., IL-6, IL-8, IL-10, IL-4, IFN- gamma, and TNF-alpha). Culture supernatants are harvested at various time points as appropriate for the assay (e.g., 24, 48 hours, or later time points) to determine the effects that the hybrid nuclease-transferrin molecules have on cytokine production. IFN-alpha production is measured using, e.g., anti-human IFN-alpha antibodies and standard curve reagents available from PBL interferon source (Piscataway, NJ). Similar assays are performed using human lymphocyte subpopulations (isolated monocytes, B cells, pDCs, T cells, etc.); purified using, e.g., commercially available magnetic bead based isolation kits available from Miltenyi Biotech (Auburn, CA). Multi-color flow cytometry can be used to assess the effects of the hybrid nuclease- transferrin molecules on immune cell activation by measuring the expression of lymphocyte activation receptors such as CD5, CD23, CD69, CD80, CD86, and CD25 in PBMCs or isolated cell subpopulations at various time points after stimulation using routine art-recognized methods. The efficacy of hybrid nuclease-transferrin molecules can also be tested by incubating SLE patient serum with normal human pDCs to activate IFN output, as described in, e.g., Ahlin et al., Lupus 2012:21:586-95; Mathsson et al., Clin Expt Immunol 2007;147:513- 20; and Chiang et al., J Immunol 2011;186:1279-1288. Without being bound by theory, circulating nucleic acid-containing immune complexes in SLE patient sera facilitate nucleic acid antigen entry into pDC endosomes via Fc receptor-mediated endocytosis, followed by binding of nucleic acids to and activation of endosomal TLRs 7, 8, and 9. To assess the impact of the hybrid nuclease-transferrin molecules, SLE patient sera or plasma is pretreated with the hybrid nuclease-transferrin molecules, followed by addition to cultures of pDC cells isolated from healthy volunteers. Levels of IFN-α produced are then determined at multiple time points. By degrading nucleic-acid containing immune complexes, effective hybrid nuclease-transferrin molecules are expected to reduce the quantity of IFN-α produced. The effectiveness of hybrid nuclease-transferrin molecules is demonstrated by comparing the results of an assay from cells treated with a hybrid nuclease-transferrin molecules disclosed herein to the results of the assay from cells treated with control formulations. After treatment, the levels of the various markers (e.g., cytokines, cell-surface receptors, proliferation) described above are generally improved in an effective hybrid nuclease- transferrin molecule treated group relative to the marker levels existing prior to the treatment, or relative to the levels measured in a control group. Methods of treatment The hybrid nuclease-transferrin molecules of the invention are particularly effective in the treatment of autoimmune disorders or abnormal immune responses. In this regard, it will be appreciated that the hybrid nuclease-transferrin molecules of the present invention may be used to control, suppress, modulate, treat, or eliminate unwanted immune responses to both external and autoantigens. In another aspect, a hybrid nuclease-transferrin molecule is adapted for preventing (prophylactic) or treating (therapeutic) a disease or disorder, such as an autoimmune disease, in a mammal by administering an hybrid nuclease-transferrin molecule in a therapeutically effective amount or a sufficient amount to the mammal in need thereof, wherein the disease is prevented or treated. Any route of administration suitable for achieving the desired effect is contemplated by the invention (e.g., intravenous, intramuscular, subcutaneous). Treatment of the disease condition may result in a decrease in the symptoms associated with the condition, which may be long-term or short-term, or even a transient beneficial effect. Numerous disease conditions are suitable for treatment with the hybrid nuclease- transferrin molecules of the invention. For example, in some aspects, the disease or disorder is an autoimmune disease or cancer. In some such aspects, the autoimmune disease is insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, an experimental form of uveoretinitis, Hashimoto’s thyroiditis, primary myxoedema, thyrotoxicosis, pernicious anaemia, autoimmune atrophic gastritis, Addison’s disease, premature menopause, male infertility, juvenile diabetes, Goodpasture’s syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune haemolytic anaemia, idiopathic leucopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerative colitis, Sjogren’s syndrome, scleroderma, Wegener’s granulomatosis, polymyositis, dermatomyositis, discoid LE, SLE, or connective tissue disease. In a specific embodiment, a hybrid nuclease-transferrin molecule is used to prevent or treat SLE or Sjogren’s syndrome. The effectiveness of a hybrid nuclease-transferrin molecule is demonstrated by comparing the IFN-alpha levels, IFN-alpha response gene levels, autoantibody titers, kidney function and pathology, and/or circulating immune complex levels in mammals treated with a hybrid nuclease-transferrin molecule disclosed herein to mammals treated with control formulations. For example, a human subject in need of treatment is selected or identified (e.g., a patient who fulfills the American College of Rheumatology criteria for SLE, or a patient who fulfills the American-European Consensus Sjogren’s Classification Criteria). The subject can be in need of, e.g., reducing a cause or symptom of SLE or Sjogren’s syndrome. The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit. At time zero, a suitable first dose of a hybrid nuclease-transferrin molecule is

administered to the subject. The hybrid nuclease-transferrin molecule is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated, e.g., by measuring IFN-alpha levels, IFN- alpha response gene levels, autoantibody titers, kidney function and pathology, and/or circulating immune complex levels. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs. After treatment, the subject's IFN-alpha levels, IFN-alpha response gene levels, autoantibody titers, kidney function and pathology, and/or circulating immune complex levels are lowered and/or improved relative to the levels existing prior to the treatment, or relative to the levels measured in a similarly afflicted but untreated/control subject. In another example, a rodent subject in need of treatment is selected or identified (see, e.g., Example 7). The identification of the subject can occur in a laboratory setting or elsewhere. At time zero, a suitable first dose of a hybrid nuclease-transferrin molecule is administered to the subject. The hybrid nuclease-transferrin molecule is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated, e.g., by measuring IFN-alpha levels, IFN- alpha response gene levels, autoantibody titers, kidney function and pathology, and/or circulating immune complex levels. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs. After treatment, the subject's IFN-alpha levels, IFN-alpha response gene levels, autoantibody titers, kidney function and pathology, and/or circulating immune complex levels are lowered and/or improved relative to the levels existing prior to the treatment, or relative to the levels measured in a similarly afflicted but untreated/control subject. Another aspect of the present invention is to use gene therapy methods for treating or preventing disorders, diseases, and conditions with one or more hybrid nuclease- transferrin molecules. The gene therapy methods relate to the introduction of hybrid nuclease-transferrin molecule nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal in need thereof to achieve expression of the polypeptide or polypeptides of the present invention. This method can include introduction of one or more polynucleotides encoding a hybrid nuclease-transferrin molecule polypeptide of the present invention operably coupled to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. In gene therapy applications, hybrid nuclease-transferrin molecule genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product.“Gene therapy” includes both conventional gene therapies where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. The oligonucleotides can be modified to enhance their uptake, e.g., by substituting their negatively charged phosphodiester groups by uncharged groups. EXAMPLES Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. The practice of the present invention will employ, unless otherwise indicated,

conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3^rd Ed. (Plenum Press) Vols A and B (l 992). EXAMPLE 1

Generating hybrid nuclease-transferrin molecule encoding expression vectors Various embodiments of the hybrid nuclease-transferrin molecules of the invention are shown in Figure 1, with amino acid sequences of each presented in Table 1. As exemplary hybrid nuclease-transferrin molecules, hybrid nuclease-transferrin molecules with the configurations shown in Figure 1 are constructed. Specifically, starting from the amino acid sequence of the hybrid nuclease-transferrin molecules, polynucleotides encoding the hybrid nuclease-transferrin molecules are directly synthesized using codon optimization by Genescript (Genescript, Piscatawy, N.J.) to allow for optimal expression in mammalian cells. The process of optimization involves, e.g., avoiding regions of very high (>80%) or very low (<30%) GC content when possible, and avoiding cis-acting sequence motifs, such as internal TATA-boxes, chi-sites and ribosomal entry sites, AT- rich or GC-rich sequence stretches, RNA instability motifs, repeat sequences and RNA secondary structures, and cryptic splice donor and acceptor sites in higher eukaryotes. DNAs encoding the hybrid nuclease-transferrin molecules are cloned into the pcDNA3.1+ mammalian expression vector. Hybrid nuclease-transferrin molecules with the following configurations are generated. Hybrid nuclease-transferrin molecule #1 (SEQ ID NO: 3) has the configuration HST- RNase1, wherein a wild-type, human RNase1 domain (SEQ ID NO: 60) is operably coupled to the C-terminus of wild-type HST (SEQ ID NO: 1). Hybrid nuclease-transferrin molecule #2 (SEQ ID NO: 4) has the configuration RNase1- HST, wherein a wild-type, human RNase1 domain is operably coupled to the N-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #3 (SEQ ID NO: 5) has the configuration HST- (Gly₄Ser)3-RNase1, wherein a wild-type, human RNase1 domain is operably coupled via a (Gly₄Ser)3 linker to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #4 (SEQ ID NO: 6) has the configuration RNase1- (Gly₄Ser)3-HST, wherein a wild-type, human RNase1 domain is operably coupled via a (Gly₄Ser)3 linker to the N-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #5 (SEQ ID NO: 7) has the configuration RNase1- HST-RNase1, wherein a first wild-type, human RNase1 domain is operably coupled to the N-terminus of wild-type HST, and a second wild-type, human RNase1 domain is operably coupled to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #6 (SEQ ID NO: 8) has the configuration RNase1- (Gly₄Ser)3-HST-(Gly₄Ser)3-RNase1, wherein a first wild-type, human RNase1 domain is operably coupled via a first (Gly₄Ser)3 sequence to the N-terminus of wild-type HST, and a second wild-type, human RNase1 domain is operably coupled via a second (Gly₄Ser)3 sequence to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #7 (SEQ ID NO: 9) has the configuration RNase1- HST-DNase1 A114F, wherein a wild-type, human RNase1 domain is operably coupled to the N-terminus of wild-type HST, and a mutant, human DNase1 A114F domain (SEQ ID NO: 52) is operably coupled to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #8 (SEQ ID NO: 10) has the configuration DNase1 A114F-HST-RNase1, wherein a mutant, human DNase1 A114F domain is operably coupled to the N-terminus of wild-type HST, and a wild-type, human RNase1 domain is operably coupled to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #9 (SEQ ID NO: 11) has the configuration RNase1- (Gly₄Ser)3-HST-(Gly₄Ser)3-DNase1 A114F, wherein a wild-type, human RNase1 domain is operably coupled via a first (Gly₄Ser)3 sequence to the N-terminus of wild-type HST, and a mutant, human DNase1 A114F domain is operably coupled via a second (Gly₄Ser)3 sequence to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #10 (SEQ ID NO: 12) has the configuration DNase1 A114F-(Gly₄Ser)3-HST-(Gly₄Ser)3-RNase1, wherein a mutant, human DNase1 A114F domain is operably coupled via a first (Gly₄Ser)3 sequence to the N-terminus of wild-type HST, and a wild-type, human RNase1 domain is operably coupled via a second (Gly₄Ser)3 sequence to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #11 (SEQ ID NO: 13) has the configuration DNase1 A114F-HST, wherein a mutant, human DNase1 A114F domain is operably coupled to the N-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #12 (SEQ ID NO: 14) has the configuration HST- DNase1 A114F, wherein a mutant, human DNase1 A114F domain is operably coupled to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #13 (SEQ ID NO: 15) has the configuration DNase1 A114F-(Gly₄Ser)3-HST, wherein a mutant, human DNase1 A114F domain is operably coupled via a (Gly₄Ser)3 sequence to the N-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #14 (SEQ ID NO: 16) has the configuration HST- (Gly₄Ser)3-DNase1 A114F, wherein a mutant, human DNase1 A114F domain is operably coupled via a (Gly₄Ser)3 sequence to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #15 (SEQ ID NO: 17) has the configuration RNase1 N34S/N76S/N88S-(Gly₄Ser)3-HST, wherein a mutant, human RNase1 N34S/N76S/N88S domain (SEQ ID NO: 61) is operably coupled via a (Gly₄Ser)3 linker to the N-terminus of wild-type HST. The asparagines residues at positions 34, 76, and 88 (potential acceptors of N-linked glycosylation) of human RNase1 are mutated to serine. Hybrid nuclease-transferrin molecule #16 (SEQ ID NO: 18) has the configuration HST- (Gly₄Ser)3-RNase1 N34S/N76S/N88S, wherein a mutant, human RNase1 domain N34S/N76S/N88S is operably coupled via a (Gly₄Ser)3 linker to the C-terminus of wild- type HST. Hybrid nuclease-transferrin molecule #17 (SEQ ID NO: 19) has the configuration DNase1 N18S/N106S/A114F-(Gly₄Ser)3-HST, wherein a mutant, human DNase1 N18S/N106S/A114F domain (SEQ ID NO: 72) is operably coupled via a (Gly₄Ser)3 linker to the N-terminus of wild-type HST. The asparagine residues at positions 18 and 106 of human DNase1 are potential acceptors of N-linked glycosylation. Hybrid nuclease-transferrin molecule #18 (SEQ ID NO: 20) has the configuration HST- (Gly₄Ser)3-DNase1 N18S/N106S/A114F, wherein a mutant, human DNase1 domain N18S/N106S/A114F is operably coupled via a (Gly₄Ser)3 linker to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #19 (SEQ ID NO: 21) has the configuration RNase1 N34S/N76S/N88S-(Gly₄Ser)3-HST-(Gly₄Ser)3-DNase1 A114F, wherein a mutant, human RNase1 N34S/N76S/N88S domain is operably coupled via a first (Gly₄Ser)3 sequence to the N-terminus of wild-type HST, and a mutant, human DNase1 A114F domain is operably coupled via a second (Gly₄Ser)3 sequence to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #20 (SEQ ID NO: 22) has the configuration DNase1 A114F-(Gly₄Ser)3-HST-(Gly₄Ser)3-RNase1 N34S/N76S/N88S, wherein a mutant, human DNase1 A114F domain is operably coupled via a first (Gly₄Ser)3 sequence to the N-terminus of wild-type HST, and a mutant, human RNase1 N34S/N76S/N88S domain is operably coupled via a second (Gly₄Ser)3 sequence to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #21 (SEQ ID NO: 23) has the configuration RNase1 N34S/N76S/N88S-(Gly₄Ser)3-HST-(Gly₄Ser)3-DNase1 N18S/N106S/A114F, wherein a mutant, human RNase1 N34S/N76S/N88S domain is operably coupled via a first (Gly₄Ser)3 sequence to the N-terminus of wild-type HST, and a mutant, human DNase1 N18S/N106S/A114F domain is operably coupled via a second (Gly₄Ser)3 sequence to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #22 (SEQ ID NO: 24) has the configuration DNase1 N18S/N106S/A114F-(Gly₄Ser)3-HST-(Gly₄Ser)3-RNase1 N34S/N76S/N88S, wherein a mutant, human DNase1 N18S/N106S/A114F domain is operably coupled via a first (Gly₄Ser)3 sequence to the N-terminus of wild-type HST, and a mutant, human RNase1 N34S/N76S/N88S domain is operably coupled via a second (Gly₄Ser)3 sequence to the C-terminus of wild-type HST. Hybrid nuclease-transferrin molecule #23 (SEQ ID NO: 25) has the configuration DNase1 A114F-(Gly₄Ser)3-RNase1-HST, wherein a mutant, human DNase1 A114F domain is operably coupled via a (Gly₄Ser)3 sequence to a wild-type, human RNase1, and wherein the wild-type human RNase1 is operably coupled to the N-terminus of wild- type HST. Hybrid nuclease-transferrin molecule #24 (SEQ ID NO: 26) has the configuration HST- RNase1-(Gly₄Ser)3-DNase1 A114F, wherein a mutant, human DNase1 A114F domain is operably coupled via a (Gly₄Ser)3 sequence to a wild-type, human RNase1, and wherein the wild-type human RNase1 is operably coupled to the C-terminus of wild-type HST. The hybrid nuclease-transferrin molecules of the invention can also be generated using conventional cloning techniques well-known in the art, for example, by preparing modular cassettes of each component of the hybrid nuclease-transferrin molecule (e.g., nuclease domain, linker domain, HST) with compatible restriction enzyme sites to allow for shuttling and domain swapping. A polynucleotide encoding each component of the hybrid nuclease-transferrin molecule (e.g., RNase, DNase, HST) can be readily obtained by amplifying the component of interest using polymerase chain reaction (PCR) from an appropriate cDNA library. For example, the full length nucleotide sequences of human RNase1, DNase1, and HST can be amplified from random primed and oligo dT primed cDNA derived from commercially available human pancreatic total RNA (Ambion/Applied Biosystems, Austin, TX) using sequence specific 5’ and 3’ primers based on published sequences of the component being amplified (or as shown in Table 1). PCR amplicons are purified by agarose gel electrophoresis and subsequent application to QIAquick gel purification columns. Purified amplicons are cloned into a convenient vector for subcloning and subsequent domain swapping and shuttling (e.g., pC42.1 TOPO cloning vector; Invitrogen, Carlsbad, CA). Polynucleotides encoding mutant nuclease domains or HST variants are generated by introducing mutations into the domain of interest using commercially available kits (e.g., QuickChange^TM site-directed mutagenesis kit; Stratagene), or overlap extension PCR to introduce mutations at desired positions, followed by DNA sequencing to confirm that the intended mutations are introduced. Linkers (e.g., (Gly₄Ser)3 linkers can be generated by overlap PCR using routine methods, or through direct synthesis using commercially available services, and designed to have overhangs or be blunt to facilitate subsequent cloning to allow for fusion with other domains of interest. EXAMPLE 2

Transient expression of and stable mammalian cell lines expressing hybrid nuclease- transferrin molecules

For transient expression, expression vectors from Example 1 containing the hybrid nuclease-transferrin molecule inserts are transiently trasfected using FreeStyle^TM MAX Reagent into Chinese Hamster Ovary (CHO) cells, e.g., CHO-S cells (e.g., FreeStyle^TM CHO-S cells, Invitrogen), using the manufacturer recommended transfection protocol. CHO-S cells are maintained in FreeStyle^TM CHO Expression Medium containing 2 mM L-Glutamine and penicillin-streptomycin. Stable CHO-S cell lines expressing the hybrid nuclease-transferrin molecules are generated using routine methods known in the art. For example, CHO-S cells can be infected with a virus (e.g., retrovirus, lentivirus) comprising the nucleic acid sequences of a hybrid nuclease-transferrin molecule, as well as the nucleic acid sequences encoding a marker (e.g., GFP, surface markers selectable by magnetic beads) that is selected for using, e.g., flow cytometry or magnetic bead separation (e.g., MACSelect^TM system). Alternatively, CHO-S cells are transfected using any transfection method known in the art, such as electroporation (Lonza) or the FreeStyle^TM MAX Reagent as mentioned above, with a vector comprising the nucleic acid sequences of the hybrid nuclease- transferrin molecules and a selectable marker, followed by selection using, e.g., flow cytometry. The selectable marker can be incorporated into the same vector as that encoding the hybrid nuclease-transferrin molecules or a separate vector. Hybrid nuclease-transferrin molecules are purified from culture supernatant by capturing the molecules using a column packed with beads conjugated with, e.g., anti-HST antibody, followed by washes in column wash buffer (e.g., 90 mM Tris, 150 mM NaCl, 0.05% sodium azide) and releasing the molecules from the column using a suitable elution buffer (e.g., 0.1 M citrate buffer, pH 3.0). The eluted material is further concentrated by buffer exchange through serial spins in PBS using Centricon

concentrators, followed by filtration through 0.2 µm filter devices. The concentration of the hybrid nuclease-transferrin molecules is determined using standard

spectrophotometric methods (e.g., Bradford, BCA, Lowry, Biuret assays). EXAMPLE 3

Half-life of hybrid nuclease-transferrin molecules in mouse sera Mice are intravenously injected with a single injection of a hybrid nuclease-transferrin molecule of Example 2 at time zero. Blood sample are collected at several time points post-injection (e.g., every day over the course of seven days) and analyzed for the presence of the hybrid nuclease-transferrin molecules. The hybrid nuclease-transferrin molecules can be detected with standard ELISA assays which capture human HST from mouse serum, followed by detection of human RNase1 or human DNase1. The same blood samples can be used to measure the enzymatic activity (i.e., the nuclease activity) of the hybrid nuclease-transferrin molecules using commercially available kits (e.g., RNaseAlert^TM QC system, Ambion; DNaseAlert^TM QC System, Invitrogen; DNase ELISA kit, Abnova; ORG 590 DNase Activity, ORGENTEC Diagnostika GmbH), following the manufacturers’ instructions. EXAMPLE 4

Nuclease activity of purified hybrid nuclease-transferrin molecules

RNase activity of affinity purified hybrid nuclease-transferrin molecules or hybrid nuclease-transferrin molecules present in mouse sera are confirmed with a SRED assay. The assay involves preparing a 2% agarose gel with distilled water and poly-IC (Sigma) dissolved in distilled water at 3 mg/ml, and preparing a gel plate as follows: 1.5 ml reaction buffer (0.2M Tris-HCl pH 7.0, 40mM EDTA, and 0.1 mg/ml ethidium bromide), 1 ml Poly-IC and 0.5 ml water are placed in a tube and maintained at 50°C for 5 min.3 ml of the agarose (kept at 50°C) is added to the tube and the mixture immediately poured onto a glass plate. Sampling wells are punched in the gel and 2 µl of each serum sample or affinity purified hybrid nuclease-transferrin molecule is loaded into the wells, followed by incubating the gel at 37°C for 4 hours in the moist chamber. The gel is subsequently incubated in a buffer (20 mM sodium acetate pH5.2, 20 mg/ml ethidium bromide) on ice for 30 min. and read under UV. Alternatively, RNase activity assays are performed using commercially available kits for measuring RNase activity, e.g., the RNaseAlert^TM QC System. DNase1 activity of the hybrid nuclease-transferrin molecules containing DNase1 domains is measured using any art-recognized method, such as the PicoGreen^TM-based assay described in Tolun et al., Nucleic Acids Research 2003;31:e111, and the plasmid nicking assay, as described in Campbell et al., JBC 1980;225:3726-35. Alternatively, the DNase activity of the hybrid nuclease-transferrin molecules is measured using an oligonucleotide digestion assay. The assay involves incubating the hybrid nuclease-transferrin molecules in 30 µl reactions containing 20mM Tris (pH7.5), 5mM MgCl₂, 2mM DTT, and a 36-mer oligonucleotide substrate, allowing the reactions to proceed for 20-30 min at 37°C, subjecting samples to electrophoresis on 23% polyacrylamide DNA gels overnight, incubating gels in TBE buffer containing 0.5 µg/ml ethidium bromide, visualizing DNA with a UV transilluminator, photographing gels using a digital camera equipped with ethidium bromide filters, and analyzing images for oligonucleotide digestion with imaging software (e.g., Kodak Molecular Imaging Software). Alternatively, the oligonucleotide substrate can be substituted for nuclear DNA isolated from, e.g., HeLa cells using standard techniques, and subjected to the same steps as the oligonucleotide digestion assay to analyze chromatin digestion (i.e., a chromatin digestion assay). These assays are compatible with serum samples or affinity purified hybrid nuclease-transferrin molecules. Alternatively, DNase activity assays are performed using commercially available kits for measuring DNase activity, e.g., the DNaseAlert^TM QC System (Invitrogen) and the DNase Detection Kit (Mo Bio Laboratories, Inc., Carlsbad CA. Appropriate negative controls for such assays include, e.g., DNA or RNA alone with no hybrid nuclease-transferrin molecule, an RNA substrate for a hybrid nuclease-transferrin molecule having a DNase domain, but lacking an RNase domain, or a DNA substrate for a hybrid nuclease-transferrin molecule having an RNase domain, but lacking a DNase domain. When RNase or DNase activity is being determined from serum samples (i.e., samples obtained from mice injected with the hybrid nuclease-transferrin molecules), an appropriate control is, e.g., serum from a mouse injected with vehicle only. EXAMPLE 5

Analysis of enzyme kinetics

Michaelis constants (Km) of the hybrid nuclease-transferrin molecules of Example 2 are determined. Enzyme kinetics of purified hybrid nuclease-transferrin molecules are assayed using the RNase Alert Substrate (Ambion/IDT, San Diego, CA.) according to manufacturer's instructions, and fluorescence is assayed using a microplate reader. Fluorescence data is collected at 30 second intervals over the course of a 30 minute incubation, and analyzed using SoftmaxPro Software (Molecular Devices). Reaction rates at different substrate concentrations are measured, plotted in the form of a Lineweaver Burke plot, and analyzed for Vmax and Km. EXAMPLE 6

Efficacy of hybrid nuclease-transferrin molecules in vitro Effects of hybrid nuclease-transferrin molecules on cytokine expression

Human PBMCs are isolated from normal patients and lupus patients and cultured. The cells are treated with various stimulatory TLR ligands, costimulatory antibodies, immune complexes, and normal or autoimmune sera, with or without the hybrid nuclease- transferrin molecules of Example 2. Culture supernatant is collected at various time points (e.g., 6 hrs, 12 hrs, 24 hrs, 48 hrs, etc) and levels of a panel of cytokines, including human IL-6, IL-8, IL-10, IL-4, IFN-gamma, IFN-alpha and TNF-alpha are measured using commercially available ELISA kits from, e.g., Thermo Fisher Scientific, Inc.

Effective hybrid nuclease-transferrin molecules are expected to reduce the levels of cytokines produced by stimulated PBMCs relative to controls. Effects of hybrid nuclease-transferrin molecules on lymphocyte activation receptor expression Human PBMCs are isolated from normal patients and lupus patients and cultured. The cells are treated with various stimulatory TLR ligands, costimulatory antibodies, immune complexes, and normal or autoimmune sera, with or without the hybrid nuclease- transferrin molecules of Example 2. Cells are then subjected to multi-color flow cytometry to measure the expression of lymphocyte activation receptors CD5, CD23, CD69, CD80, CD86, and CD25 at various time points (e.g., 6 hrs, 12 hrs, 24 hrs, 48 hrs, etc.) after stimulation using routine art-recognized methods. Suitable antibodies for these receptors are commercially available from, e.g., BD/PharMingen. Effective hybrid nuclease-transferrin molecules are expected to reduce the expression of the lymphocyte activation receptors in stimulated PBMCs relative to controls. Effects of hybrid nuclease-transferrin molecules on plasmacytoid dendritic cell (pDC) interferon output pDCs from healthy volunteers are isolated using art-recognized methods or commercially available kits, such as the EasySep^TM Human EpCAM Positive Selection Kit (StemCell Technologies, Inc.). Isolated pDCs are cultured in, e.g., 96-well flat-bottom plates, at a densities ranging from 5 x 10⁴ to 2.5 x 10⁵/well in 0.1 ml in an appropriate medium (e.g., complete RPMI medium containing 10% FBS, 2 mM glutamine, 55 µM β- mercaptoethanol, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin). Cultured pDCs are activated by adding sera or plasma from individual SLE patients diluted with culture medium at a 1:5 ratio, and 0.1 ml of these samples are added to the cell-containing wells (final patient serum concentration is 10%). Cultures are incubated at 37°C for 40 hr, after which the conditioned media is harvested and assessed for IFNα content using a commercially available ELISA kit. Serum samples obtained from healthy volunteers are used as controls. To assess the impact of the hybrid nuclease-transferrin constructs, SLE patient sera or plasma is pretreated with the hybrid nuclease-transferrin constructs (1-10 µg/ml) for 30 min and added to the pDC cultures. Effective hybrid nuclease-transferrin molecules are expected to reduce the quantity of IFNα produced as a result of degrading the nucleic acid-containing ICs. ************************

While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

KSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNL

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11 RNase-linker- METPAQLLFLLLLWLPDTTGKESRAKKFQRQHMDSDSSPSSSSTYCNQMMR HST-linker-DNase RRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHIT A114F DCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDSTGGGGS

GGGGSGGGGSVPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVK KASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQ TFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPR KPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKC LKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQ VPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFK DSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCAL SHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYI AGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKS CHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMG SGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWA KNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILR QQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGE EYVKAVGNLRKCSTSSLLEACTFRRPGGGGSGGGGSGGGGSLKIAAFNIQT FGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPD TYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNRE PFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDV MLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRI VVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

12 DNase1 A114F- METPAQLLFLLLLWLPDTTGLKIAAFNIQTFGETKMSNATLVSYIVQILSR linker-HST- YDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLF linker-RNase1 VYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPL

HAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSI RLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNF QAAYGLSDQLAQAISDHYPVEVMLKGGGGSGGGGSGGGGSVPDKTVRWCAV SEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTL DAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQLR GKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADG TDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENLA NKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWEL LNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLGY EYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIEC VSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCE DTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNK INHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAF RCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVE EYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFR SETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEAC

KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRSMTQGRCKPVNTFVHEPL

VDVQNVCFQEKVTCKNGQGNCYKSSSSMHITDCRLTSGSRYPNCAYRTSPK ERHIIVACEGSPYVPVHFDASVEDST

19 DNase1 METPAQLLFLLLLWLPDTTGLKIAAFNIQTFGETKMSSATLVSYIVQILSR N18S/N106S/A114F YDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLF -linker-HST VYRPDQVSAVDSYYYDDGCEPCGSDTFNREPFIVRFFSRFTEVREFAIVPL

HAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSI RLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNF QAAYGLSDQLAQAISDHYPVEVMLKGGGGSGGGGSGGGGSVPDKTVRWCAV SEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTL DAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQLR GKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADG TDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENLA NKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWEL LNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLGY EYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIEC VSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCE DTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNK INHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAF RCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVE EYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFR SETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEAC TFRRP

20 HST-linker- METPAQLLFLLLLWLPDTTGVPDKTVRWCAVSEHEATKCQSFRDHMKSVIP DNase1 SDGPSVACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVA N18S/N106S/A114F EFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIG

LLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQ YFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPV DEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFS SPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTD ECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADA MSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVKKSASDL TWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKK DSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQN TGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKD KEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHD RNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRPGGGGSGGGGSGGGGS LKIAAFNIQTFGETKMSSATLVSYIVQILSRYDIALVQEVRDSHLTAVGKL LDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCE PCGSDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLD VQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTA TPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPV EVMLK

21 RNase1 METPAQLLFLLLLWLPDTTGKESRAKKFQRQHMDSDSSPSSSSTYCNQMMR N34S/N76S/N88S- RRSMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSSSSMHIT linker-HST- DCRLTSGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDSTGGGGS linker-DNase1 GGGGSGGGGSVPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVK A114F KASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQ

TFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPR KPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKC LKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQ VPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFK DSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCAL SHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYI AGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKS CHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMG SGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWA

KNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILR QQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGE EYVKAVGNLRKCSTSSLLEACTFRRPGGGGSGGGGSGGGGSLKIAAFNIQT FGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPD TYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNRE PFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDV MLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRI VVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

22 DNase1 A114F- METPAQLLFLLLLWLPDTTGLKIAAFNIQTFGETKMSNATLVSYIVQILSR linker-HST- YDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLF linker-RNase1 VYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPL N34S/N76S/N88S HAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSI

RLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNF QAAYGLSDQLAQAISDHYPVEVMLKGGGGSGGGGSGGGGSVPDKTVRWCAV SEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTL DAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQLR GKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADG TDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENLA NKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWEL LNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLGY EYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIEC VSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCE DTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNK INHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAF RCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVE EYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFR SETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEAC TFRRPGGGGSGGGGSGGGGSKESRAKKFQRQHMDSDSSPSSSSTYCNQMMR RRSMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSSSSMHIT DCRLTSGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDST

23 RNase1 METPAQLLFLLLLWLPDTTGKESRAKKFQRQHMDSDSSPSSSSTYCNQMMR N34S/N76S/N88S- RRSMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSSSSMHIT linker-HST- DCRLTSGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDSTGGGGS linker-DNase1 GGGGSGGGGSVPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVK N18S/N106S/A114F KASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQ

TFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPR KPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKC LKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQ VPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFK DSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCAL SHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYI AGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKS CHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMG SGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWA KNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILR QQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGE EYVKAVGNLRKCSTSSLLEACTFRRPGGGGSGGGGSGGGGSLKIAAFNIQT FGETKMSSATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPD TYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGSDTFNRE PFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDV MLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRI VVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

24 DNase1 METPAQLLFLLLLWLPDTTGLKIAAFNIQTFGETKMSSATLVSYIVQILSR N18S/N106S/A114F YDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLF -linker-HST- VYRPDQVSAVDSYYYDDGCEPCGSDTFNREPFIVRFFSRFTEVREFAIVPL

VHFDASVEDSTGGGGSGGGGSGGGGSLKIAAFNIQTFGETKMSNATLVSYI

VQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSY KERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVRE FAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRP SQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDS ALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

27 HST-RNase (w/o VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRA leader) IAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVK

KDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANF FSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAF VKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARS MGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVP PRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDE WSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVL AENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAG WNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNN KEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYEL LCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNV TDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLR KCSTSSLLEACTFRRPKESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNM TQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRL TNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDST

28 RNase-HST (w/o KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPL leader) VDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPK

ERHIIVACEGSPYVPVHFDASVEDSTVPDKTVRWCAVSEHEATKCQSFRDH MKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNN LKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAG WNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCG CSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLD NTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSK EFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTC PEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIM NGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVK KSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGC APGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKH QTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHA VVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVC LAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP

29 HST-linker-RNase VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRA (w/o leader) IAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVK

KDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANF FSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAF VKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARS MGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVP PRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDE WSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVL AENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAG WNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNN KEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYEL LCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNV TDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLR KCSTSSLLEACTFRRPGGGGSGGGGSGGGGSKESRAKKFQRQHMDSDSSPS SSSTYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGN CYKSNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDA SVEDST

30 RNase-linker-HST KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPL

leader) ERHIIVACEGSPYVPVHFDASVEDSTVPDKTVRWCAVSEHEATKCQSFRDH

MKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNN LKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAG WNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCG CSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLD NTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSK EFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTC PEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIM NGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVK KSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGC APGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKH QTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHA VVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVC LAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRPLKIAAFNIQ TFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAP DTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNR EPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLED VMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDR IVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

34 DNase A114F-HST- LKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKL RNase (w/o LDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCE leader) PCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLD

VQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTA TPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPV EVMLKVPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYL DCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYA VAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEK AVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGA GDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHT VVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHG FLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHER LKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCG LVPVLAENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAV GRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNL CEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNE KDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHL FGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKA VGNLRKCSTSSLLEACTFRRPKESRAKKFQRQHMDSDSSPSSSSTYCNQMM RRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYKSNSSMHI TDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVEDST

35 RNase-linker- KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPL HST-linker-DNase VDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPK A114F (w/o ERHIIVACEGSPYVPVHFDASVEDSTGGGGSGGGGSGGGGSVPDKTVRWCA leader) VSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVT

LDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQL RGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCAD GTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENL ANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWE LLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLG YEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIE CVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNC EDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYN KINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGA FRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPV EEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLF RSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEA CTFRRPGGGGSGGGGSGGGGSLKIAAFNIQTFGETKMSNATLVSYIVQILS

KEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYEL

LCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNV TDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLR KCSTSSLLEACTFRRPLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIA LVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRP DQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAP GDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWT SPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAY GLSDQLAQAISDHYPVEVMLK

39 a DNase A114F- LKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKL linker-HST (w/o LDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCE leader) PCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLD

VQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTA TPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPV EVMLKGGGGSGGGGSGGGGSVPDKTVRWCAVSEHEATKCQSFRDHMKSVIP SDGPSVACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVA EFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIG LLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQ YFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPV DEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFS SPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTD ECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADA MSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVKKSASDL TWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKK DSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQN TGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKD KEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHD RNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP

40 HST-linker-DNase VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRA A114F (w/o IAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVK leader) KDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANF

FSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAF VKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARS MGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVP PRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDE WSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVL AENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAG WNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNN KEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYEL LCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNV TDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLR KCSTSSLLEACTFRRPGGGGSGGGGSGGGGSLKIAAFNIQTFGETKMSNAT LVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPL GRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRF TEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGC SYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGA VVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

41 RNase1 KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRSMTQGRCKPVNTFVHEPL N34S/N76S/N88S- VDVQNVCFQEKVTCKNGQGNCYKSSSSMHITDCRLTSGSRYPNCAYRTSPK linker-HST (w/o ERHIIVACEGSPYVPVHFDASVEDSTGGGGSGGGGSGGGGSVPDKTVRWCA leader) VSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVT

LDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQL RGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCAD GTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENL ANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWE LLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLG YEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIE

CVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNC EDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYN KINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGA FRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPV EEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLF RSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEA CTFRRP

42 HST-linker- VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRA RNase1 IAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVK N34S/N76S/N88S KDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANF (w/o leader) FSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAF

VKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARS MGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVP PRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDE WSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVL AENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAG WNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNN KEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYEL LCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNV TDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLR KCSTSSLLEACTFRRPGGGGSGGGGSGGGGSKESRAKKFQRQHMDSDSSPS SSSTYCNQMMRRRSMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGN CYKSSSSMHITDCRLTSGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDA SVEDST

43 DNase1 LKIAAFNIQTFGETKMSSATLVSYIVQILSRYDIALVQEVRDSHLTAVGKL N18S/N106S/A114F LDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCE -linker-HST (w/o PCGSDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLD leader) VQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTA

TPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPV EVMLKGGGGSGGGGSGGGGSVPDKTVRWCAVSEHEATKCQSFRDHMKSVIP SDGPSVACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVA EFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIG LLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQ YFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPV DEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFS SPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTD ECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADA MSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVKKSASDL TWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKK DSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQN TGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKD KEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHD RNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRP

44 HST-linker- VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRA DNase1 IAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVK N18S/N106S/A114F KDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANF (w/o leader) FSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAF

VKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARS MGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVP PRMDAKMYLGYEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDE WSVNSVGKIECVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVL AENYNKSDNCEDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAG WNIPMGLLYNKINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNN KEGYYGYTGAFRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYEL LCLDGTRKPVEEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNV TDCSGNFCLFRSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLR

KCSTSSLLEACTFRRPGGGGSGGGGSGGGGSLKIAAFNIQTFGETKMSSAT LVSYIVQILSRYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPL GRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGSDTFNREPFIVRFFSRF TEVREFAIVPLHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGC SYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGA VVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK

45 RNase1 KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRSMTQGRCKPVNTFVHEPL N34S/N76S/N88S- VDVQNVCFQEKVTCKNGQGNCYKSSSSMHITDCRLTSGSRYPNCAYRTSPK linker-HST- ERHIIVACEGSPYVPVHFDASVEDSTGGGGSGGGGSGGGGSVPDKTVRWCA linker-DNase1 VSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVT A114F (w/o LDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQL leader) RGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANFFSGSCAPCAD

GTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAFVKHSTIFENL ANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARSMGGKEDLIWE LLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVPPRMDAKMYLG YEYVTAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIE CVSAETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNC EDTPEAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYN KINHCRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGA FRCLVEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPV EEYANCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLF RSETKDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEA CTFRRPGGGGSGGGGSGGGGSLKIAAFNIQTFGETKMSNATLVSYIVQILS RYDIALVQEVRDSHLTAVGKLLDNLNQDAPDTYHYVVSEPLGRNSYKERYL FVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPFIVRFFSRFTEVREFAIVP LHAAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSS IRLWTSPTFQWLIPDSADTTATPTHCAYDRIVVAGMLLRGAVVPDSALPFN FQAAYGLSDQLAQAISDHYPVEVMLK

46 DNase1 A114F- LKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKL linker-HST- LDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCE linker-RNase1 PCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLD N34S/N76S/N88S VQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTA (w/o leader) TPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPV

EVMLKGGGGSGGGGSGGGGSVPDKTVRWCAVSEHEATKCQSFRDHMKSVIP SDGPSVACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVA EFYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIG LLYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQ YFGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPV DEYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFS SPHGKDLLFKDSAHGFLKVPPRMDAKMYLGYEYVTAIRNLREGTCPEAPTD ECKPVKWCALSHHERLKCDEWSVNSVGKIECVSAETTEDCIAKIMNGEADA MSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTPEAGYFAIAVVKKSASDL TWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINHCRFDEFFSEGCAPGSKK DSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCLVEKGDVAFVKHQTVPQN TGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYANCHLARAPNHAVVTRKD KEACVHKILRQQQHLFGSNVTDCSGNFCLFRSETKDLLFRDDTVCLAKLHD RNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFRRPGGGGSGGGGSGGGGS KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRSMTQGRCKPVNTFVHEPL VDVQNVCFQEKVTCKNGQGNCYKSSSSMHITDCRLTSGSRYPNCAYRTSPK ERHIIVACEGSPYVPVHFDASVEDST

47 RNase1 KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRSMTQGRCKPVNTFVHEPL N34S/N76S/N88S- VDVQNVCFQEKVTCKNGQGNCYKSSSSMHITDCRLTSGSRYPNCAYRTSPK linker-HST- ERHIIVACEGSPYVPVHFDASVEDSTGGGGSGGGGSGGGGSVPDKTVRWCA linker-DNase1 VSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRAIAANEADAVT N18S/N106S/A114F LDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVKKDSGFQMNQL

56 Human Trex1 MGPGARRQGRIVQGRPEMCFCPPPTPLPPLRILTLGTHTPTPCSSPGSAAG

TYPTMGSQALPPGPMQTLIFFDMEATGLPFSQPKVTELCLLAVHRCALESP PTSQGPPPTVPPPPRVVDKLSLCVAPGKACSPAASEITGLSTAVLAAHGRQ CFDDNLANLLLAFLRRQPQPWCLVAHNGDRYDFPLLQAELAMLGLTSALDG AFCVDSITALKALERASSPSEHGPRKSYSLGSIYTRLYGQSPPDSHTAEGD VLALLSICQWRPQALLRWVDAHARPFGTIRPMYGVTASARTKPRPSAVTTT AHLATTRNTSPSLGESRGTKDLPPVKDPGALSREGLLAPLGLLAILTLAVA TLYGLSLATPGE

57 Human DNase2 MIPLLLAALLCVPAGALTCYGDSGQPVDWFVVYKLPALRGSGEAAQRGLQY alpha KYLDESSGGWRDGRALINSPEGAVGRSLQPLYRSNTSQLAFLLYNDQPPQP (NP_001366.1) SKAQDSSMRGHTKGVLLLDHDGGFWLVHSVPNFPPPASSAAYSWPHSACTY

GQTLLCVSFPFAQFSKMGKQLTYTYPWVYNYQLEGIFAQEFPDLENVVKGH HVSQEPWNSSITLTSQAGAVFQSFAKFSKFGDDLYSGWLAAALGTNLQVQF WHKTVGILPSNCSDIWQVLNVNQIAFPGPAGPSFNSTEDHSKWCVSPKGPW TCVGDMNRNQGEEQRGGGTLCAQLPALWKAFQPLVKNYQPCNGMARKPSRA YKI

58 human DNase2 MKQKMMARLLRTSFALLFLGLFGVLGAATISCRNEEGKAVDWFTFYKLPKR beta QNKESGETGLEYLYLDSTTRSWRKSEQLMNDTKSVLGRTLQQLYEAYASKS

NNTAYLIYNDGVPKPVNYSRKYGHTKGLLLWNRVQGFWLIHSIPQFPPIPE EGYDYPPTGRRNGQSGICITFKYNQYEAIDSQLLVCNPNVYSCSIPATFHQ ELIHMPQLCTRASSSEIPGRLLTTLQSAQGQKFLHFAKSDSFLDDIFAAWM AQRLKTHLLTETWQRKRQELPSNCSLPYHVYNIKAIKLSRHSYFSSYQDHA KWCISQKGTKNRWTCIGDLNRSPHQAFRSGGFICTQNWQIYQAFQGLVLYY ESCK

59 Precursor mouse MSLHPASPRLASLLLFILALHDTLALRLCSFNVRSFGASKKENHEAMDIIV DNase1L1-3 KIIKRCDLILLMEIKDSSNNICPMLMEKLNGNSRRSTTYNYVISSRLGRNT

YKEQYAFVYKEKLVSVKTKYHYHDYQDGDTDVFSREPFVVWFHSPFTAVKD FVIVPLHTTPETSVKEIDELVDVYTDVRSQWKTENFIFMGDFNAGCSYVPK KAWQNIRLRTDPKFVWLIGDQEDTTVKKSTSCAYDRIVLCGQEIVNSVVPR SSGVFDFQKAYDLSEEEALDVSDHFPVEFKLQSSRAFTNNRKSVSLKKRKK GNRS

60 Mature human KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQGRCKPVNTFVHEPL RNase1 VDVQNVCFQEKVTCKNGQGNCYKSNSSMHITDCRLTNGSRYPNCAYRTSPK

ERHIIVACEGSPYVPVHFDASVEDST

61 Mature human KESRAKKFQRQHMDSDSSPSSSSTYCNQMMRRRSMTQGRCKPVNTFVHEPL RNase1 VDVQNVCFQEKVTCKNGQGNCYKSSSSMHITDCRLTSGSRYPNCAYRTSPK N34S/N76S/N88S ERHIIVACEGSPYVPVHFDASVEDST

62 Precursor human MALEKSLVRLLLLVLILLVLGWVQPSLGKESRAKKFQRQHMDSDSSPSSSS RNase1 TYCNQMMRRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGQGNCYK

SNSSMHITDCRLTNGSRYPNCAYRTSPKERHIIVACEGSPYVPVHFDASVE DST

63 N domain (full MRLAVGALLVCAVLGLCLAVPDKTVRWCAVSEHEATKCQSFRDHMKSVIPS length) DGPSVACVKKASYLDCIRAIAANEADAVTLDAGLVYDAYLAPNNLKPVVAE

FYGSKEDPQTFYYAVAVVKKDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGL LYCDLPEPRKPLEKAVANFFSGSCAPCADGTDFPQLCQLCPGCGCSTLNQY FGYSGAFKCLKDGAGDVAFVKHSTIFENLANKADRDQYELLCLDNTRKPVD EYKDCHLAQVPSHTVVARSMGGKEDLIWELLNQAQEHFGKDKSKEFQLFSS PHGKDLLFKDSAHGFLKVPPRMDA

64 C domain (full TAIRNLREGTCPEAPTDECKPVKWCALSHHERLKCDEWSVNSVGKIECVSA length) ETTEDCIAKIMNGEADAMSLDGGFVYIAGKCGLVPVLAENYNKSDNCEDTP

EAGYFAIAVVKKSASDLTWDNLKGKKSCHTAVGRTAGWNIPMGLLYNKINH CRFDEFFSEGCAPGSKKDSSLCKLCMGSGLNLCEPNNKEGYYGYTGAFRCL VEKGDVAFVKHQTVPQNTGGKNPDPWAKNLNEKDYELLCLDGTRKPVEEYA NCHLARAPNHAVVTRKDKEACVHKILRQQQHLFGSNVTDCSGNFCLFRSET KDLLFRDDTVCLAKLHDRNTYEKYLGEEYVKAVGNLRKCSTSSLLEACTFR RP 65 N domain (of VPDKTVRWCAVSEHEATKCQSFRDHMKSVIPSDGPSVACVKKASYLDCIRA HST) IAANEADAVTLDAGLVYDAYLAPNNLKPVVAEFYGSKEDPQTFYYAVAVVK

KDSGFQMNQLRGKKSCHTGLGRSAGWNIPIGLLYCDLPEPRKPLEKAVANF FSGSCAPCADGTDFPQLCQLCPGCGCSTLNQYFGYSGAFKCLKDGAGDVAF VKHSTIFENLANKADRDQYELLCLDNTRKPVDEYKDCHLAQVPSHTVVARS MGGKEDLIWELLNQAQEHFGKDKSKEFQLFSSPHGKDLLFKDSAHGFLKVP PRMDA

66 Lactoferrin MKLVFLVLLFLGALGLCLAGRRRSVQWCAVSQPEATKCFQWQRNMRKVRGP precursor PVSCIKRDSPIQCIQAIAENRADAVTLDGGFIYEAGLAPYKLRPVAAEVYG

TERQPRTHYYAVAVVKKGGSFQLNELQGLKSCHTGLRRTAGWNVPIGTLRP FLNWTGPPEPIEAAVARFFSASCVPGADKGQFPNLCRLCAGTGENKCAFSS QEPYFSYSGAFKCLRDGAGDVAFIRESTVFEDLSDEAERDEYELLCPDNTR KPVDKFKDCHLARVPSHAVVARSVNGKEDAIWNLLRQAQEKFGKDKSPKFQ LFGSPSGQKDLLFKDSAIGFSRVPPRIDSGLYLGSGYFTAIQNLRKSEEEV AARRARVVWCAVGEQELRKCNQWSGLSEGSVTCSSASTTEDCIALVLKGEA DAMSLDGGYVYTAGKCGLVPVLAENYKSQQSSDPDPNCVDRPVEGYLAVAV VRRSDTSLTWNSVKGKKSCHTAVDRTAGWNIPMGLLFNQTGSCKFDEYFSQ SCAPGSDPRSNLCALCIGDEQGENKCVPNSNERYYGYTGAFRCLAENAGDV AFVKDVTVLQNTDGNNNEAWAKDLKLADFALLCLDGKRKPVTEARSCHLAM APNHAVVSRMDKVERLKQVLLHQQAKFGRNGSDCPDKFCLFQSETKNLLFN DNTECLARLHGKTTYEKYLGPQYVAGITNLKKCSTSPLLEACEFLRK

67 Mouse MRLLSVTFWLLLSLRTVVCVMEVQWCTISDAEQQKCKDMSEAFQGAGIRPS Melanotransferri LLCVQGNSADHCVQLIKEQKADAITLDGGAIYEAGKEHGLKPVVGEVYDQD n precursor IGTSYYAVAVVRRNSNVTINTLKGVKSCHTGINRTVGWNVPVGYLVESGHL

SVMGCDVLKAVGDYFGGSCVPGTGETSHSESLCRLCRGDSSGHNVCDKSPL ERYYDYSGAFRCLAEGAGDVAFVKHSTVLENTDGNTLPSWGKSLMSEDFQL LCRDGSRADITEWRRCHLAKVPAHAVVVRGDMDGGLIFQLLNEGQLLFSHE DSSFQMFSSKAYSQKNLLFKDSTLELVPIATQNYEAWLGQEYLQAMKGLLC DPNRLPHYLRWCVLSAPEIQKCGDMAVAFSRQNLKPEIQCVSAESPEHCME QIQAGHTDAVTLRGEDIYRAGKVYGLVPAAGELYAEEDRSNSYFVVAVARR DSSYSFTLDELRGKRSCHPYLGSPAGWEVPIGSLIQRGFIRPKDCDVLTAV SQFFNASCVPVNNPKNYPSALCALCVGDEKGRNKCVGSSQERYYGYSGAFR CLVEHAGDVAFVKHTTVFENTNGHNPEPWASHLRWQDYELLCPNGARAEVD QFQACNLAQMPSHAVMVRPDTNIFTVYGLLDKAQDLFGDDHNKNGFQMFDS SKYHSQDLLFKDATVRAVPVREKTTYLDWLGPDYVVALEGMLSQQCSGAGA AVQRVPLLALLLLTLAAGLLPRVL

68 Lactoferrin MKLVFLVLLFLGALGLCLAGRRRSVQWCAVSQPEATKCFQWQRNMRKVRGP splice variant PVSCIKRDSPIQCIQAIAENRADAVTLDGGFIYEAGLAPYKLRPVAAEVYG

69 Region of splice EDCIALKGEADA

variance for

lactoferrin

70 Linker GGSG

71 Linker GSAT

72 Mature human LKIAAFNIQTFGETKMSSATLVSYIVQILSRYDIALVQEVRDSHLTAVGKL DNase1 LDNLNQDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCE N18S/N106S/A114F PCGSDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLD

VQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTA TPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPV EVMLK

73 (Gly₄Ser)3 linker GGGGSGGGGSGGGGS

74 Gaussia MGVKVLFALICIAVAEA

luciferase

signal peptide

75 Linker LEA(EAAAK)₄ALEA(EAAAK)₄

76 O-linked CXXGG-T/S-C

glycosylation

consensus

77 O-linked NST-E/D-A

glycosylation

consensus

78 O-linked NITQS

glycosylation

consensus

79 O-linked QSTQS

glycosylation

consensus

80 O-linked D/EFT-R/K-V

glycosylation

consensus

81 O-linked C-E/D-SN

glycosylation

consensus

82 O-linked GGSC-K/R

glycosylation

consensus

83 VK3 light chain METPAQLLFLLLLWLPDTTG

signal peptide

84 NLG linker VDGAAASPVNVSSPSVQDI

85 linker LEA(EAAAK)₄ALEA(EAAAK)₄ALE

86 Mature human LKIAAFNIQTFGRTKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKL DNase1 LDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCE

PCGNDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLD

E13R/N74K/A114F

VQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTA

/ T205K KPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPV

EVMLK

87 Mature human LKIAAFNIQTFGRTKMSSATLVSYIVQILSRYDIALVQEVRDSHLTAVGKL DNase1 LDNLNQDAPDTYHYVVSEPLGRKSYKERYLFVYRPDQVSAVDSYYYDDGCE

PCGSDTFNREPFIVRFFSRFTEVREFAIVPLHAAPGDAVAEIDALYDVYLD

E13R/N74K/A114F

VQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTA

/ KPTHCAYDRIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPV T205K/N18S/N106 EVMLK

S

Claims

We claim: 1. A a polypeptide comprising a first nuclease domain and a transferrin, or a variant or fragment thereof that has transferrin activity, wherein the first nuclease domain is operably coupled to the transferrin, or variant or fragment thereof.

2. The polypeptide of claim 1, wherein the first nuclease domain is operably coupled to the N-terminus of the transferrin, or variant or fragment thereof.

3. The polypeptide molecule of claim 1, wherein the first nuclease domain is operably coupled to the C-terminus of the transferrin, or variant or fragment thereof.

4. The polypeptide of claim 2 or 3, wherein the first nuclease domain is operably coupled to the transferrin, or variant or fragment thereof, via a linker domain.

5. The polypeptide of any one of claims 2-4, wherein the first nuclease domain is an RNase.

6. The polypeptide of any one of claims 2-4, wherein the first nuclease domain is a DNase.

7. The polypeptide of claim 1, further comprising a second nuclease domain operably coupled to the transferrin, or variant or fragment thereof.

8. The polypeptide of claim 7, wherein the first nuclease domain is operably coupled to the N-terminus of the transferrin, or variant or fragment thereof, and the second nuclease domain is operably coupled to the C-terminus of the transferrin, or variant or fragment thereof.

9. The polypeptide of claim 8, wherein the first and second nuclease domains are operably coupled to the N- and C-terminus, respectively, of the transferrin, or variant or fragment thereof, via a linker.

10. The polypeptide of claim 7, wherein the first nuclease molecule is operably coupled to the second nuclease domain via a linker, and the second nuclease domain is operably coupled to the transferrin, or variant or fragment thereof.

11. The polypeptide of claim 10, wherein the second nuclease domain is operably coupled to the N-terminus of the transferrin, or variant or fragment thereof.

12. The polypeptide of claim 10, wherein the second nuclease domain is operably coupled to the C-terminus of the transferrin, or variant or fragment thereof.

13. The polypeptide of any one of claims 7-9, wherein the first and second nuclease domains each comprise an RNase.

14. The polypeptide of any one of claims 7-12, wherein the first nuclease domain comprises a DNase and the second nuclease domain comprises an RNase.

15. The polypeptide of any one of claims 7-12, wherein the first nuclease domain comprises an RNase and the second nuclease domain comprises a DNase.

16. The polypeptide of any one of claims 1-5 and 7-15, wherein the RNase is a wild type human RNase, such as a human pancreatic RNase1 (SEQ ID NO: 60), or a mutant RNase, such as an aglycosylated, underglycosylated, or deglycosylated RNase 1, such as human RNase1 N34S/N76S/N88S (SEQ ID NO: 61).

17. The polypeptide of claim 16, wherein the RNase is wild type human RNase1 (SEQ ID NO: 60).

18. The polypeptide of claim 16, wherein the RNase is human RNase1 N34S/N76S/N88S (SEQ ID NO: 61).

19. The polypeptide of claim 16, which degrades circulating RNA and RNA in immune complexes, or inhibits interferon-α production, or both.

20. The polypeptideof claim 19, wherein the activity of the RNase is not less than about 2- to 10-fold less than the activity of a control RNase molecule.

21. The polypeptide of claim 20, wherein the activity of the RNase is about equal to the activity of a control RNase molecule.

22. The polypeptide of any one of claims 1-4, 12, 14, and 15, wherein the DNase is a wild type human DNase, such as a human pancreatic DNase1 (SEQ ID NO: 51), or a mutant DNase, such as human DNase1 A114F (SEQ ID NO: 52) or an aglycosylated, underglycosylated, or deglycosylated human DNase, such as mutant, human DNase1 N18S/N106S/A114F (SEQ ID NO: 72).

23. The polypeptide of claim 22, wherein the activity of the DNase is not less than about 2- to 10-fold less than the activity of a control DNase molecule.

24. The polypeptide of claim 23, wherein the activity of the DNase is about equal to the activity of a control DNase molecule.

25. The polypeptide of any one of claims 4-6 or 9-24, wherein the linker domain is a polypeptide linker, such as a gly-ser linker.

26. The polypeptide of any of the preceding claims, wherein the transferrin, or variant or fragment thereof, increases the serum half-life and/or activity of the hybrid nuclease-transferrin molecule relative to a hybrid nuclease- transferrin molecule that does not contain the transferrin, or variant or fragment thereof.

27. The polypeptide of claim 26, whrein the transferrin, or variant or fragment thereof, is derived from human, cow, pig, sheep, dog, rabbit, rat, mouse, hamster, echnida, platypus, chicken, frog, hornworm, monkey, horse, or bovine transferrin.

28. The polypeptide of claim 27, wherein the transferrin, or variant or fragment thereof, is derived from human serum transferrin.

29. The polypeptide of claim 28, wherein the transferrin is more than 80% identical to the corresponding amino acid sequence set forth in SEQ ID NO: 1.

30. The polypeptide of claim 29, wherein the transferrin is more than 85% identical to the corresponding amino acid sequence set forth in SEQ ID NO: 1.

31. The polypeptide of claim 30, wherein the transferrin, or variant or fragment thereof, is more than 90% identical to the corresponding amino acid sequence set forth in SEQ ID NO: 1.

32. The polypeptide of claim 31, wherein the transferrin, or variant or fragment thereof, is more than 95% identical to the corresponding amino acid sequence set forth in SEQ ID NO: 1.

33. The polypeptide of claim 28, wherein the fragment of the transferrin or variant is at least 20 amino acids, at least 40 amino acids, at least 60 amino acids, at least 80 amino acides, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, atleast 300 amino acids, at least 400 amino acids, or at least 500 amino acids in length.

34. The polypeptide of claim 28, wherein the variant or fragment of the human serum transferrin binds to the transferrin receptor with a higher affinity than that for a corresponding wild type human serum transferrin or fragment thereof.

35. A polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 3-50, or a hybrid nuclease-transferrin molecule comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 3-50.

36. A composition comprising the polypeptide of any of the preceding claims and a carrier.

37. A nucleic acid molecule encoding the polypeptide according to claim 1.

38. A recombinant expression vector comprising a nucleic acid molecule according to claim 37.

39. A host cell transformed with the recombinant expression vector according to claim 38.

40. A method of making the polypeptide of any one of claims 1-35, comprising: providing a host cell comprising a nucleic acid sequence that encodes the polypeptide; and maintaining the host cell under conditions in which the polypeptide is expressed.

41. A method for treating or preventing a condition associated with an abnormal immune response, comprising administering to a subject an effective amount of a polypeptide of any one of claims 1-35.

42. The method of claim 41, wherein the condition is an autoimmune disease.

43. The method of claim 42, wherein the autoimmune disease is selected from the group consisting of insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, an experimental form of

uveoretinitis, Hashimoto’s thyroiditis, primary myxoedema, thyrotoxicosis, pernicious anaemia, autoimmune atrophic gastritis, Addison’s disease, premature menopause, male infertility, juvenile diabetes, Goodpasture’s syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune haemolytic anaemia, idiopathic leucopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerative colitis, Sjogren’s syndrome, scleroderma, Wegener’s granulomatosis, polymyositis, dermatomyositis, discoid LE, systemic lupus erythematosus (SLE), and connective tissue disease.

44. The method of claim 43, wherein the autoimmune disease is SLE.

45. The method of claim 43, wherein the autoimmune disease is Sjogren’s syndrome 46. A method of treating SLE comprising administering to a subject a hybrid nuclease-transferrin molecule containing composition in an amount effective to degrade immune complexes containing RNA, DNA or both RNA and DNA, wherein the composition comprises a pharmaceutically acceptable carrier and a polypeptide of any one of claims 1-35. 47. A method of treating Sjogren’s syndrome comprising administering to a subject a hybrid nuclease-transferrin molecule containing composition in an amount effective to degrade immune complexes containing RNA, DNA or both RNA and DNA, wherein the composition comprises a pharmaceutically acceptable carrier and a polypeptide of any one of claims 1-35.