METHODS OF USING DNASE1-LIKE 3 IN THERAPY

Abstract

The present disclosure provides D1L3 enzymes having complete or partial C-terminal deletions of the basic domain (BD), which have substantially enhanced chromatin-degrading activity. In various aspects, the invention provides chromatinase enzyme therapy, which is optionally provided by delivering polynucleotides encoding chromatinases such as D1L3, or by delivering host cells expressing and secreting the same.

Description

PRIORITY

[0001] This application is a continuation-in-part of U.S. application Ser. No. 16/697,502, filed Nov. 27, 2019, which is a continuation of International Application No. PCT/US2019/055178, filed Oct. 8, 2019, which claims the benefit of: U.S. Provisional Application No. 62/846,904, filed May 13, 2019; U.S. Provisional Application No. 62/808,601, filed Feb. 21, 2019; U.S. Provisional Application No. 62/779,104, filed Dec. 13, 2018; U.S. Provisional Application No. 62/775,563, filed Dec. 5, 2018; and U.S. Provisional Application No. 62/742,682, filed Oct. 8, 2018, each of which is hereby incorporated by reference in its entirety. This application further claims the benefit of U.S. Provisional Application No. 62/978,976, filed Feb. 20, 2020, which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Inflammation is an essential host response to control invading microbes and heal damaged tissues. Uncontrolled and persistent inflammation causes tissue injury in a plethora of inflammatory disorders. Neutrophils are the predominant leukocytes in acute inflammation. During infections, neutrophils generate neutrophil extracellular traps (NETs), lattices of DNA-filaments decorated with toxic histones and enzymes that immobilize and neutralize bacteria. However, inappropriately released NETs may harm host cells due to their cytotoxic, proinflammatory, and prothrombotic activity.

[0003] Two endogenous extracellular DNA-degrading enzymes, DNASE1 (D1) and DNASE1-LIKE 3 (D1L3), limit collateral damage during homeostatic inflammatory responses. D1 and D1L3 are evolutionarily conserved and found in a variety of species including, humans, primates, and rodents. D1 is predominantly expressed in the gastrointestinal tract and exocrine glands, whereas hematopoietic cells, namely macrophages and dendritic cells produce D1L3. While D1L3 has much higher activity for degrading extracellular chromatin and NETs (as compared to D1, which has little to no chromatin-degrading activity), wild-type D1L3 does not have the physical, enzymatic, or pharmacodynamic properties suitable for enzyme replacement therapy.

[0004] Therapies providing high DNA- or chromatin-degrading activity are needed for treating conditions characterized by pathological accumulation of extracellular chromatin, including NETs.

SUMMARY

[0005] D1L3 features a 23-amino acid long C-terminal tail, which contains 9 basic amino acids and is thus known as the Basic Domain (BD). The BD is unique to D1L3 and is not present in D1. The BD contains a nuclear localization signal (NLS) that targets the enzyme to the nucleus during apoptosis. While it has been widely considered that the BD is critical for the biologic activity of D1L3 in the extracellular space, this disclosure surprisingly shows that deletion of the C-terminal tail in fact stimulates chromatinase activity of D1L3. In accordance with aspects of the invention, the D1L3 enzymes described herein are more suitable and/or effective for therapy and/or are more amenable to large-scale manufacturing. The D1L3 enzymes disclosed herein have benefits for systemic therapy. Such benefits include longer exposure (e.g., slower elimination, longer circulatory half-life), extended duration of pharmacodynamic action, and improved chromatin-degrading activity.

[0006] In various embodiments, the invention provides a DNASE1-LIKE 3 (D1L3) enzyme comprising an amino acid sequence that has at least 70% sequence identity to D1L3 Isoform 1 (SEQ ID NO:4) or D1L3 Isoform 2 (SEQ ID NO:5) lacking the BD, and wherein the D1L3 enzyme has a deletion of at least three amino acids from the BD. Amino acid deletions of the Basic Domain of D1L3 improve its chromatin-degrading activity. Further, increasing deletions of the 23-amino acid BD directly correlate with increasing chromatin-degrading activity, including activity for degrading mono-nucleosomes.

[0007] Amino acid deletions of the BD can be anywhere in the BD. For example, deletions can be independently selected from the N-terminal side of the BD, from the C-terminal side of the BD, and internal to the BD. In some embodiments, one or more amino acid deletions are within the NLS of the BD.

[0008] In some embodiments, the D1L3 enzyme is fused to a carrier protein, optionally by means of an amino acid linker. The carrier protein is generally a half-life extending moiety, such as albumin, transferrin, an Fc, or elastin-like protein, or a variant thereof.

[0009] In some embodiments, the D1L3 enzyme is fused to an albumin amino acid sequence or domain. Albumin can be joined to the D1L3, optionally with an interposed linker, at the N-terminus and/or the C-terminus of the D1L3 enzyme. In some embodiments, the D1L3 enzyme comprises an albumin sequence fused to the N-terminus of the mature D1L3 enzyme with an interposed amino acid linker. The peptide linker may be a flexible linker, a rigid linker, or in some embodiments a physiologically-cleavable linker. An exemplary fusion protein for use in systemic therapy is represented by SEQ ID NO: 47, which includes an N-terminal albumin amino acid sequence, a flexible linker of 31 amino acids, and a mature D1L3 amino acid sequence having a full deletion of the BD.

[0010] In still other aspects, the invention provides a DNASE1-LIKE 3 (D1L3) enzyme comprising an amino acid sequence that has at least 70% sequence identity to D1L3 Isoform 1 (SEQ ID NO:4) or D1L3 Isoform 2 (SEQ ID NO:5), and wherein the D1L3 enzyme has a single amino acid truncation of the BD. D1L3 enzymes having a single amino acid truncation from the BD have surprisingly high DNase activity.

[0011] The invention in some aspects provides pharmaceutical compositions comprising the D1L3 enzyme described herein, or optionally a polynucleotide encoding the D1L3 enzyme, or a transfection or expression vector comprising the same, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be formulated for any administration route, including topical, parenteral, or pulmonary administration.

[0012] In some aspects, the invention provides mammalian host cells (e.g., human host cells), as well as methods of making and using the same. The host cells comprise a heterologous polynucleotide encoding a chromatinase enzyme operably linked to a promoter. The host cells delivered to a subject express and secrete the encoded chromatinase enzyme. In these aspects, challenges in manufacturing chromatinases such as D1L3 at large scale are avoided. Further, by expressing and delivering D1L3 through heterologous expression in a white blood cell such as a T cell, D1L3 therapy can be localized in part to areas of inflammation or tissue destruction or cell apoptosis. Further, since D1L3 has a circulation half-life of less than about 30 minutes, the cell therapy described herein provides for a sustained therapy, with as few as one, two, three, or four treatments in some embodiments. In some embodiments, the therapy is provided to a subject for treatment of cancer (e.g., leukemia) or viral infection, including infection of the lower respiratory tract.

[0013] In other aspects, the invention provides a method for treating a subject in need of extracellular DNA or chromatin degradation, extracellular trap (ET) degradation and/or neutrophil extracellular trap (NET) degradation. The method comprises administering a therapeutically effective amount of the D1L3 enzyme or composition described herein (including host cell compositions). In various embodiments, the present invention provides a method for treating, preventing, or managing diseases or conditions characterized by the presence or accumulation of NETs or extracellular chromatin.

[0014] In certain embodiments, the present invention pertains to the treatment of diseases or conditions characterized by deficiency of D1L3, or a deficiency of D1. In some cases, the subject has a mutation (i.e., a loss of function mutation) in the Dnase1l3 gene or the Dnase1 gene. In some embodiments, such subjects manifest with an autoimmune disease. In some cases, the subject has an acquired inhibitor of D1 and/or of D1L3. Such subjects can also have an autoimmune or inflammatory disease, such as SLE or systemic sclerosis.

[0015] In some embodiments, the subject has a loss of function mutation in one or both D1L3 genes, and may exhibit symptoms of SLE, or may be further diagnosed with clinical SLE. In such embodiments, the subject may receive systemic therapy with a BD-deleted D1L3 described herein. For example, therapeutically effective amounts of the fusion protein represented by SEQ ID NO:47, or other fusion between albumin and a BD-deleted D1L3, are administered once or twice weekly, or once or twice monthly.

[0016] Other aspects and embodiments of the invention will be apparent from the following examples.

BRIEF DESCRIPTION OF FIGURES

[0017] FIG. 1 shows the enzymatic activity of increasing amounts of DNASE1 (D1) and DNASE1-LIKE 3 (D1L3) as characterized by the degradation of high-molecular weight (HMW)-chromatin from HEK293 cell purified nuclei. D1 has limited activity for degrading chromatin as compared to D1L3.

[0018] FIG. 2 shows a schematic representation of a building block substitution technology used for creating chimeric DNase enzymes. Summarily, building block substitution involves protein-protein sequence alignment of a donor DNase (e.g. DNASE1) and a recipient DNase (e.g. DNASE1L3), identification of variable amino acid blocks (building block) between conserved anchors, and substitution of a segment of cDNA encoding a building block from the recipient DNase with a segment of cDNA between the anchors in the donor DNase, thereby creating chimeric DNase enzymes.

[0019] FIG. 3 shows 14 of the 63 D1L3-D1 chimeras (#50-#63), SEQ ID NOs: 8-21, respectively, generated using the building block substitution method.

[0020] FIG. 4 shows the chromatin-degrading activities of D1L3-D1 chimeras (#50 to #63) shown in FIG. 3. As shown, the D1L3-D1 chimera #63, which harbors a Q282_S305delinsK mutation (involving deletion of C-terminal basic domain (BD) and substitution of Glutamine 282 of D1L3 with a Lysine) (SEQ ID NO: 21), exhibits hyperactivity for chromatin degradation as compared to wild-type D1L3.

[0021] FIG. 5A shows a western blot of culture supernatants using a monoclonal antibody that targets MGDFNAGCSYV (SEQ ID NO:42) (Anti-D1/D1L3), a peptide sequence that is shared by D1 and D1L3. Mobility shift confirms the C-terminal deletion in the Q282_S305delinsK mutant. FIG. 5B shows the results of a titration experiment performed to compare the chromatin-degrading activities of wild type D1L3 and the BD-deleted D1L3. The results demonstrate that the BD-deleted D1L3 had approximately 5 to 10-fold higher enzymatic activity on HMW-chromatin compared to wild type D1L3. FIG. 5C is a graph showing chromatinase activity of wild type D1L3 and the BD-deleted D1L3 as a function of enzyme concentration.

[0022] FIG. 6 shows sequence alignment of C-termini of the S305del, K303_S305del, V294_S305del, K291_S305del, R285_S305del, and S283_S305del deletion mutants (SEQ ID NOs: 22-27, respectively), which lack 1, 3, 12, 15, 21, and 23 C-terminal amino acids, respectively.

[0023] FIG. 7 shows a western blot of CHO cell culture supernatants after transient transfection of deletion mutants using the anti-D1/D1L3 antibody.

[0024] FIG. 8A compares the enzymatic activities of the indicated deletion mutant enzymes with the wild type D1L3 on ultra-large chromatin. FIG. 8B shows a graph illustrating chromatinase activity of D1L3 as a function length of C-terminal deletion.

[0025] FIG. 9A shows the ability to degrade ultra-large chromatin into mononucleosomes by the indicated deletion mutant enzymes in comparison with the wild type D1L3. FIG. 9B shows a graph illustrating the degradation of mononucleosomes by the D1L3 mutants as a function length of C-terminal deletion.

[0026] FIG. 10 shows C-terminal amino acid sequences of recombinantly expressed wild-type D1L3 in Pichia pastoris to identify frequent cleavage sites. Amino acid sequencing of purified wild-type D1L3 identified three C-terminal deletion mutants: K291_S305del, K292_S305del, and S293_S305del. The C-terminus of wild-type D1L3 was not detected. In parallel, the chromatin degrading activity in the different concentrations of purified protein was analyzed and compared to purified DNASE1 (D1) and the Basic Domain Deleted DNASE1L3 (BDD-D1L3) with a F275Y/F279_K280delinsVM/Q282_S305delinsK mutation. The figure shows DNA analyzed by agarose gel electrophoresis.

[0027] FIG. 11 shows that the addition of dextran sulfate to CHO medium improves protein yield. Stable pools of CHO cells expressing wild-type D1L3 were incubated in standard CHO medium or CHO medium supplemented with dextran sulfate. Supernatants were analyzed by Western Blot (WB) using an anti-DNASE1L3 antibody. The figure shows that D1L3 expresses poorly in CHO cells with low yield. Addition of dextran sulfate increases the yield, but does not prevent production fragmentation.

[0028] FIGS. 12A-B show that D1L3 has a propensity to misfold when expressed in CHO cells. FIG. 12A illustrates a simple expression vector for D1L3 expression using the native secretory signal peptide. Supernatants of stable pools were analyzed by Western Blot using an anti-DNASE1L3 antibody, and FIG. 12B shows the presence of high molecular weight aggregates under non-reducing conditions, which are resolved under reducing conditions.

[0029] FIG. 13 is an alignment of human D1 (SEQ ID NO: 1) and human D1L3 (SEQ ID NO: 4) amino acid sequences, with conserved and non-conserved cysteine residues shown.

[0030] FIG. 14 shows the location of NLS2 sequence superimposed with a sequence alignment of C-termini of the S305del, K303_S305del, V294_S305del, K291_S305del, R285_S305del, and S283_S305del deletion mutants, which lack 1, 3, 12, 15, 21, and 23 C-terminal amino acids, respectively.

[0031] FIG. 15 shows a western blot of wild type D1L3 and BD-deleted D1L3 expressed in furin-overexpressing CHO cells. CHO cells without overexpression of furin were included as control. Data suggest that cleavage of the C-terminal tail or a portion thereof could act as a natural activation signal for D1L3.

[0032] FIG. 16 shows the expression of D1L3 in white blood cell lineages. T cells do not naturally express D1L3 mRNA.

[0033] FIG. 17 shows a schematic for D1L3 therapy in connection with T cell therapy. T cells express basic-specific PCSK enzymes that result in the secretion of active enzyme.

[0034] FIG. 18 compares the enzymatic activities of the indicated deletion mutant enzymes with the wild type D1L3 on DNA.

DETAILED DESCRIPTION OF THE INVENTION

[0035] The present disclosure is based, in part, on the discovery that D1L3 enzymes having complete or partial C-terminal deletions of the basic domain (BD) have substantially enhanced chromatin-degrading activity. In accordance with aspects of the invention, the D1L3 enzymes described herein are more suitable and/or effective for therapy and/or are more amenable to large-scale manufacturing. In some embodiments, the enzymes disclosed herein have benefits for systemic therapy, including cell therapy in which a host cell expresses a heterologous chromatinase such as D1L3. Such benefits include longer exposure (e.g., slower elimination, longer circulatory half-life), extended duration of pharmacodynamic action, and improved chromatin-degrading activity.

[0036] In the description that follows, certain conventions will be followed regarding the usage of terminology. As used herein and in the claims, the singular forms "a," "an," and "the" include the singular and the plural reference unless the context clearly indicates otherwise.

[0037] The term "chromatinase" refers to a class of deoxyribonuclease enzyme that exhibits more than a negligible ability to cut, cleave or digest chromatin, i.e., DNA associated with one or more histone proteins. Human DNASE1L3 is a chromatinase. DNASE1L3 variants disclosed herein are chromatinases. Not all DNASE enzymes are chromatinases. For example, human DNASE1 has essentially no ability to specifically cut, cleave, or digest chromatin and is not a chromatinase.

[0038] As used herein, unless stated to the contrary, the term "D1L3" when referring to the wild-type sequence, includes either D1L3 Isoform 1 (SEQ ID NO:4) or D1L3 Isoform 2 (SEQ ID NO:5).

[0039] When referring to sequence identity with wild-type DNase enzymes, and unless stated otherwise, sequences refer to mature enzymes lacking the signal peptide. Further, unless stated otherwise, amino acid positions are numbered with respect to the full-translated DNase sequence, including signal peptide, for clarity. Accordingly, for example, reference to sequence identity to the enzyme of SEQ ID NO: 4 (human D1L3, Isoform 1) refers to a percent identity with the mature enzyme having M21 at the N-terminus. Polynucleotides encoding enzymes may also encode the signal peptide to effect secretion from host cells and processing of the signal peptide.

[0040] As used herein with reference to a drug, "half-life" refers to the elimination half-life of the concentration of the drug in an animal, as measured in a matrix of interest, e.g., serum or plasma. The skilled person will understand that not all drugs exhibit first-order kinetics or do so during all phases of elimination. In such cases, the skilled person will understand that the terms "half-life extension" or "extended half-life" are expressions that refer to a slower rate of elimination.

[0041] As used herein, "neutrophil extracellular trap" and the acronym "NET" refer to a network of extracellular fibers comprising nuclear contents, e.g., DNA bound to histone proteins that are released from an immune cell, typically a neutrophil, in a programmed fashion.

[0042] Unless otherwise specified, a "nucleotide sequence" or "nucleic acid" encoding an amino acid sequence includes all degenerate versions that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns. In some embodiments, a polynucleotide encoding a chromatinase does not have introns.

[0043] The terms "about" and "approximately" include an amount that is .+-.10% of an associated numerical value.

[0044] D1L3 features a 23-amino acid long C-terminal tail, which contains 9 basic amino acids and is thus known as Basic Domain (BD). The BD is unique to D1L3 and is not present in D1. The BD contains a nuclear localization signal (NLS) that targets the enzyme to the nucleus during apoptosis. While it has been widely considered that the BD is critical for the biologic activity of D1L3 in the extracellular space, this disclosure surprisingly shows that deletion of the C-terminal tail in fact stimulates chromatinase activity of D1L3.

[0045] In various embodiments, the invention provides a DNASE1-LIKE 3 (D1L3) enzyme comprising an amino acid sequence that has at least 70% sequence identity to D1L3 Isoform 1 (SEQ ID NO:4) or D1L3 Isoform 2 (SEQ ID NO:5) lacking the BD (i.e., at least 70% sequence identity with amino acids 21 to 282 of SEQ ID NO: 4, or amino acids 21 to 252 of SEQ ID NO:5), and wherein the D1L3 enzyme has a deletion of at least three amino acids from the BD. Amino acid deletions of the Basic Domain of D1L3 improve its chromatin-degrading activity. Further, increasing deletions of the 23-amino acid BD directly correlate with increasing chromatin-degrading activity, including activity for degrading mono-nucleosomes.

[0046] In various embodiments, the amino acid deletions from the BD are at the C-terminus of the BD. For example, the D1L3 enzyme may have a deletion of at least the five C-terminal amino acids of BD. In some embodiments, the D1L3 enzyme has a deletion of at least the eight C-terminal amino acids of the BD. In some embodiments, the D1L3 enzyme has a deletion of at least the ten C-terminal amino acids of the BD. In some embodiments, the D1L3 enzyme has a deletion of at least the twelve C-terminal amino acids of the BD. In some embodiments, the D1L3 enzyme has a deletion of at least the fifteen C-terminal amino acids of the BD. In some embodiments, the D1L3 enzyme has a deletion of at least the eighteen C-terminal amino acids of the BD. In some embodiments, the D1L3 enzyme has a deletion of at least the twenty one C-terminal amino acids of the BD. In some embodiments, the D1L3 enzyme has a deletion of at least the twenty three C-terminal amino acids of the BD.

[0047] Alternatively, deletions of the BD (from three to 23 amino acids) can be anywhere in the BD, and not necessarily from the C-terminus of the BD. For example, in various embodiments, the D1L3 enzyme has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids deleted from the BD. In some embodiments, the D1L3 enzyme has a deletion of at least 5, or at least 8, or at least 12, or at least 15, or at least 18, or at least 21 amino acids from the BD. These deletions can be independently selected from the N-terminal side of the BD, from the C-terminal side of the BD, and internal to the BD. In some embodiments, one or more amino acid deletions are within the NLS within the BD.

[0048] In addition to deletions of one or more amino acids, the BD may further comprise amino acid substitutions, which may further impact chromatin-degrading activity. For example, the D1L3 enzyme may have from 1 to 20 amino acid substitutions of BD amino acids, in addition to a deletion of at least three amino acids. In some embodiments, the BD contains a substitution of at least three amino acids, or at least five amino acids, or at least 10 amino acids. In some embodiments, at least two amino acid substitutions are in the NLS of the BD.

[0049] In some embodiments, the D1L3 enzyme has a deletion of one or more additional amino acids from the C-terminus, in addition to a deletion of the BD. For example, the D1L3 enzyme may have a deletion of an additional one to fifty amino acids, or from one to twenty amino acids, or from one to ten amino acids, or from one to five amino acids from the C-terminal amino acids of SEQ ID NO:4 or SEQ ID NO:5, in addition to the deletion of the BD.

[0050] In some embodiments, after partial or complete deletion of the BD as described, from 1 to 10 amino acids, or from 1 to 5 amino acids may be added to the C-terminus that do not impact chromatin-degrading activity.

[0051] In various embodiments, the D1L3 enzyme amino acid sequence has at least 80% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD. In some embodiments, the D1L3 amino acid sequence has at least 85% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD. In some embodiments, the D1L3 amino acid sequence has at least 90% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD. In such embodiments, the amino acid sequence may have at least 95% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD, or at least 97% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD. In some embodiments, the D1L3 amino acid sequence has 100% sequence identity with the enzyme of SEQ ID NO:4 or SEQ ID NO:5 lacking the BD.

[0052] In still other aspects, the invention provides a DNASE1-LIKE 3 (D1L3) enzyme comprising an amino acid sequence that has at least 70% sequence identity to D1L3 Isoform 1 (SEQ ID NO:4) or D1L3 Isoform 2 (SEQ ID NO:S), and wherein the D1L3 enzyme has a single amino acid truncation of the BD. D1L3 enzymes having a single amino acid truncation from the BD have surprisingly high DNase activity.

[0053] In some embodiments, the D1L3 enzyme comprises additional modifications outside the BD, and which can provide additional advantages, including advantages in stability and compatibility with expression systems. Such modifications are disclosed in US 2020/0024585, PCT/US2019/055178, or PCT/US2020/016490, each of which are hereby incorporated by reference in its entirety.

[0054] In some embodiments, the D1L3 enzyme comprises at least one building block substitution from D1 (SEQ ID NO:1), DNASE-1-LIKE 1 (D1L1) (SEQ ID NO:2), or DNASE-1-LIKE 2 (SEQ ID NO:3). These building block substitutions are disclosed in PCT/US2020/016490, which is hereby incorporated by reference in its entirety.

[0055] In some embodiments, the D1L3 sequence or domain contains a building block substitution from D1 defined by amino add sequences, which can be selected from: M1_A20delinsMRGMKLLGALLALAALLQGAVS, M21_S25delinsLKIAA, V28_S30delinsIQT, E33_S34delinsET, Q36_I45delinsMSNATLVSYI, K47_K50delinsQILS, C52Y, I54_M58delinsIALVQ, I60_K61delinsVR, S63_I70delinsSHLTAVGK, M72_K74delinsLDN, R77_T84delinsQDAPDT, N86H, V88_I89delinsVV, S91_R92delinsEP, N96_T97delinsNS, Q101R, A103L, L105V, K107_L110delinsRPDQ, V113_S116delinsAVDS, H118Y, H120D, Y122_A127delinsGCEPCGN, V129T, S131N, 135F_136VdelinsAI, W138R, Q140_H143delinsFSRF, A145_D148delinsAVKD, V150A, I152A, T156_T157delinsAA, E159_S161delinsGDA, K163A, E167A, V169_E170delinsYD, T173L, K176_R178delinsQEK, K180_A181 delinsGL, N183_F186delinsDVML, P198_A201delinsRPSQ, K203_N204delinsSS, R208W, D210S, R212T, V214Q, G218P, Q220_E221delins SA, V225_S228delinsATP, N230H, L238_R239delinsVA, Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256N, K259_A260delinsAA, K262G, T264_E267delinsSDQL, L269_V271delinsQAI, F275Y, F279_K280delinsVM, and Q282_S305delinsK.

[0056] In some embodiments, the D1L3 enzyme is fused to a carrier protein, optionally by means of an amino acid linker. The carrier protein is generally a half-life extending moiety, such as albumin, transferrin, an Fc, XTEN, or elastin-like protein, or a variant thereof. See, e.g., U.S. Pat. No. 9,458,218, which is hereby incorporated by reference in its entirety.

[0057] In some embodiments, the D1L3 enzyme is fused to an albumin amino acid sequence or domain, i.e., a human albumin or a fragment or variant thereof. See, for example, WO 2015/066550 and U.S. Pat. No. 9,221,896, which are hereby incorporated by reference in their entirety. Albumin can be joined to the D1L3, optionally with an interposed linker, at the N-terminus and/or the C-terminus of the D1L3 enzyme. An exemplary albumin amino acid sequence is provided by SEQ ID NO: 39. In some embodiments, the D1L3 enzyme comprises an albumin sequence fused to the N-terminus of the mature D1L3 enzyme with an interposed amino acid linker.

[0058] In some embodiments, the albumin amino acid sequence or domain of the fusion protein has at least about 75%, or at least about 80%, or at least about 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the reference albumin sequence defined by SEQ ID NO: 39. In some embodiments, the albumin amino acid sequence or domain comprises or consists of the reference albumin sequence defined by SEQ ID NO: 39. In various embodiments, the albumin amino acid sequence binds to the neonatal Fc receptor (FcRn), e.g., human FcRn. The albumin amino acid sequence may be a variant of wild-type HSA (e.g., as represented by SEQ ID NO: 39). In various embodiments, albumin variants may have from one to twenty, or from one to ten amino acid modifications independently selected from deletions, substitutions, and insertions with respect to SEQ ID NO: 39. In some embodiments, the albumin amino acid sequence is any mammalian albumin amino acid sequence. Various modification to the albumin sequence that enhance its ability to serve as a circulation half-life extending carrier are known, and such modifications can be employed with the present invention. Exemplary modifications to the albumin amino acid sequence are described in U.S. Pat. Nos. 8,748,380, 10,233,228, and 10,501,524, which are each hereby incorporated by reference in their entireties.

[0059] In some embodiments, the albumin amino acid sequence or domain is a fragment of full-length albumin, as represented by SEQ ID NO: 39. The term "fragment," when used in the context of albumin, refers to any fragment of full-length albumin or a variant thereof (as described above) that extends the half-life of a D1L3 enzyme to which it is fused or conjugated, relative to the corresponding non-fused D1L3. In some embodiments, a fragment of an albumin can refer to an amino acid sequence comprising a fusion of multiple domains of albumin (see, e.g., WO2011/124718), such as domains I and III, and domains II and III. Generally, a fragment of albumin has at least about 100 amino acids or at least about 200 or at least about 300 amino acids of the full-length sequence. In various embodiments, the albumin fragment maintains the ability to bind human FcRn.

[0060] In some embodiments, the D1L3 enzyme is fused at the N-terminus to an albumin amino acid sequence, through a peptide linker. The peptide linker may be a flexible linker, a rigid linker, or in some embodiments a physiologically-cleavable linker (e.g., a protease-cleavable linker). In some embodiments, the linker is 5 to 100 amino acids in length, or is 5 to 50 amino acids in length. In some embodiments, the linker is from about 10 to about 35 amino acids in length, or from about 15 to about 35 amino acids.

[0061] Flexible linkers are predominately or entirely composed of small, non-polar or polar residues such as Gly, Ser and Thr. An exemplary flexible linker comprises (Gly.sub.ySer).sub.nS.sub.z linkers, where y is from 1 to 10 (e.g., from 1 to 5), n is from 1 to about 10, and z is 0 or 1. In some embodiments, n is from 3 to about 8, or from 3 to about 6. In exemplary embodiments, y is from 2 to 4, and n is from 3 to 8. Due to their flexibility, these linkers are unstructured. More rigid linkers include polyproline or poly Pro-Ala motifs and .alpha.-helical linkers. An exemplary .alpha.-helical linker is A(EAAAK).sub.nA, where n is as defined above (e.g., from 1 to 10, or 3 to 6). Generally, linkers can be predominately composed of amino acids selected from Gly, Ser, Thr, Ala, and Pro. Exemplary linker sequences contain at least 10 amino acids, and may be in the range of 15 to 35 amino acids. Exemplary linker designs are provided as SEQ ID NOS: 31 to 38.

[0062] In some embodiments, the variant comprises a linker, wherein the amino acid sequence of the linker is predominately glycine and serine residues, or consists essentially of glycine and serine residues. In some embodiments, the ratio of Ser and Gly in the linker is, respectively, from about 1:1 to about 1:10, from about 1:2 to about 1:6, or about 1:4. Exemplary linker sequences comprise or consist of S(GGS).sub.4GSS (SEQ ID NO: 36), S(GGS).sub.9GSS (SEQ ID NO: 37), (GGS).sub.9GSS (SEQ ID NO: 38). In some embodiments, the linker has at least 10 amino acids, or at least 15 amino acids, or at least 20 amino acids, or at least 25 amino acids, or at least 30 amino acids. For example, the linker may have a length of from 15 to 40 amino acids. In various embodiments, longer linkers of at least 15 amino acids can provide improvements in yield upon expression in Pichia pastoris. See PCT/US2019/055178, which is hereby incorporated by reference in its entirety.

[0063] An exemplary fusion protein for use in systemic therapy is shown as SEQ ID NO: 47, which includes an N-terminal albumin amino acid sequence, a flexible linker of 31 amino acids, and a mature D1L3 amino acid sequence having a full deletion of the BD.

[0064] In other embodiments, the linker is a physiologically-cleavable linker, such as a protease-cleavable linker. For example, the protease may be a coagulation pathway protease, such as activated Factor XII. In certain embodiments, the linker comprises the amino acid sequence of Factor XI (SEQ ID NO: 40) and/or prekallikrein (SEQ ID NO: 41) or a physiologically cleavable fragment thereof. In selected embodiments, the linker amino acid sequence from Factor XI contains all or parts of SEQ ID NO: 40 (e.g., parts of SEQ ID NO: 40, including modifications of SEQ ID NO: 40 that allow for cleavage by Factor XIIa). In some embodiments, the linker amino acid sequence from prekallikrein contains all or parts of SEQ ID NO: 41 (e.g., parts of SEQ ID NO: 41, including modifications of SEQ ID NO: 41 that allow for cleavage by Factor XIIa). In other embodiments, the linker includes a peptide sequence that is targeted for cleavage by a neutrophil specific protease, such as neutrophil elastase, cathepsin G, and proteinase 3.

[0065] The chromatin- and/or NET-degrading activity of a D1L3 enzyme variant, e.g., comprising a deletion of one or more amino acids of the BD, can be measured in vitro, for example by incubation of the enzyme with chromatin or NETs. Chromatin or NETs can be obtained in some embodiments from purified nuclei or ex vivo blood or neutrophils induced to form NETs. Alternatively, the chromatin- and/or NET-degrading activity of an enzyme can be measured in vivo, for example by administering the enzyme to a subject, wherein the subject produces or is induced to produce extracellular DNA, chromatin or NETs, and measuring the effect of the enzyme on concentrations of DNA, chromatin, or NET levels in a matrix, e.g. serum, preferably with a parallel negative control, or by temporally comparing the concentrations before and after administration of the enzyme.

[0066] In some embodiments, the fusion protein is synthesized with a signal peptide. The signal peptide may be removed during secretion from the host cell. With respect to expression in Pichia pastoris, the alpha-mating factor (.alpha.MF) pre-pro secretion leader from Saccharomyces cerevisiae (SEQ ID NO: 28) may be used for expression. In other embodiments, the signal peptide and propeptide of HSA, which consists of a signal sequence of 24 amino acids (MKWVTFISLLFLFSSAYSRGVFRR; SEQ ID NO: 29) may be used. In some embodiments, the human DNASE1L3 Signal Peptide (Q13609) (SEQ ID NO: 30) is used for expression. These elements are cleaved during expression, and are not present in the D1L3 enzyme product.

[0067] The invention in some aspects provides pharmaceutical compositions comprising the D1L3 enzyme described herein, or optionally a polynucleotide encoding the D1L3 enzyme, or a transfection or expression vector comprising the same, and a pharmaceutically acceptable carrier.

[0068] In some embodiments, delivery of polynucleotides is used for therapy. Encoding polynucleotides can be delivered as mRNA or as DNA constructs using known procedures, e.g., electroporation or cell squeezing, and/or vectors (including viral vectors). mRNA polynucleotides can include known modifications (mmRNA) to avoid activation of the innate immune system. See WO 2014/028429, which is hereby incorporated by reference in its entirety. In some embodiments, the polynucleotide is delivered to the body of a subject.

[0069] In some embodiments, the polynucleotide is delivered into a cell in vitro, and the cell is delivered to the body of a subject. The cell can be, for example, a white blood cell (e.g., a T cell or macrophage), an endothelial cell, an epithelial cell, a hepatocyte, or a stem cell (e.g., LT-HSC). In these aspects, the invention provides mammalian host cells (e.g., human host cells) (as well as methods of making and using the same) that comprise a heterologous polynucleotide encoding a chromatinase enzyme operably linked to a promoter. The host cell expresses and secretes the chromatinase enzyme. In these aspects, challenges in manufacturing chromatinases such as D1L3 at large scale are avoided. Further, by expressing and delivering D1L3 through heterologous expression in a white blood cell such as a T cell, D1L3 therapy can be localized in part to areas of inflammation or tissue destruction or cell apoptosis. Further, since D1L3 has a circulation half-life of less than about 30 minutes, the cell therapy described herein provides for a sustained therapy, with as few as one, two, three, or four treatments. In various embodiments, a subject can be treated with ten or fewer administrations of the cellular therapy, or with four or fewer treatments of the cellular therapy. While T cells (and other host cells) can be engineered to express D1L3 having whole or partial deletions of the C-terminal BD (as described herein), because T cells express PCSK types 3, 5, 6, and 7 (including Furin, PCSK3), expression of wild type D1L3 can be activated by T cells through cleavage within the C-terminal BD. Exemplary T cells include CD4+ T cells or CD8+ T cells (e.g., CTLs). In some embodiments, the T cell is a regulatory T cell (T.sub.reg). T cells such as gamma delta T cells or Chimeric Antigen Receptor (CAR)-T cells can be employed in certain embodiments. In some embodiments, the CAR-T cells are directed against CD19. In some embodiments, D1L3 C-terminal basic domain processing is induced when the T cell is activated (e.g., by activation of the TCR or CAR). Exemplary T cells can comprise memory T cells, such as (in order of proliferative capacity) T memory stem cells, central memory T cells, or effector memory T cells. In some embodiments, the T cells are predominately terminally differentiated T cells.

[0070] Exemplary T cells for chromatinase cell therapy may recognize (through the TCR or CAR) a cancer-associated antigen, such as a leukemia-associated antigen, or an antigen of a solid tumor. In some embodiments, the T cell recognizes a viral antigen, including but not limited to an oncovirus. Exemplary oncoviruses include Epstein-Barr virus, human papilloma virus, hepatitis B or C virus, human herpes virus (e.g., HSV8), and human T lymphotrophic virus. In some embodiments, the T cell recognizes a coronavirus antigen, such as SARS-CoV-2.

[0071] In some embodiments, the host cell (e.g., a T cell) secretes D1L3 enzyme having a deletion of at least 12 amino acids of the C-terminal BD. In some embodiments, the secreted D1L3 enzyme includes enzymes having deletions of one or more of: K291_S305 del, K292_S305 del, K293_S305 del, with respect to SEQ ID NO:4. In these or other embodiments, the polynucleotide encodes a D1L3 enzyme having a deletion of one or more amino acids of the C-terminal BD, such as at least three or at least five amino acids of the C-terminal BD. In some embodiments, the polynucleotide encodes a D1L3 enzyme having a deletion of at least 12 amino acids of the BD, or a complete deletion of the BD.

[0072] In some embodiments, the polynucleotide encodes a D1L3 enzyme having an inactivation or mutation of one or more of the NLS1 and the NLS2. D1L3 features two nuclear localization sites (NLS1, NLS2), which may target the enzyme to the nucleus during apoptosis. NLS1 is located near the N-terminus (about amino acid positions 80 to 96 with respect to SEQ ID NO:4). NLS2 (amino acid positions 291 to 304 with respect to SEQ ID NO:4) is embedded within the C-terminal BD. In some embodiments, the NLS inactivation is by deletion of all or part of NLS1 and/or NLS2. In some embodiments, the NLS is inactivated by substitution and/or deletion of amino acids within NLS1 and/or NLS2. In some embodiments, NLS2 is deleted, entirely or partially.

[0073] In certain embodiments, the D1L3 enzyme contains one or more, e.g., 1, 2, 3, 4, 5, or more amino acid substitutions, additions (e.g., insertions), or deletions in the NLS1. In certain embodiments, the D1L3 enzyme contains one or more, e.g., 1, 2, 3, 4, 5, or more amino acid substitutions, additions, or deletions in the NLS2.

[0074] In these or other embodiments, the polynucleotide may express the D1L3 fused to a carrier protein as described (e.g., albumin), which is optionally linked at the N-terminus of D1L3 enzyme through a flexible or cleavable linker (as described).

[0075] A vector generally comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Exemplary vectors include autonomously replicating plasmids or a virus (e.g. AAV vectors). The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0076] In some embodiments, the polynucleotide or cell therapy may employ expression vectors, which comprise the nucleic acid encoding the chromatinase (e.g., D1L3) operably linked to an expression control region that is functional in the host cell. The expression control region is capable of driving expression of the operably linked encoding nucleic acid such that the chromatinase is produced in a human cell transformed with the expression vector. Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that influence expression of an operably linked nucleic acid. An expression control region of an expression vector is capable of expressing operably linked encoding nucleic acid in a human cell. In an embodiment, the expression control region confers regulatable expression to an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. In various embodiments, the chromatinase expression is inducible or repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.

[0077] Expression systems functional in human cells are well known in the art, and include viral systems. Generally, a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA. A promoter will have a transcription-initiating region, which is usually placed proximal to the 5' end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.

[0078] Where appropriate, gene delivery agents such as, e.g., integration sequences can also be employed. Numerous integration sequences are known in the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), FIp (Broach, et al., Cell, 29:227-234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth et al., J. Mol. Biol. 335:667-678, 2004), sleeping beauty, transposases of the mariner family, and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). In addition, direct and targeted genetic integration strategies may be used to insert nucleic acid sequences including CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editing technologies.

[0079] The pharmaceutical composition may be formulated for any administration route, including topical, parenteral, or pulmonary administration. In various embodiments, the composition is formulated for intravenous, intradermal, intramuscular, intraperitoneal, intraarticular, subcutaneous, or intraarterial. In some embodiments, the composition is formulated for intravenous or subcutaneous administration. In some embodiments, the composition comprises an effective amount of host cells (expressing a chromatinase such as D1L3) for delivery (e.g., by infusion). An effective amount of host cells to be delivered by the composition can be determined by one of skill in art, and may include, for example, at least about 10.sup.6 cells, at least about 10.sup.7 cells, at least about 10.sup.8 cells, or at least about 10.sup.9 cells.

[0080] In other aspects, the present technology provides a method for treating a subject in need of extracellular chromatin degradation, extracellular trap (ET) degradation and/or neutrophil extracellular trap (NET) degradation. The method comprises administering a therapeutically effective amount of the D1L3 enzyme or composition described herein. Exemplary indications where a subject is in need of extracellular chromatin degradation (including ET or NET degradation) are disclosed in PCT/US18/47084, the disclosure of which is hereby incorporated by reference. In some embodiments, the method comprising administering the isolated host cells described herein (expressing a chromatinase for secretion) to the subject. In some embodiments, the subject is at risk of occlusions involving extracellular chromatin, including chromatin released by cancer cells and injured endothelial cells, among others. Thus, in exemplary embodiments, the subject has cancer (e.g., leukemia or solid tumor). In some embodiments, the subject has metastatic cancer.

[0081] Subjects receiving therapy for cancer (including but not limited to T cell therapies) are at risk of tumor lysis syndrome, which occurs when tumor cells release their contents (including chromatin) into the bloodstream. Tumor lysis syndrome is a complication during the treatment of cancer, where large amounts of tumor cells are killed at the same time. Tumor lysis syndrome occurs commonly after the treatment of lymphomas and leukemias.

[0082] In still other embodiments, the subject has an inflammatory disease of the respiratory tract, such as the lower respiratory tract. Exemplary diseases include bacterial and viral infections. In some embodiments, the subject has Acute Respiratory Distress Syndrome (ARDS), Acute Lung Injury (ALI), or pneumonia. Exemplary viral infections include RSV and coronavirus infection (such as SARS, or SARS-CoV-2, e.g., COVID-19 as well as variants thereof).

[0083] Neutrophils, the predominant leukocytes in acute inflammation, generate neutrophil extracellular traps (NETs), lattices of high-molecular weight chromatin filaments decorated with biologically active proteins and peptides, which immobilize bacteria in wounds. Systemic accumulation of NETs harms tissues and organs due to their cytotoxic, proinflammatory, and prothrombotic activity. Indeed, NETs are frequently associated with inflammatory, ischemic, and autoimmune conditions, including Systemic Lupus Erythematosus (SLE).

[0084] In various embodiments, the present invention provides a method for treating, preventing, or managing diseases or conditions characterized by the presence or accumulation of NETs. Such diseases or conditions include, but are not limited to, diseases associated with chronic neutrophilia, neutrophil aggregation and leukostasis, thrombosis and vascular occlusion, ischemia-reperfusion injury, surgical and traumatic tissue injury, an acute or chronic inflammatory reaction or disease, an autoimmune disease, cardiovascular disease, metabolic disease, systemic inflammation, inflammatory diseases of the respiratory tract, renal inflammatory diseases, inflammatory diseases related to transplanted tissue (e.g. graft-versus-host disease) and cancer (including leukemia).

[0085] In some embodiments, the subject has SLE. The discovery of NETs raised the speculation that neutrophils may be the predominant source of autoantigens (i.e. dsDNA, chromatin) in SLE (Brinkmann, et al. Neutrophil Extracellular Traps Kill Bacteria. Science, 303(5663): 1532-1545 (2004). Indeed, autoantibodies such as anti-dsDNA, -histone, and -nucleosome antibodies bind to NETs, forming pathological ICs. Hakkim, et al., Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis, Proceedings of the National Academy of Sciences 107: 9813-9818 (2010). The accumulation of NET-IC breaks immune tolerance via activation of adaptive immune cells that lead to the production of autoantibodies against NET components, forming a vicious cycle of inflammation and autoimmunity. Gupta and Kaplan, The role of neutrophils and NETosis in autoimmune and renal diseases. Nat Rev Nephrol. 12(7): 402-13 (2016). Therefore, reducing accumulation of NETs can break the cycle and thus provide an attractive therapeutic strategy for SLE.

[0086] In certain embodiments, the present invention pertains to the treatment of diseases or conditions characterized by deficiency of D1L3, or a deficiency of D1. In some cases, the subject has a mutation (e.g., a loss of function mutation) in the Dnase1l3 gene or the Dnase1 gene. Such subjects can manifest with an autoimmune disease, such as: systemic lupus erythematosus (SLE), lupus nephritis, scleroderma or systemic sclerosis, rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, and urticarial vasculitis. In some cases, the subject has an acquired inhibitor of D1 (e.g., anti-DNase1-antibody and actin) and/or D1L3 (e.g., anti-Dnase1l3-antibody). Such subjects can also have an autoimmune or inflammatory disease (e.g., SLE, systemic sclerosis).

[0087] In some embodiments, the subject has or is at risk of NETs occluding ductal systems. For example, the D1L3 enzymes or compositions disclosed herein can be administered to a subject to treat pancreatitis, cholangitis, conjunctivitis, mastitis, dry eye disease, obstructions of vas deferens, or renal diseases.

[0088] In some embodiments, the subject has or is at risk of NETs accumulating on endothelial surfaces (e.g. surgical adhesions), the skin (e.g. wounds/scarring), or in synovial joints (e.g. gout and arthritis, e.g., rheumatoid arthritis). The D1L3 enzymes and compositions described herein can be administered to a subject to treat a condition characterized by an accumulation of NETs on an endothelial surface such as, but not limited to, a surgical adhesion.

[0089] Other diseases and conditions associated with NETs, which the D1L3 enzymes or compositions disclosed herein may be used to treat or prevent, include: ANCA-associated vasculitis, asthma, chronic obstructive pulmonary disease, a neutrophilic dermatosis, dermatomyositis, burns, cellulitis, meningitis, encephalitis, otitis media, pharyngitis, tonsillitis, pneumonia, endocarditis, cystitis, pyelonephritis, appendicitis, cholecystitis, pancreatitis, uveitis, keratitis, disseminated intravascular coagulation, acute kidney injury, acute respiratory distress syndrome, shock liver, hepatorenal syndrome, myocardial infarction, stroke, ischemic bowel, limb ischemia, testicular torsion, preeclampsia, eclampsia, and solid organ transplant (e.g., kidney, heart, liver, and/or lung transplant). Furthermore, the D1L3 enzymes or compositions disclosed herein can be used to prevent a scar or contracture, e.g., by local application to skin, in an individual at risk thereof, e.g., an individual with a surgical incision, laceration, or burn.

[0090] In various embodiments, the subject has a disease that is or has been treated with wild-type Dnases, including D1 and streptodornase. Such diseases or conditions include thrombosis, stroke, sepsis, lung injury, atherosclerosis, viral infection, sickle cell disease, myocardial infarction, ear infection, wound healing, liver injury, endocarditis, liver infection, pancreatitis, primary graft dysfunction, limb ischemia reperfusion, kidney injury, blood clotting, alum-induced inflammation, hepatorenal injury, pleural exudations, hemothorax, intrabiliary blood clots, post pneumatic anemia, ulcers, otolaryngological conditions, oral infections, minor injuries, sinusitis, post-operative rhinoplasties, infertility, bladder catheter, wound cleaning, skin reaction test, pneumococcal meningitis, gout, leg ulcers, cystic fibrosis, Kartegener's syndrome, asthma, lobar atelectasis, chronic bronchitis, bronchiectasis, lupus, primary ciliary dyskinesia, bronchiolitis, empyema, pleural infections, cancer, dry eyes disease, lower respiratory tract infections, chronic hematomas, Alzheimer's disease, and obstructive pulmonary disease.

[0091] In some embodiments, the subject has a loss of function mutation in one or both D1L3 genes, and may exhibit symptoms of SLE, or may be further diagnosed with clinical SLE. In such embodiments, the subject may receive systemic therapy with a BD-deleted D1L3 described herein, such as the fusion protein represented by SEQ ID NO:47. In various embodiments, therapeutically effective amounts of the fusion protein represented by SEQ ID NO:47, or other fusion between albumin and a BD-deleted D1L3, are administered once or twice weekly, or once or twice monthly.

[0092] Other aspects and embodiments of the invention will be apparent from the following examples.

EXAMPLES

Example 1: Creating Chimeric DNase Enzymes

[0093] In this Example, chimeric DNase enzymes were created to evaluate the potential to create novel DNase enzymes for therapy against disorders caused by the accumulation of extracellular chromatin, including NETs. To produce variants of D1L3, transient transfection of in vitro expression systems [e.g. Chinese hamster ovary (CHO) cells or HEK293 cells] was used. Enzymatic activity in culture supernatants was characterized using the degradation of high-molecular weight (HMW)-chromatin (i.e. purified nuclei from HEK293 cells) as a readout. In brief, HMW-chromatin was first incubated with the D1L3 variants, followed by DNA isolation and visualization via agarose gel electrophoresis (AGE). As shown in FIG. 1, it was observed that, unlike D1, D1L3 degrades HMW-chromatin (app. 50-300,000,000 base pairs) specifically and efficiently into nucleosomes (app. 180 base pairs), the basic units of chromatin fibers, whereas no such effect was observed in samples with D1.

[0094] We aimed to identify the regions of D1L3 that are responsible for its chromatin degrading activity. Sequence alignments of human D1 and human D1L3 were performed. The sequence alignments showed that 44% of the amino acids in human D1 and human D1L3 are identical. Without being bound by theory, it was speculated that the capacity of human D1L3 to degrade chromatin is mediated by amino acids that are not present in D1. Thus, only the variable amino acids (56% non-shared amino acids) were mutated to generate DL3 variants. The method used to transfer enzymatic properties from D1 to D1L3 (building block-technology) is schematically represented in FIG. 2. The following cardinal steps characterize the building block substitution approach:

[0095] (1) Provide protein-protein alignment of donor (DNASE1) and recipient DNase (DNASE1L3);

[0096] (2) Identify variable amino acid or amino acid sequence for transfer (building block);

[0097] (3) Identify conserved amino acids in donor and recipient DNase that are located up and downstream of building blocks, respectively ("anchors");

[0098] (4) Replace the cDNA sequences encoding building block sequences, which are flanked by the C- and N-terminal anchors from a recipient DNase, with the cDNA sequence between the corresponding anchors from donor DNase;

[0099] (5) Synthesize a cDNA encoding the chimeric DNase. Prepare an expression vector capable of expressing the chimeric DNase, which harbors the cDNA of the chimeric DNase, operably linked to a promoter, terminator and/or other regulatory sequences of interest.

[0100] Chimeric DNase-encoding polynucleotides can be introduced and expressed into a recipient organism/cell of interest, which is preferably deficient in both donor and recipient DNase (e.g. CHO cells or Dnase1.sup.-/-Dnase1l3.sup.-/- mice).

Example 2: Biochemical Characterization of the Chimeric Enzymes

[0101] Using the building block substitution approach, 63 D1L3-D1 chimeras were generated (FIG. 3). These chimeras included D1L3-variants with the following building block mutations (BB): M1_A20delinsMRGMKLLGALLALAALLQGAVS, M21_S25delinsLKIAA, V28_S30delinsIQT, E33_S34delinsET, Q36_I45delinsMSNATLVSYI, K47_K50delinsQILS, C52Y, I54_M58delinsIALVQ, I60_K61delinsVR, S63_I70delinsSHLTAVGK, M72_K74delinsLDN, R77_T84delinsQDAPDT, N86H, V88_I89delinsVV, S91_R92delinsEP, N96_T97delinsNS, Q101R, A103L, L105V, K107_L110delinsRPDQ, V113_S116delinsAVDS, H118Y, H120D, Y122_A127delinsGCEPCGN, V129T, S131N, 135F_136VdelinsAI, W138R, Q140_H143delinsFSRF, A145_D148delinsAVKD, V150A, I152A, T156_T157delinsAA, E159_S161delinsGDA, K163A, E167A, V169_E170delinsYD, T173L, K176_R178delinsQEK, K180_A181delinsGL, N183_F186delinsDVML, P198_A201delinsRPSQ, K203_N204delinsSS, R208W, D210S, R212T, V214Q, G218P, Q220_E221delinsSA, V225_S228delinsATP, N230H, L238_R239delinsVA, Q241_S246delinsMLLRGA, K250D, N252_V254delinsALP, D256N, K259_A260delinsAA, K262G, T264_E267delinsSDQL, L269_V271delinsQAI, F275Y, F279_K280delinsVM, and Q282_S305delinsK.

[0102] The D1L3-variants were transiently expressed in CHO cells and culture supernatants were screened for the activity to degrade high molecular weight (HMW)-chromatin. The reaction mixtures of HMW-chromatin degradation assay were examined by agarose gel electrophoresis (AGE) to assess the activity of the D1L3-D1 chimera. As shown in FIG. 4, these assays led to the identification of Q282_S305delinsK (i.e. BB #63), which confers hyperactivity on D1L3.

[0103] The mutation Q282_S305delinsK causes a complete deletion of the BD domain and the substitution of Q282 (glutamine at position 282) of D1L3 with a K (Lysine). As shown in FIG. 5A, the deletion of the C-terminal BD was confirmed using Western Blotting of culture supernatants using a monoclonal antibody that targets MGDFNAGCSYV (SEQ ID NO:42) (Anti-D1/D1L3), a peptide sequence that is shared by D1 and D1L3. The data further illustrated similar protein concentration of wild-type and mutated D1L3, indicating a lack of toxicity or altered protease sensitivity of the Q282_S305delinsK mutant D1L3 protein in the expression system (FIG. 5A).

[0104] To compare the enzymatic activity of wild type and Q282_S305delinsK mutant D1L3 proteins, a titration experiment was performed: high molecular weight chromatin was digested with increasing amounts of the enzymes, and reaction products were resolved by agarose gel electrophoresis (AGE). As shown in FIG. 5B, titration of culture supernatants showed that BD-deleted D1L3 had approximately 5- to 10-fold higher activity to degrade HMW-chromatin. The extent of chromatinase activity of wild type and Q282_S305delinsK mutant D1L3 proteins was calculated based on the quantitation of degraded chromatin (FIG. 5C). Collectively, the data suggest that the BD domain of D1L3 is not required for chromatin degradation, but rather suppresses enzymatic activity.

Example 3: Comparison of Different C-Terminal Truncation Mutants

[0105] Since the Q282_S305delinsK mutation lacking the BD domain showed approximately 5- to 10-fold higher chromatinase activity compared to wild type D1L3 protein, the effect of extent of C-terminal deletion was evaluated. Whether full or partial truncation of BD of D1L3 is required to enable chromatin degradation was explored. Deletion mutants S305del, K303_S305del, V294_S305del, K291_S305del, R285_S305del, and S283_S305del, which lack 1, 3, 12, 15, 21, and 23 C-terminal amino acids, respectively were designed (FIG. 6). CHO and HEK293 cells were transiently transfected with vectors expressing wild-type and truncation mutants. Culture supernatants containing D1L3 and the variants were collected and tested by Western Blot using the aforementioned anti-D1/D1L3 antibody. As shown in FIG. 7, wild-type D1L3 as well as variants of smaller molecular weight could be detected. The observed mobility shift, inter alia, confirmed the deletion of BD. Furthermore, the western blots showed that all C-terminal truncation mutants and the wild-type D1L3 exhibited similar protein expression levels.

[0106] The effect of deleting all or part of the BD on chromatin degrading activity was determined by incubating culture supernatants with intact chromatin from isolated nuclei. In the first set of experiments, the culture supernatants of cells expressing wild-type and truncation mutants were diluted 10-fold with incubation buffer and then incubated with high molecular weight chromatin. Analysis of DNA fragmentation by AGE revealed that deletion of 3 or less amino acids caused only a minor increase in enzymatic activity, whereas the deletion of 12 or more amino acids strongly accelerated the degradation of ultra-large chromatin into mono-nucleosomes (FIG. 8A). To quantify chromatin degrading activity, the concentration of ultra-large chromatin, defined as DNA-fragments of >1,500 base pairs, was determined using image analysis of agarose gel electrophoresis (AGE). As shown in FIG. 8B, this analysis confirmed the correlation of BD-deletion and enzymatic activity.

[0107] In a second set of experiments, undiluted culture supernatants of cells expressing wild-type and truncation mutants were mixed with intact chromatin from isolated nuclei. Under these conditions, ultra-large chromatin was completely degraded into mono-nucleosomes by D1L3 samples (FIG. 9A). Next, mono-nucleosomes, defined as DNA-fragments between 100 and 300 base pairs, were quantified using image analysis. As shown in FIG. 9B, a linear correlation was observed between the deletion of C-terminal amino acids and the activity of D1L3 to degrade mono-nucleosomes. These data showed that enzymatic activity for degrading mononucleosomes increased with increasing length of deletion from three C-terminal amino acids to 23 C-terminal amino acids (FIG. 9B). Taken together, the data illustrate that the entire BD of D1L3 has distinct inhibitory effects on degradation of ultra-large chromatin and mono-nucleosomes.

Example 4: Engineering D1L3 for Large-Scale Manufacturing

[0108] Nearly 70% of all biologics are produced using Chinese Hamster Ovary (CHO) cells. Indeed, wild-type DNASE1 (D1; dornase alpha) is typically produced in CHO cells. Despite significant advantages in cell line development and large-scale production using CHO cells, there still remains a significant challenge in the production of Dnase enzymes due to a considerable degree of variability and no reliable methods for predicting or modeling cell growth characteristics. Importantly, CHO cells were not able to stably produce hyperactive variants of D1, which prevented their clinical manufacturing, and the manufacturing properties of other DNASE1-protein family members, including DNASE1-LIKE 3 (D1L3), are largely unknown.

[0109] Using CHO and microbial expression systems, several challenges were identified in manufacturing of D1L3, including low production yield, proteolytic degradation, protein misfolding, and erroneous or undesired glycosylation. These are described below.

[0110] Pichia pastoris was evaluated as an alternative, microbial expression system to CHO cells. Higher expression levels were generally observed with BDD-D1L3, when compared to wild-type D1L3. Here, we purified and characterized wild-type D1L3 and BDD-D1L3 from Pichia pastoris fermentation supernatants (FIG. 10). Unexpectedly, we observed that wild-type D1L3 was proteolytically truncated within the BD at the amino acid positions K291, K291, or 5293, leading to a heterogeneous mix of D1L3 variants after purification. Unlike wild-type D1L3, expression of BDD-D1L3 due to three building block substitutions (F275Y, F279_K280delinsVM, Q282_S205delinsK) generated a pure protein.

[0111] The chromatinase activity of both D1L3 purifications was compared, where it was observed that the heterogeneous mix of D1L3 variants with BD truncations at positions K291, K291, or 5293 had approximately 10-fold lower chromatinase activity compared to the D1L3 variant with a full BD deletion due to F275Y/F279_K280delinsVM/Q282_S205delinsK (FIG. 10). Collectively, the data illustrate that the proteolytic cleavage of the BD can occur naturally in some microbial and mammalian expression systems, and removal of the BD appears to activate D1L3 activity to degrade chromatin.

[0112] We attempted to develop a stable CHO cell line producing wild-type D1L3. The cell lines were cultured in bioreactors using standard CHO culture medium. Specifically, FIG. 11 shows a Western Blot of human D1L3 expressed and secreted by CHO cells in a bioreactor under cGMP-compatible conditions. Samples were collected at different time points (t1-t3). Very low levels of D1L3 and D1L3 fragments were detected. The data suggest that low production yield of D1L3 is a challenge in manufacturing of D1L3.

[0113] Higher production levels of wild-type D1L3 may be achieved by the addition of polyanions to the culture medium. Such polyanions can comprise one or more of heparin, dextran sulfate, ferric citrate, and ethylenediaminetetraacetic acid, and represent the biologically active ingredient in "anti-cell clumping reagents". Specifically, dextran sulfate was added to the CHO culture medium and an increase was observed in D1L3 as well as D1L3 fragments (FIG. 11). The data illustrate that polyanions can increase production yield of D1L3, but did not prevent proteolytic degradation.

[0114] The potential to engineer a protease-resistant D1L3 was also investigated. Wild-type D1L3 contains 50 arginine and lysine residues, which makes the enzyme particularly susceptible to proteases like trypsin, thrombin, and plasmin. In this example, trypsin and plasmin cleavage sites were identified in D1L3 and mutated in an attempt to identify protease-resistance variants of D1L3.

[0115] In brief, purified D1L3 was digested with trypsin. D1L3 fragments were isolated, and the amino acid sequence of the fragments determined using combinations of liquid chromatography (LC) and mass spectrometry (MS). It was identified that trypsin cleaved D1L3 at the following arginine and lysine residues: R22, R29, R51, R66, R80, R81, R95, K99, R115, K147, K163, K180, R208, R212, R235, R239, K250, and K262. These arginine and lysine residues might be substituted with small amino acids such as alanine, valine, and serine or with amino acids that have similar properties according to the Grantham's distance score (e.g. histidine, glutamine, and glutamate). D1, which is protease resistant, features arginine and lysine residues corresponding to R51, R95, K99, and R235, suggesting that these residues are not primarily responsible for proteolytic degradation of D1L3.

[0116] Building Block Protein Engineering was applied to transfer the following Building Blocks from D1 to replace Building Blocks of D1L3 that contain the trypsin cleavage sites: R22 (Mutation: M21_R22delinsLK), R29 (V28_S30delinsIQT), R66 (N64_I70delinsHLTAVGK), R80 (R77_I83delinsQDAPD), R81 (R77_I83delinsQDAPD), R115 (V113_R115delinsAVD), K163 (K163A), K180 (K180_A181delinsGL), R208 (R208W) MR212 (R212T), R239 (L238_R239delinsVA), K250 (K250D), and K262 (K262G).

[0117] Plasmin is a plasma protease that is generated by activation of its zymogen plasminogen. Plasminogen activator inhibitor 1 (PAI-1) inhibits the activation of plasmin. Interestingly, PAI-1 increases the enzymatic activity of D1L3 in serum, suggesting that plasmin may proteolytically inactivate D1L3. However, the plasmin cleavage sites in D1L3 have not been identified.

[0118] In silico analysis showed that the amino acid combination lysine-alanine (KA) or arginine-alanine (RA) might be preferably cleaved by the protease plasmin or proteases that have plasmin-like activity. D1L3 contains a total of four putative plasmin-cleavage sites: (Site 1) K180/A181 (K160/A161 without signal peptide), (Site 2) K200/A201 (K180/A181 without signal peptide), (Site 3) K259/A260 (K239/A240 without signal peptide), and (Site 4) R285/A286 (R270/A250 without signal peptide). Using a paired alignment of D1 and D1L3, we found that none of the plasmin cleavage sites are present in D1. The data are in line with the fact that D1 activity is resistant to inactivation by serum proteases, such as thrombin and plasmin. Building Block Protein Engineering was applied to transfer the following Building Blocks from D1 to replace Building Blocks of D1L3 that contain the plasmin cleavage sites: (Site 1) K180_A181delinsGL, (Site 2) P198_A201delinsRPSQ, and (Site 3) K259A. R285/A286 (Site 4) is located in a C-terminal extension that is absent in D1. Consequently, we generated a D1L3 variant in which all four putative plasmin cleavage sites were mutated: K180_A181delinsGL, P198_A201delinsRPSQ, K259A, and R285A. Next, we analyzed chromatin degradation by the D1L3 variant and observed potent chromatin degrading activity in the mutated D1L3. Collectively, the data show that four arginine and lysine residues, K180, K200, K259, and R285, can be mutated to reduce the risk of proteolytic degradation without compromising enzymatic activity.

[0119] Next, purified D1L3 was digested with purified plasmin. D1L3 fragments were isolated, and the amino acid sequence of the fragments determined using combinations of LC and MS. We identified that plasmin cleaved D1L3 at the following arginine and lysine residues: R22, R29, K45, K47, K74, R81, R92, K107, K176, R212, R226, R227, K250, K259, and K262. These arginine and lysine residues can be substituted with small amino acids such as alanine, valine, and serine or with amino acids that have similar properties according to the Grantham's distance score (e.g. histidine, glutamine, and glutamate). D1, which is protease resistant, features a lysine residue corresponding to K45, suggesting that this residue is not primarily responsible for proteolytic degradation of D1L3 by plasmin. Building Block Protein Engineering was applied to transfer the following Building Blocks from D1 to replace Building Blocks of D1L3 that contain the trypsin cleavage sites in silico: R22 (Mutation: M21_R22delinsLK), R29 (V28_S30delinsIQT), K47 (K47_K50delinsQILS), K74 (M72_K74delinsLDN), R81 (R77_I83delinsQDAPD), R92 (S91_R92delinsEP), K107 (K107_L110delinsRPDQ), K176 (K176_R178delinsQEK), R212 (R212T), K226 (V225_S228delinsATP), K227 (V225_S228delinsATP), K250 (K250D), K259 (K259A), and K262 (K262G).

[0120] Finally, recombinantly expressed wild-type D1L3 was isolated and its C-terminus sequenced. Three different amino acid sequences were identified ending in S290, K291, and K292. The data identify lysine residues 291 and 292 as prominent proteolytic cleavage sites of D1L3 during large-scale manufacturing.

[0121] We observed fragmentation of D1L3 after heterologous expression in Pichia pastoris. Analysis of the fragments characterized paired basic amino acids, arginine (R) and lysine (K) residues, as proteolytic cleavage sites. A similar degradation pattern was observed after expressing D1L3 in CHO cells. These observations suggest that Pichia pastoris and CHO cells share homologous proteases that cleave D1L3 at paired basic amino acids, and the effect was more significant in CHO cells.

[0122] It was determined that the paired basic amino acid cleaving enzyme (PACE) contributed to the DNASE1L3 fragmentation. PACE, also known as Furin (Uniprot ID: P09958), is expressed in humans and mammals. Pichia pastoris expresses two enzymes, which target paired basic amino acids, namely Aspartic proteinase 3 (Gene: Ysp1; Uniprot ID: P32329) and Kexin (Gene: Kex2; Uniprot ID: P13134).

[0123] In addition, mutations of paired basic amino acids in DNASE1L3 and DNASE1L3 variants enable their expression in CHO and Pichia pastoris with reduced fragmentation. Analysis of DNASE1L3 fragments identified feature paired basic amino acid at positions: K50/R51, R80/R81, K114/R115, K199/K200, K226/K227, K291/K292, R297/K298/K299, and K303/R304.

[0124] Kexin preferably cleaves after KR and RR residues. DNASE1L3 features at K50/R51, R80/R81, K114/R115, and K303/R304 are 4 KEX2-cleavage sites. Amino acid substitutions of these residues render DNASE1L3 resistant to KEX2 and enable the expression of DNASE1L3 and DNASE1L3 variants in Pichia pastoris and in CHO cells. These amino acid substitutions can be conservative, e.g. R51K, R81K, R115K, and R304K.

[0125] During cGMP-compatible expression of D1L3 in CHO cells, the accumulation of high-molecular weight aggregates of D1L3 was observed, pointing towards an additional challenge for clinical manufacturing D1L3. The high molecular weight aggregates were observed by a lower extent in Pichia pastoris.

[0126] The application of reducing conditions to proteins of bioreactor material dissolved D1L3 aggregates. The data illustrate that D1L3 aggregate formation is caused by intra- and/or inter-molecular cross-linking via disulfide bridges during protein expression. Specifically, as shown in FIG. 12, the gel was run under non-reducing conditions and shows the accumulation of high-molecular weight aggregates of D1L3 over time. The gel was run under reducing conditions and no aggregates were detected. The data illustrate that erroneous intra- and inter-molecular disulfide bonds cause misfolding of human D1L3 under manufacturing conditions.

[0127] FIG. 13 shows an amino acid sequence alignment of human D1 (SEQ ID NO: 1) and human D1L3 (SEQ ID NO: 4). The signal peptide, conserved amino acids, variable amino acids, non-conserved cysteine residues, and conserved cysteine residues are highlighted. Mutations in non-conserved cysteine residues will reduce the possibilities of intra- and inter-molecular disulfide bonds during protein expression. Analysis of the amino acid sequence of D1L3 showed the presence of five cysteine (C) residues: C24, C52, C68, C194, and C231. The cysteine residues C194 and C231 are conserved among all members of the DNASE1-protein family and form disulfide bonds that are required for enzymatic activity of DNASE1. Accordingly, as disclosed herein, mutation of these cysteine residues reduces the cross-linking via disulfide bridges and thus increases the yield of protein production.

[0128] Cysteine residues can be substituted with other small amino acids, namely alanine (A), serine (S), and glycine (G), among others. Such substitutions cause the following amino acid mutations C24A/S/G, C52A/S/G, C68A/S/G, C194A/S/G, and C231A/S/G. In addition, Building Blocks that comprise the conserved cysteine residues can be replaced by Building Blocks from a donor DNase of the DNASE1-protein family (e.g. D1 and D1L3). The following Building Blocks from D1 were used to replace the Building Blocks of D1L3 that contain the non-conserved cysteine residues C24, C52, and C68: C24_S25delinsAA, C52Y, and N64_I70delinsHLTAVGK. The chromatin degrading activity of D1L3 variants was quantified, as described in PCT/US18/4708. Both conventional amino acids substitutions (C24A, C52A) and building block substitutions (C24_S25delinsAA, C52Y) caused a complete absence of chromatin degradation, indicating that C24 and C52 are required for D1L3 activity. Importantly, mutation of cysteine C68, either by conventional amino acid substitution (C68A) or by BB mutation (N64_I70delinsHLTAVGK), resulted in a D1L3 variant with chromatin degrading activity. Amino acid sequence alignment showed that cysteine C68 is not conserved among other DNASE1-protein family members, supporting the notion that C68 is not required for enzymatic activity. Furthermore, it was observed that the amino acid substitution of highly conserved cysteine C194 with alanine (C194A), but not the mutation of the highly conserved cysteine C231 with alanine (C231A), resulted in an enzymatically active D1L3 variant. Thus, cysteine C68 and C194 can be mutated to reduce the risk of erroneous disulfide bonds during D1L3 production.

Example 5: The Paired Basic Amino Acid Cleaving Enzyme Furin Regulates D1L3

[0129] The BD domain contains an NLS and three paired basic amino acids that are potentially responsible for the inhibitory effects on enzymatic activity (FIG. 14). The NLS is located between amino acids L286 to S302 [LRKKTKS; Reference: Q13609 (Uniprot)] and therefore absent in all identified hyperactive D1L3 mutants that lack 12 amino acids or more.

[0130] It was hypothesized that the three sets of paired basic amino acids (K291/K292, K298/K299, and K303/R304) and may serve as proteolytic cleavage sites of the Paired Basic Amino Acid Cleaving Enzyme (PACE). Furin is a well-characterized PACE, which is involved in the maturation of pro-enzymes. To test the possibility of furin generating active D1L3, and to understand the possible role of furin in activation of chromatinase activity of D1L3, furin-overexpressing CHO cells were transiently transfected with wild-type and BD-deleted D1L3 (S283_S305del mutant). CHO cells without overexpression of furin were included as control. Culture supernatants were collected and tested by western blot using an antibody that targets the N-terminus of D1L3. As shown in FIG. 15, two bands of D1L3 were detected in furin-overexpressing CHO cells. The top band corresponded to D1L3 expressed by CHO cells without overexpression of furin, whereas the lower band corresponded to the BD-deleted D1L3. The data suggest that furin may directly or indirectly delete portions of the BD and thus cause the maturation of D1L3 into its enzymatically active form.

[0131] The results disclosed here demonstrate that if D1L3 were expressed in a mammalian cell that provides functional expression of a PACE, such as furin, hyperactive D1L3 could be conveniently administered by cell therapy. Cell therapy is one manner to overcome the numerous challenges in producing D1L3 at large scale, as well as the short half-life of wild-type D1L3, which has a half-life in circulation of less than 30 minutes. Further, white blood cells that migrate to sites of inflammation, cell necrosis and apoptosis, and which can penetrate diseased tissues, could provide an elegant localized chromatinase therapy.

[0132] Mammalian T cells do not naturally express D1L3 (FIG. 16). However, T cells express several basic-specific Proprotein Convertase Subtilisin/Kexin (PCSK) enzymes, including PCSK types 3, 5, 6, and 7. In fact, activated T-cells specifically induce Furin (PCSK3) expression. See Pesu M, et al., T-cell-expressed proprotein convertase furin is essential for maintenance of peripheral immune tolerance, Nature 455, 246-250 (2008). Consequently, the expression of DNASE1L3, including wild-type D1L3 which has low activity) in T-cells will lead to the secretion of an enzymatically hyperactive DNASE1L3 (FIG. 17). This hyperactive variant may be produced at higher levels when the T cells are in an activated state. Further, because T cell therapy, including CAR-T cells, produce large amounts of extracellular chromatin during their action against diseased cells (e.g., cancer or virally-infected cells), D1L3 expression by effector T cells (either CD8+ or CD4+ T cells, or CAR-T cell) can avoid or lessen adverse effects of T cell therapy, such as Tumor Lysis Syndrome. Further still, the pathological presence of NETs in these inflammatory environments can be substantially reduced. Thus, T-cells that express and secret heterologous DNASE1L3 may be used for cell-based therapies in patients in need of extracellular chromatin and/or NET degradation, such as patients with cancer, tumor lysis syndrome, and patients with pneumonia, acute lung injury, or acute respiratory distress syndrome (including COVID-19).

Example 6: Single Amino Acid Deletion Generated D1L3 with Chromatin and DNA-Degrading Activity

[0133] We tested the activity of the BD-deleted D1L3 mutants to degrade protein-free DNA. In brief, the D1L3-variants were transiently expressed in CHO cells and culture supernatants were incubated with a commercially available DNA-probe, which becomes fluorescent upon cleavage by a DNASE, i.e. DNASEAlert. Surprisingly, we observed a robust increase in DNASE activate upon deletion of the C-terminal serine residue (e.g., S305 of SEQ ID NO:4) (FIG. 18). The deletion of a single amino acid--the C-terminal serine (S305)--generated a DNASE1L3 variant that has chromatinase and high DNASE activity (SEQ ID NO: 22).

TABLE-US-00001 Wild-Type Human DNASES DNASE1 (NP_005212.2): Signal Peptide, Mature Protein: SEQ ID NO: 1 MRGMKLLGALLALAALLQGAVSLKIAAFNIQTFGETKMSNATLVSYIVQILSRYDIALVQEVRDSHLTAVGKLL- DNLN QDAPDTYHYVVSEPLGRNSYKERYLFVYRPDQVSAVDSYYYDDGCEPCGNDTFNREPAIVRFFSRFTEVREFAI- VPLH AAPGDAVAEIDALYDVYLDVQEKWGLEDVMLMGDFNAGCSYVRPSQWSSIRLWTSPTFQWLIPDSADTTATPTH- CAYD RIVVAGMLLRGAVVPDSALPFNFQAAYGLSDQLAQAISDHYPVEVMLK DNASE1-LIKE1 (NP_006721.1): Signal Peptide; Mature Protein: SEQ ID NO: 2 MHYPTALLFLILANGAQAFRICAFNAQRLTLAKVAREQVMDTLVRILARCDIMVLQEVVDSSGSAIPLLLRELN- RFDG SGPYSTLSSPQLGRSTYMETYVYFYRSHKTQVLSSYVYNDEDDVFAREPFVAQFSLPSNVLPSLVLVPLHTTPK- AVEK ELNALYDVFLEVSQHWQSKDVILLGDFNADCASLTKKRLDKLELRTEPGFHWVIADGEDTTVRASTHCTYDRVV- LHGE RCRSLLHTAAAFDFPTSFQLTEEEALNISDHYPVEVELKLSQAHSVQPLSLTVLLLLSLLSPQLCPAA DNASE1-LIKE2 (NP_001365.1): Signal Peptide, Mature Protein: SEQ ID NO: 3 MGGPRALLAALWALEAAGTAALRIGAFNIQSFGDSKVSDPACGSIIAKILAGYDLALVQEVRDPDLSAVSALME- QINS VSEHEYSFVSSQPLGRDQYKEMYLFVYRKDAVSVVDTYLYPDPEDVFSREPFVVKFSAPGTGERAPPLPSRRAL- TPPP LPAAAQNLVLIPLHAAPHQAVAEIDALYDVYLDVIDKWGTDDMLFLGDFNADCSYVRAQDWAAIRLRSSEVFKW- LIPD SADTTVGNSDCAYDRIVACGARLRRSLKPQSATVHDFQEEFGLDQTQALAISDHFPVEVTLKFHR DNASE1-LIKE 3; Isoform 1 (NP_004935.1): Signal Peptide, Mature Protein: SEQ ID NO: 4 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS DNASE1-LIKE 3, Isoform 2 (NP_001243489.1): Signal Peptide; Mature Protein: SEQ ID NO: 5 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRE KLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWKAE- NFIF MGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQKA- YKLT EEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS DNASE2A (O00115): Signal Peptide; Mature Protein: SEQ ID NO: 6 MIPLLLAALLCVPAGALTCYGDSGQPVDWFVVYKLPALRGSGEAAQRGLQYKYLDESSGGWRDGRALINSPEGA- VGRS LQPLYRSNTSQLAFLLYNDQPPQPSKAQDSSMRGHTKGVLLLDHDGGFWLVHSVPNFPPPASSAAYSWPHSACT- YGQT LLCVSFPFAQFSKMGKQLTYTYPWVYNYQLEGIFAQEFPDLENVVKGHHVSQEPWNSSITLTSQAGAVFQSFAK- FSKF GDDLYSGWLAAALGTNLQVQFWHKTVGILPSNCSDIWQVLNVNQIAFPGPAGPSFNSTEDHSKWCVSPKGPWTC- VGDM NRNQGEEQRGGGTLCAQLPALWKAFQPLVKNYQPCNGMARKPSRAYKI DNASE2B (Q8WZ79): Signal Peptide; Mature Protein: SEQ ID NO: 7 MKQKMMARLLRTSFALLFLGLFGVLGAATISCRNEEGKAVDWFTFYKLPKRQNKESGETGLEYLYLDSTTRSWR- KSEQ LMNDTKSVLGRTLQQLYEAYASKSNNTAYLIYNDGVPKPVNYSRKYGHTKGLLLWNRVQGFWLIHSIPQFPPIP- EEGY DYPPTGRRNGQSGICITFKYNQYEAIDSQLLVCNPNVYSCSIPATFHQELIHMPQLCTRASSSEIPGRLLTTLQ- SAQG QKFLHFAKSDSFLDDIFAAWMAQRLKTHLLTETWQRKRQELPSNCSLPYHVYNIKAIKLSRHSYFSSYQDHAKW- CISQ KGTKNRWTCIGDLNRSPHQAFRSGGFICTQNWQIYQAFQGLVLYYESCK Chimeras of Human DNASE1L3 and DNASE1 D1L3 + D1-BB#50: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 8 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTATPTNC- AYDR IVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#51: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 9 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTH- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#52: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 10 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVVAGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#53: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 11 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGMLLRGAVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#54: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 12 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPDSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#55: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 13 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNALPDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#56: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 14 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFNFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#57: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 15 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQAAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#58: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 16 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYGLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#59: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 17 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLSDQLALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS

D1L3 + D1-BB#60: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 18 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEAQAISDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#61: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 19 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHYPVEFKLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#62: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 20 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEVMLQSSRAFTNSKKSVTLRKKTKSKRS D1L3 + D1-BB#63: Signal Peptide; Mature Protein (Building block transferred from D1): SEQ ID NO: 21 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLK C-terminal deletion mutants of Human DNASE1L3 S305del: Signal Peptide; Mature Protein: SEQ ID NO: 22 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKSKR K303_S305del: Signal Peptide; Mature Protein: SEQ ID NO: 23 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKSVTLRKKTKS V294_S305del: Signal Peptide; Mature Protein: SEQ ID NO: 24 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNSKKS K291_S305del: Signal Peptide; Mature Protein: SEQ ID NO: 25 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSSRAFTNS R285_S305del: Signal Peptide; Mature Protein: SEQ ID NO: 26 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQSS S283_S305del: Signal Peptide; Mature Protein: SEQ ID NO: 27 MSRELAPLLLLLLSIHSALAMRICSFNVRSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEK- LNRN SRRGITYNYVISSRLGRNTYKEQYAFLYKEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVII- PLHT TPETSVKEIDELVEVYTDVKHRWKAENFIFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTN- CAYD RIVLRGQEIVSSVVPKSNSVFDFQKAYKLTEEEALDVSDHFPVEFKLQ SIGNAL PEPTIDES Alpha mating factor (P01149): SEQ ID NO: 28 MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIA- AKEE GVS Human Albumin Secretory Signal Peptide + Propeptide (P02768): SEQ ID NO: 29 MKWVTFISLLFLFSSAYSRGVFRR Human DNASE1L3 Signal Peptide (Q13609): SEQ ID NO: 30 MSRELAPLLLLLLSIHSALA LINKER SEQUENCES SEQ ID NO: 31 GGGGS SEQ ID NO: 32 GGGGSGGGGSGGGGS SEQ ID NO: 33 APAPAPAPAPAPAP SEQ ID NO: 34 AEAAAKEAAAKA SEQ ID NO: 35 SGGSGSS SEQ ID NO: 36 SGGSGGSGGSGGSGSS SEQ ID NO: 37 SGGSGGSGGSGGSGGSGGSGGSGGSGGSGSS SEQ ID NO: 38 GGSGGSGGSGGSGGSGGSGGSGGSGGSGS OTHER SEQUENCES Human Serum Albumin (Mature Protein): SEQ ID NO: 39 DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKL- CTVA TLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAP- ELLF FAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAE- VSKL VTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAAD- FVES KDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN- LIKQ NCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLH- EKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE- QLKA VMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL Human Factor XI: SEQ ID NO: 40 MIFLYQVVHFILFTSVSGECVTQLLKDTCFEGGDITTVFTPSAKYCQVVCTYHPRCLLFTFTAESPSEDPTRWF- TCVL KDSVTETLPRVNRTAAISGYSFKQCSHQISACNKDIYVDLDMKGINYNSSVAKSAQECQERCTDDVHCHFFTYA- TRQF PSLEHRNICLLKHTQTGTPTRITKLDKVVSGFSLKSCALSNLACIRDIFPNTVFADSNIDSVMAPDAFVCGRIC- THHP GCLFFTFFSQEWPKESQRNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRHSIPVFCHSSFYHDTDFLGEELDI- VAAK SHEACQKLCTNAVRCQFFTYTPAQASCNEGKGKCYLKLSSNGSPTKILHGRGGISGYTLRLCKMDNECTTKIKP- RIVG GTASVRGEWPWQVTLHITSPTQRHLCGGSIIGNQWILTAAHCFYGVESPKILRVYSGILNQSEIKEDTSFFGVQ- EIII HDQYKMAESGYDIALLKLETTVNYTDSQRPICLPSKGDRNVIYTDCWVTGWGYRKLRDKIQNTLQKAKIPLVTN- EECQ KRYRGHKITHKMICAGYREGGKDACKGDSGGPLSCKHNEVWHLVGITSWGEGCAQRERPGVYTNVVEYVDWILE- KTQA V Human prekallikrein: SEQ ID NO: 41 MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTFHPRCLLFSFLPASSINDMEKR- FGCF LKDSVTGILPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKVSSVEECQKRCTNNIRCQFFSY- ATQT FHKAEYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSDVDVARVLTPDAFVCRTI- CTYH PNCLFFTFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYSLLICKRTLPEPCHSKIYPGVDFGGEELN- VTFV KGVNVCQETCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLRLCNTGDNSVCTIK- TSTR

IVGGINSSWGEWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGILNLSDITKDTPFS- QIKE IIIHQNYKVSEGNHDIALIKLQAPLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNILQKVNIPL- VTNE ECQKRYQDYKITQRMVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPGVYTKVAEYMDW- ILEK TQSSDGKAQMQSPA DNase epitope SEQ ID NO: 42 MGDFNAGCSYV ACTIVATABLE LINKER SEQUENCES FXIIa-susceptible linker (Factor XI peptide): SEQ ID NO: 43 CTTKIKPRIVGGTASVRGEWPWQVT FXIIa-susceptible linker SEQ ID NO: 44 GGGGSPRIGGGGS FXIIa-susceptible linker (Prekallikrein peptide): SEQ ID NO: 45 VCTIKTSTRIVGGINSSWGEWPWQVS FXIIa-susceptible linker (Prekallikrein peptide): SEQ ID NO: 46 STRIVGG FUSIONS BETWEEN ALBUMIN AND BD-DELETED D1L3 SEQ ID NO: 47 Albumin-DNASE1L3 Variant-Fusion Protein. (Albumin, DNASE1L3 Variant): DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKL- CTVA TLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAP- ELLF FAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAE- VSKL VTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAAD- FVES KDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN- LIKQ NCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLH- EKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKE- QLKA VMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLSGGSGGSGGSGGSGGSGGSGGSGGSGGSGSSMRIC- SFNV RSFGESKQEDKNAMDVIVKVIKRCDIILVMEIKDSNNRICPILMEKLNRNSRRGITYNYVISSRLGRNTYKEQY- AFLY KEKLVSVKRSYHYHDYQDGDADVFSREPFVVWFQSPHTAVKDFVIIPLHTTPETSVKEIDELVEVYTDVKHRWK- AENF IFMGDFNAGCSYVPKKAWKNIRLRTDPRFVWLIGDQEDTTVKKSTNCAYDRIVLRGQEIVSSVVPKSNSVFDFQ- KAYK LTEEEALDVSDHFPVEFKLQ

Sequence CWU 1

1

471282PRTHomo sapiens 1Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu1 5 10 15Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp65 70 75 80Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 140Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly145 150 155 160Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly225 230 235 240Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270Asp His Tyr Pro Val Glu Val Met Leu Lys 275 2802302PRTHomo sapiens 2Met His Tyr Pro Thr Ala Leu Leu Phe Leu Ile Leu Ala Asn Gly Ala1 5 10 15Gln Ala Phe Arg Ile Cys Ala Phe Asn Ala Gln Arg Leu Thr Leu Ala 20 25 30Lys Val Ala Arg Glu Gln Val Met Asp Thr Leu Val Arg Ile Leu Ala 35 40 45Arg Cys Asp Ile Met Val Leu Gln Glu Val Val Asp Ser Ser Gly Ser 50 55 60Ala Ile Pro Leu Leu Leu Arg Glu Leu Asn Arg Phe Asp Gly Ser Gly65 70 75 80Pro Tyr Ser Thr Leu Ser Ser Pro Gln Leu Gly Arg Ser Thr Tyr Met 85 90 95Glu Thr Tyr Val Tyr Phe Tyr Arg Ser His Lys Thr Gln Val Leu Ser 100 105 110Ser Tyr Val Tyr Asn Asp Glu Asp Asp Val Phe Ala Arg Glu Pro Phe 115 120 125Val Ala Gln Phe Ser Leu Pro Ser Asn Val Leu Pro Ser Leu Val Leu 130 135 140Val Pro Leu His Thr Thr Pro Lys Ala Val Glu Lys Glu Leu Asn Ala145 150 155 160Leu Tyr Asp Val Phe Leu Glu Val Ser Gln His Trp Gln Ser Lys Asp 165 170 175Val Ile Leu Leu Gly Asp Phe Asn Ala Asp Cys Ala Ser Leu Thr Lys 180 185 190Lys Arg Leu Asp Lys Leu Glu Leu Arg Thr Glu Pro Gly Phe His Trp 195 200 205Val Ile Ala Asp Gly Glu Asp Thr Thr Val Arg Ala Ser Thr His Cys 210 215 220Thr Tyr Asp Arg Val Val Leu His Gly Glu Arg Cys Arg Ser Leu Leu225 230 235 240His Thr Ala Ala Ala Phe Asp Phe Pro Thr Ser Phe Gln Leu Thr Glu 245 250 255Glu Glu Ala Leu Asn Ile Ser Asp His Tyr Pro Val Glu Val Glu Leu 260 265 270Lys Leu Ser Gln Ala His Ser Val Gln Pro Leu Ser Leu Thr Val Leu 275 280 285Leu Leu Leu Ser Leu Leu Ser Pro Gln Leu Cys Pro Ala Ala 290 295 3003299PRTHomo sapiens 3Met Gly Gly Pro Arg Ala Leu Leu Ala Ala Leu Trp Ala Leu Glu Ala1 5 10 15Ala Gly Thr Ala Ala Leu Arg Ile Gly Ala Phe Asn Ile Gln Ser Phe 20 25 30Gly Asp Ser Lys Val Ser Asp Pro Ala Cys Gly Ser Ile Ile Ala Lys 35 40 45Ile Leu Ala Gly Tyr Asp Leu Ala Leu Val Gln Glu Val Arg Asp Pro 50 55 60Asp Leu Ser Ala Val Ser Ala Leu Met Glu Gln Ile Asn Ser Val Ser65 70 75 80Glu His Glu Tyr Ser Phe Val Ser Ser Gln Pro Leu Gly Arg Asp Gln 85 90 95Tyr Lys Glu Met Tyr Leu Phe Val Tyr Arg Lys Asp Ala Val Ser Val 100 105 110Val Asp Thr Tyr Leu Tyr Pro Asp Pro Glu Asp Val Phe Ser Arg Glu 115 120 125Pro Phe Val Val Lys Phe Ser Ala Pro Gly Thr Gly Glu Arg Ala Pro 130 135 140Pro Leu Pro Ser Arg Arg Ala Leu Thr Pro Pro Pro Leu Pro Ala Ala145 150 155 160Ala Gln Asn Leu Val Leu Ile Pro Leu His Ala Ala Pro His Gln Ala 165 170 175Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Ile Asp 180 185 190Lys Trp Gly Thr Asp Asp Met Leu Phe Leu Gly Asp Phe Asn Ala Asp 195 200 205Cys Ser Tyr Val Arg Ala Gln Asp Trp Ala Ala Ile Arg Leu Arg Ser 210 215 220Ser Glu Val Phe Lys Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Val225 230 235 240Gly Asn Ser Asp Cys Ala Tyr Asp Arg Ile Val Ala Cys Gly Ala Arg 245 250 255Leu Arg Arg Ser Leu Lys Pro Gln Ser Ala Thr Val His Asp Phe Gln 260 265 270Glu Glu Phe Gly Leu Asp Gln Thr Gln Ala Leu Ala Ile Ser Asp His 275 280 285Phe Pro Val Glu Val Thr Leu Lys Phe His Arg 290 2954305PRTHomo sapiens 4Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser3055275PRTHomo sapiens 5Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Glu Lys Leu65 70 75 80Val Ser Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp 85 90 95Ala Asp Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro 100 105 110His Thr Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro 115 120 125Glu Thr Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp 130 135 140Val Lys His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe145 150 155 160Asn Ala Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg 165 170 175Leu Arg Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp 180 185 190Thr Thr Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu 195 200 205Arg Gly Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val 210 215 220Phe Asp Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp225 230 235 240Val Ser Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala 245 250 255Phe Thr Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser 260 265 270Lys Arg Ser 2756360PRTHomo sapiens 6Met Ile Pro Leu Leu Leu Ala Ala Leu Leu Cys Val Pro Ala Gly Ala1 5 10 15Leu Thr Cys Tyr Gly Asp Ser Gly Gln Pro Val Asp Trp Phe Val Val 20 25 30Tyr Lys Leu Pro Ala Leu Arg Gly Ser Gly Glu Ala Ala Gln Arg Gly 35 40 45Leu Gln Tyr Lys Tyr Leu Asp Glu Ser Ser Gly Gly Trp Arg Asp Gly 50 55 60Arg Ala Leu Ile Asn Ser Pro Glu Gly Ala Val Gly Arg Ser Leu Gln65 70 75 80Pro Leu Tyr Arg Ser Asn Thr Ser Gln Leu Ala Phe Leu Leu Tyr Asn 85 90 95Asp Gln Pro Pro Gln Pro Ser Lys Ala Gln Asp Ser Ser Met Arg Gly 100 105 110His Thr Lys Gly Val Leu Leu Leu Asp His Asp Gly Gly Phe Trp Leu 115 120 125Val His Ser Val Pro Asn Phe Pro Pro Pro Ala Ser Ser Ala Ala Tyr 130 135 140Ser Trp Pro His Ser Ala Cys Thr Tyr Gly Gln Thr Leu Leu Cys Val145 150 155 160Ser Phe Pro Phe Ala Gln Phe Ser Lys Met Gly Lys Gln Leu Thr Tyr 165 170 175Thr Tyr Pro Trp Val Tyr Asn Tyr Gln Leu Glu Gly Ile Phe Ala Gln 180 185 190Glu Phe Pro Asp Leu Glu Asn Val Val Lys Gly His His Val Ser Gln 195 200 205Glu Pro Trp Asn Ser Ser Ile Thr Leu Thr Ser Gln Ala Gly Ala Val 210 215 220Phe Gln Ser Phe Ala Lys Phe Ser Lys Phe Gly Asp Asp Leu Tyr Ser225 230 235 240Gly Trp Leu Ala Ala Ala Leu Gly Thr Asn Leu Gln Val Gln Phe Trp 245 250 255His Lys Thr Val Gly Ile Leu Pro Ser Asn Cys Ser Asp Ile Trp Gln 260 265 270Val Leu Asn Val Asn Gln Ile Ala Phe Pro Gly Pro Ala Gly Pro Ser 275 280 285Phe Asn Ser Thr Glu Asp His Ser Lys Trp Cys Val Ser Pro Lys Gly 290 295 300Pro Trp Thr Cys Val Gly Asp Met Asn Arg Asn Gln Gly Glu Glu Gln305 310 315 320Arg Gly Gly Gly Thr Leu Cys Ala Gln Leu Pro Ala Leu Trp Lys Ala 325 330 335Phe Gln Pro Leu Val Lys Asn Tyr Gln Pro Cys Asn Gly Met Ala Arg 340 345 350Lys Pro Ser Arg Ala Tyr Lys Ile 355 3607361PRTHomo sapiens 7Met Lys Gln Lys Met Met Ala Arg Leu Leu Arg Thr Ser Phe Ala Leu1 5 10 15Leu Phe Leu Gly Leu Phe Gly Val Leu Gly Ala Ala Thr Ile Ser Cys 20 25 30Arg Asn Glu Glu Gly Lys Ala Val Asp Trp Phe Thr Phe Tyr Lys Leu 35 40 45Pro Lys Arg Gln Asn Lys Glu Ser Gly Glu Thr Gly Leu Glu Tyr Leu 50 55 60Tyr Leu Asp Ser Thr Thr Arg Ser Trp Arg Lys Ser Glu Gln Leu Met65 70 75 80Asn Asp Thr Lys Ser Val Leu Gly Arg Thr Leu Gln Gln Leu Tyr Glu 85 90 95Ala Tyr Ala Ser Lys Ser Asn Asn Thr Ala Tyr Leu Ile Tyr Asn Asp 100 105 110Gly Val Pro Lys Pro Val Asn Tyr Ser Arg Lys Tyr Gly His Thr Lys 115 120 125Gly Leu Leu Leu Trp Asn Arg Val Gln Gly Phe Trp Leu Ile His Ser 130 135 140Ile Pro Gln Phe Pro Pro Ile Pro Glu Glu Gly Tyr Asp Tyr Pro Pro145 150 155 160Thr Gly Arg Arg Asn Gly Gln Ser Gly Ile Cys Ile Thr Phe Lys Tyr 165 170 175Asn Gln Tyr Glu Ala Ile Asp Ser Gln Leu Leu Val Cys Asn Pro Asn 180 185 190Val Tyr Ser Cys Ser Ile Pro Ala Thr Phe His Gln Glu Leu Ile His 195 200 205Met Pro Gln Leu Cys Thr Arg Ala Ser Ser Ser Glu Ile Pro Gly Arg 210 215 220Leu Leu Thr Thr Leu Gln Ser Ala Gln Gly Gln Lys Phe Leu His Phe225 230 235 240Ala Lys Ser Asp Ser Phe Leu Asp Asp Ile Phe Ala Ala Trp Met Ala 245 250 255Gln Arg Leu Lys Thr His Leu Leu Thr Glu Thr Trp Gln Arg Lys Arg 260 265 270Gln Glu Leu Pro Ser Asn Cys Ser Leu Pro Tyr His Val Tyr Asn Ile 275 280 285Lys Ala Ile Lys Leu Ser Arg His Ser Tyr Phe Ser Ser Tyr Gln Asp 290 295 300His Ala Lys Trp Cys Ile Ser Gln Lys Gly Thr Lys Asn Arg Trp Thr305 310 315 320Cys Ile Gly Asp Leu Asn Arg Ser Pro His Gln Ala Phe Arg Ser Gly 325 330 335Gly Phe Ile Cys Thr Gln Asn Trp Gln Ile Tyr Gln Ala Phe Gln Gly 340 345 350Leu Val Leu Tyr Tyr Glu Ser Cys Lys 355 3608304PRTArtificial SequenceSynthetic Sequence 8Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Ala Thr Pro Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly Gln225 230 235 240Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp Phe 245 250 255Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser Asp 260

265 270His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr Asn 275 280 285Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg Ser 290 295 3009305PRTArtificial SequenceSynthetic Sequence 9Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr His Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30510305PRTArtificial SequenceSynthetic Sequence 10Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Val Ala Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30511305PRTArtificial SequenceSynthetic Sequence 11Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Met Leu Leu Arg Gly Ala Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30512305PRTArtificial SequenceSynthetic Sequence 12Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Asp Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30513305PRTArtificial SequenceSynthetic Sequence 13Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ala Leu Pro Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30514305PRTArtificial SequenceSynthetic Sequence 14Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asn 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30515305PRTArtificial SequenceSynthetic Sequence 15Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Ala Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30516305PRTArtificial SequenceSynthetic Sequence 16Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Gly Leu Thr Glu Glu Glu Ala Leu Asp Val Ser

260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30517305PRTArtificial SequenceSynthetic Sequence 17Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Ser Asp Gln Leu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30518305PRTArtificial SequenceSynthetic Sequence 18Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Gln Ala Ile Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30519305PRTArtificial SequenceSynthetic Sequence 19Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Tyr Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30520305PRTArtificial SequenceSynthetic Sequence 20Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Val Met Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300Ser30521282PRTArtificial SequenceSynthetic Sequence 21Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Lys 275 28022304PRTHomo sapiens 22Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 30023302PRTHomo sapiens 23Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser 290 295 30024293PRTHomo sapiens 24Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser Lys Lys Ser

29025290PRTHomo sapiens 25Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285Asn Ser 29026284PRTHomo sapiens 26Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser 275 28027282PRTHomo sapiens 27Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg65 70 75 80Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr145 150 155 160Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly225 230 235 240Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270Asp His Phe Pro Val Glu Phe Lys Leu Gln 275 2802881PRTArtificial SequenceSynthetic Sequence 28Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser1 5 10 15Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40 45Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val65 70 75 80Ser2924PRTHomo sapiens 29Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Arg Gly Val Phe Arg Arg 203020PRTHomo sapiens 30Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His1 5 10 15Ser Ala Leu Ala 20315PRTArtificial SequenceSynthetic Sequence 31Gly Gly Gly Gly Ser1 53215PRTArtificial SequenceSynthetic Sequence 32Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 153314PRTArtificial SequenceSynthetic Sequence 33Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro1 5 103412PRTArtificial SequenceSynthetic Sequence 34Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala1 5 10357PRTArtificial SequenceSynthetic Sequence 35Ser Gly Gly Ser Gly Ser Ser1 53616PRTArtificial SequenceSynthetic Sequence 36Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser Ser1 5 10 153731PRTArtificial SequenceSynthetic Sequence 37Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser1 5 10 15Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser Ser 20 25 303829PRTArtificial SequenceSynthetic Sequence 38Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly1 5 10 15Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser 20 2539585PRTHomo sapiens 39Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 58540625PRTHomo sapiens 40Met Ile Phe Leu Tyr Gln Val Val His Phe Ile Leu Phe Thr Ser Val1 5 10 15Ser Gly Glu Cys Val Thr Gln Leu Leu Lys Asp Thr Cys Phe Glu Gly 20 25 30Gly Asp Ile Thr Thr Val Phe Thr Pro Ser Ala Lys Tyr Cys Gln Val 35 40 45Val Cys Thr Tyr His Pro Arg Cys Leu Leu Phe Thr Phe Thr Ala Glu 50 55 60Ser Pro Ser Glu Asp Pro Thr Arg Trp Phe Thr Cys Val Leu Lys Asp65 70 75 80Ser Val Thr Glu Thr Leu Pro Arg Val Asn Arg Thr Ala Ala Ile Ser 85 90 95Gly Tyr Ser Phe Lys Gln Cys Ser His Gln Ile Ser Ala Cys Asn Lys 100 105 110Asp Ile Tyr Val Asp Leu Asp Met Lys Gly Ile Asn Tyr Asn Ser Ser 115 120 125Val Ala Lys Ser Ala Gln Glu Cys Gln Glu Arg Cys Thr Asp Asp Val 130 135 140His Cys His Phe Phe Thr Tyr Ala Thr Arg Gln Phe Pro Ser Leu Glu145 150 155 160His Arg Asn Ile Cys Leu Leu Lys His Thr Gln Thr Gly Thr Pro Thr 165 170 175Arg Ile Thr Lys Leu Asp Lys Val Val Ser Gly Phe Ser Leu Lys Ser 180 185 190Cys Ala Leu Ser Asn Leu Ala Cys Ile Arg Asp Ile Phe Pro Asn Thr 195 200 205Val Phe Ala Asp Ser Asn Ile Asp Ser Val Met Ala Pro Asp Ala Phe 210 215 220Val Cys Gly Arg Ile Cys Thr His His Pro Gly Cys Leu Phe Phe Thr225 230 235 240Phe Phe Ser Gln Glu Trp Pro Lys Glu Ser Gln Arg Asn Leu Cys Leu 245 250 255Leu Lys Thr Ser Glu Ser Gly Leu Pro Ser Thr Arg Ile Lys Lys Ser 260 265 270Lys Ala Leu Ser Gly Phe Ser Leu Gln Ser Cys Arg His Ser Ile Pro 275 280 285Val Phe Cys His Ser Ser Phe Tyr His Asp Thr Asp Phe Leu Gly Glu 290 295 300Glu Leu Asp Ile Val Ala Ala Lys Ser His Glu Ala Cys Gln Lys Leu305 310 315 320Cys Thr Asn Ala Val Arg Cys Gln Phe Phe Thr Tyr Thr Pro Ala Gln 325 330 335Ala Ser Cys Asn Glu Gly Lys Gly Lys Cys Tyr Leu Lys Leu Ser Ser 340 345 350Asn Gly Ser Pro Thr Lys Ile Leu His Gly Arg Gly Gly Ile Ser Gly 355 360 365Tyr Thr Leu Arg Leu Cys Lys Met Asp Asn Glu Cys Thr Thr Lys Ile 370 375 380Lys Pro Arg Ile Val Gly Gly Thr Ala Ser Val Arg Gly Glu Trp Pro385 390 395 400Trp Gln Val Thr Leu His Thr Thr Ser Pro Thr Gln Arg His Leu Cys 405 410 415Gly Gly Ser Ile Ile Gly Asn Gln Trp Ile Leu Thr Ala Ala His Cys 420 425 430Phe Tyr Gly Val Glu Ser Pro Lys Ile Leu Arg Val Tyr Ser Gly Ile 435 440 445Leu Asn Gln Ser Glu Ile Lys Glu Asp Thr Ser Phe Phe Gly Val Gln 450 455 460Glu Ile Ile Ile His Asp Gln Tyr Lys Met Ala Glu Ser Gly Tyr Asp465 470 475 480Ile Ala Leu Leu Lys Leu Glu Thr Thr Val Asn Tyr Thr Asp Ser Gln 485 490 495Arg Pro Ile Cys Leu Pro Ser Lys Gly Asp Arg Asn Val Ile Tyr Thr 500 505 510Asp Cys Trp Val Thr Gly Trp Gly Tyr Arg Lys Leu Arg Asp Lys Ile 515 520 525Gln Asn Thr Leu Gln Lys Ala Lys Ile Pro Leu Val Thr Asn Glu Glu 530 535 540Cys Gln Lys Arg Tyr Arg Gly His Lys Ile Thr His Lys Met Ile Cys545 550 555 560Ala Gly Tyr Arg Glu Gly Gly Lys Asp Ala Cys Lys Gly Asp Ser Gly 565 570 575Gly Pro Leu Ser Cys Lys His Asn Glu Val Trp His Leu Val Gly Ile 580 585 590Thr Ser Trp Gly Glu Gly Cys Ala Gln Arg Glu Arg Pro Gly Val Tyr 595 600 605Thr Asn Val Val Glu Tyr Val Asp Trp Ile Leu Glu Lys Thr Gln Ala 610 615 620Val62541638PRTHomo sapiens 41Met Ile Leu Phe Lys Gln Ala Thr Tyr Phe Ile Ser Leu Phe Ala Thr1 5 10 15Val Ser Cys Gly Cys Leu Thr Gln Leu Tyr Glu Asn Ala Phe Phe Arg 20 25 30Gly Gly Asp Val Ala Ser Met Tyr Thr Pro Asn Ala Gln Tyr Cys Gln 35 40 45Met Arg Cys Thr Phe His Pro Arg Cys Leu Leu Phe Ser Phe Leu Pro 50 55 60Ala Ser Ser Ile Asn Asp Met Glu Lys Arg Phe Gly Cys Phe Leu Lys65 70 75 80Asp Ser Val Thr Gly

Thr Leu Pro Lys Val His Arg Thr Gly Ala Val 85 90 95Ser Gly His Ser Leu Lys Gln Cys Gly His Gln Ile Ser Ala Cys His 100 105 110Arg Asp Ile Tyr Lys Gly Val Asp Met Arg Gly Val Asn Phe Asn Val 115 120 125Ser Lys Val Ser Ser Val Glu Glu Cys Gln Lys Arg Cys Thr Asn Asn 130 135 140Ile Arg Cys Gln Phe Phe Ser Tyr Ala Thr Gln Thr Phe His Lys Ala145 150 155 160Glu Tyr Arg Asn Asn Cys Leu Leu Lys Tyr Ser Pro Gly Gly Thr Pro 165 170 175Thr Ala Ile Lys Val Leu Ser Asn Val Glu Ser Gly Phe Ser Leu Lys 180 185 190Pro Cys Ala Leu Ser Glu Ile Gly Cys His Met Asn Ile Phe Gln His 195 200 205Leu Ala Phe Ser Asp Val Asp Val Ala Arg Val Leu Thr Pro Asp Ala 210 215 220Phe Val Cys Arg Thr Ile Cys Thr Tyr His Pro Asn Cys Leu Phe Phe225 230 235 240Thr Phe Tyr Thr Asn Val Trp Lys Ile Glu Ser Gln Arg Asn Val Cys 245 250 255Leu Leu Lys Thr Ser Glu Ser Gly Thr Pro Ser Ser Ser Thr Pro Gln 260 265 270Glu Asn Thr Ile Ser Gly Tyr Ser Leu Leu Thr Cys Lys Arg Thr Leu 275 280 285Pro Glu Pro Cys His Ser Lys Ile Tyr Pro Gly Val Asp Phe Gly Gly 290 295 300Glu Glu Leu Asn Val Thr Phe Val Lys Gly Val Asn Val Cys Gln Glu305 310 315 320Thr Cys Thr Lys Met Ile Arg Cys Gln Phe Phe Thr Tyr Ser Leu Leu 325 330 335Pro Glu Asp Cys Lys Glu Glu Lys Cys Lys Cys Phe Leu Arg Leu Ser 340 345 350Met Asp Gly Ser Pro Thr Arg Ile Ala Tyr Gly Thr Gln Gly Ser Ser 355 360 365Gly Tyr Ser Leu Arg Leu Cys Asn Thr Gly Asp Asn Ser Val Cys Thr 370 375 380Thr Lys Thr Ser Thr Arg Ile Val Gly Gly Thr Asn Ser Ser Trp Gly385 390 395 400Glu Trp Pro Trp Gln Val Ser Leu Gln Val Lys Leu Thr Ala Gln Arg 405 410 415His Leu Cys Gly Gly Ser Leu Ile Gly His Gln Trp Val Leu Thr Ala 420 425 430Ala His Cys Phe Asp Gly Leu Pro Leu Gln Asp Val Trp Arg Ile Tyr 435 440 445Ser Gly Ile Leu Asn Leu Ser Asp Ile Thr Lys Asp Thr Pro Phe Ser 450 455 460Gln Ile Lys Glu Ile Ile Ile His Gln Asn Tyr Lys Val Ser Glu Gly465 470 475 480Asn His Asp Ile Ala Leu Ile Lys Leu Gln Ala Pro Leu Asn Tyr Thr 485 490 495Glu Phe Gln Lys Pro Ile Cys Leu Pro Ser Lys Gly Asp Thr Ser Thr 500 505 510Ile Tyr Thr Asn Cys Trp Val Thr Gly Trp Gly Phe Ser Lys Glu Lys 515 520 525Gly Glu Ile Gln Asn Ile Leu Gln Lys Val Asn Ile Pro Leu Val Thr 530 535 540Asn Glu Glu Cys Gln Lys Arg Tyr Gln Asp Tyr Lys Ile Thr Gln Arg545 550 555 560Met Val Cys Ala Gly Tyr Lys Glu Gly Gly Lys Asp Ala Cys Lys Gly 565 570 575Asp Ser Gly Gly Pro Leu Val Cys Lys His Asn Gly Met Trp Arg Leu 580 585 590Val Gly Ile Thr Ser Trp Gly Glu Gly Cys Ala Arg Arg Glu Gln Pro 595 600 605Gly Val Tyr Thr Lys Val Ala Glu Tyr Met Asp Trp Ile Leu Glu Lys 610 615 620Thr Gln Ser Ser Asp Gly Lys Ala Gln Met Gln Ser Pro Ala625 630 6354211PRTArtificial SequenceSynthetic Sequence 42Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val1 5 104325PRTArtificial SequenceSynthetic Sequence 43Cys Thr Thr Lys Ile Lys Pro Arg Ile Val Gly Gly Thr Ala Ser Val1 5 10 15Arg Gly Glu Trp Pro Trp Gln Val Thr 20 254413PRTArtificial SequenceSynthetic Sequence 44Gly Gly Gly Gly Ser Pro Arg Ile Gly Gly Gly Gly Ser1 5 104526PRTArtificial SequenceSynthetic Sequence 45Val Cys Thr Thr Lys Thr Ser Thr Arg Ile Val Gly Gly Thr Asn Ser1 5 10 15Ser Trp Gly Glu Trp Pro Trp Gln Val Ser 20 25467PRTArtificial SequenceSynthetic Sequence 46Ser Thr Arg Ile Val Gly Gly1 547878PRTArtificial SequenceSynthetic Sequence 47Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465 470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550 555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala Ser Gln Ala Ala Leu Gly Leu Ser Gly Gly Ser Gly Gly Ser 580 585 590Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 595 600 605Gly Ser Gly Gly Ser Gly Ser Ser Met Arg Ile Cys Ser Phe Asn Val 610 615 620Arg Ser Phe Gly Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val625 630 635 640Ile Val Lys Val Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile 645 650 655Lys Asp Ser Asn Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn 660 665 670Arg Asn Ser Arg Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg 675 680 685Leu Gly Arg Asn Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu 690 695 700Lys Leu Val Ser Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp705 710 715 720Gly Asp Ala Asp Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln 725 730 735Ser Pro His Thr Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr 740 745 750Thr Pro Glu Thr Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr 755 760 765Thr Asp Val Lys His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly 770 775 780Asp Phe Asn Ala Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn785 790 795 800Ile Arg Leu Arg Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln 805 810 815Glu Asp Thr Thr Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile 820 825 830Val Leu Arg Gly Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn 835 840 845Ser Val Phe Asp Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala 850 855 860Leu Asp Val Ser Asp His Phe Pro Val Glu Phe Lys Leu Gln865 870 875

Claims

1. An isolated human host cell comprising a heterologous polynucleotide encoding a chromatinase enzyme operably linked to a promoter, wherein the host cell expresses and secretes the chromatinase enzyme.

2. The isolated host cell of claim 1, wherein the chromatinase is DNASE1-LIKE 3 (D1L3).

3. The isolated host cell of claim 2, wherein the secreted D1L3 enzyme has at least a partial deletion of the C-terminal basic domain.

4. The isolated host cell of claim 3, wherein the secreted D1L3 enzyme has a deletion of at least 12 amino acids of the C-terminal basic domain.

5. The isolated host cell of claim 4, wherein the secreted D1L3 enzyme includes enzymes having deletions of one or more of: K291_S305 del, K292_S305 del, K293_S305 del, with respect to SEQ ID NO:4.

6. The isolated host cell of any one of claims 1 to 5, wherein the host cell is a white blood cell, an endothelial cell, an epithelial cell, a hepatocyte, or a stem cell.

7. The isolated host cell of claim 6, wherein the host cell is a white blood cell.

8. The isolated host cell of claim 7, wherein the host cell is a macrophage.

9. The isolated host cell of claim 7, wherein the host cell is a T cell.

10. The isolated host cell of claim 9, wherein the T cell is CD8+ or CD4+.

11. The isolated host cell of claim 9, wherein the host cell is a Chimeric Antigen Receptor (CAR) T cell.

12. The isolated host cell of any one of claims 9 to 11, wherein D1L3 C-terminal basic domain processing is induced when the T cell is activated.

13. The isolated host cell of any one of claims 9 to 12, wherein the T cell recognizes a cancer-associated antigen.

14. The isolated host cell of claim 13, wherein the T cell recognizes a leukemia antigen.

15. The isolated host cell of any one of claims 9 to 12, wherein the T cell recognizes a viral antigen.

16. The isolated host cell of claim 15, wherein the T cell recognizes a coronavirus antigen.

17. The isolated host cell of claim 16, wherein the coronavirus is SARS-CoV-2.

18. The isolated host cell of any one of claims 1 to 17, wherein the heterologous polynucleotide is an mRNA or a modified mRNA (mmRNA).

19. The isolated host cell of any one of claims 1 to 17, wherein the heterologous polynucleotide is DNA.

20. The isolated host cell of any one of claims 1 to 19, wherein the D1L3 enzyme comprises an amino acid sequence that has at least 70% sequence identity to the enzyme of SEQ ID NO:4 or SEQ ID NO:5 lacking the C-terminal Basic Domain (BD).

21. The isolated host cell of claim 10, wherein the amino acid sequence has at least 80% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD.

22. The isolated host cell of claim 21, wherein the amino acid sequence has at least 85% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD.

23. The isolated host cell of claim 21, wherein the amino acid sequence has at least 90% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD.

24. The isolated host cell of claim 21, wherein the amino acid sequence has at least 95% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the BD.

25. The isolated host cell of claim 21, wherein the amino acid sequence is the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:5.

26. The isolated host cell of any one of claims 1 to 25, wherein the polynucleotide encodes a D1L3 enzyme having a deletion of one or more amino acids of the C-terminal basic domain, and optionally at least five amino acids of the C-terminal basic domain.

27. The isolated host cell of any one of claims 1 to 26, wherein the polynucleotide encodes a D1L3 enzyme having an inactivation of one or more of the NLS1 and the NLS2.

28. The isolated host cell of claim 27, wherein the polynucleotide encodes a D1L3 enzyme having an inactivation of the NLS1.

29. The isolated host cell of claim 27, wherein the polynucleotide encodes a D1L3 enzyme having an inactivation of NLS2.

30. The isolated host cell of claim 27, wherein the polynucleotide encodes a D1L3 having an inactivation of both NLS1 and NLS2.

31. The isolated host cell of any one of claims 26 to 30, wherein the D1L3 enzyme includes one or more amino acid substitutions and/or deletions within the NLS1 and/or NLS2.

32. The isolated host cell of any one of claims 1 to 31, wherein the D1L3 enzyme is fused to a carrier protein.

33. The isolated host cell of claim 32, wherein the carrier protein is albumin, which is optionally linked at the N-terminus of the D1L3 protein through a flexible or cleavable linker.

34. A method for preparing the host cell of any one of claims 1 to 33, comprising introducing the polynucleotide encoding the chromatinase to a host cell in vitro.

35. The method of claim 34, wherein the polynucleotide is introduced to the cell using a viral vector.

36. The method of claim 34, wherein the polynucleotide is introduced to the cell using a gene-editing system selected from CRISPR-Cas9, zinc finger nuclease, or TALENS.

37. A method for treating a subject in need of extracellular chromatin degradation, the method comprising administering the isolated host cell of any one of claims 1 to 33, or the host cell prepared by the method of any one of claims 34 to 36, to the subject.

38. The method of claim 37, wherein the subject is in need of extracellular trap (ET) degradation, neutrophil extracellular trap (NET) degradation.

39. The method of claim 37 or 38, wherein the subject is at risk of vascular occlusion involving extracellular chromatin.

40. The method of any one of claims 37 to 39, wherein the extracellular chromatin includes chromatin released by cancer cells or injured endothelial cells.

41. The method of any one of claims 37 to 40, wherein the subject has a loss of function mutation in one or both D1L3 genes.

42. The method of any one of claims 37 to 41, wherein the subject has a condition selected from chronic neutrophilia, neutrophil aggregation or leukostasis, thrombosis or vascular occlusion, ischemia-reperfusion injury, surgical or traumatic tissue injury, an acute or chronic inflammatory reaction or disease, an autoimmune disease, inflammatory disease of the respiratory tract, renal inflammatory disease, inflammatory disease related to transplanted tissue, and cancer.

43. The method of claim 42, wherein the subject has cancer.

44. The method of claim 43, wherein the cancer is leukemia.

45. The method of claim 43, wherein the cancer is a solid tumor.

46. The method of claim 45, wherein the tumor is metastatic.

47. The method of any one of claims 43 to 46, wherein the subject has or is at risk of tumor lysis syndrome.

48. The method of claim 47, wherein the subject has an inflammatory disease of the respiratory tract.

49. The method of claim 48, wherein the subject has an infection of the lower respiratory tract.

50. The method of claim 48 or 49, wherein the subject has a viral infection.

51. The method of any one of claims 48 to 50, wherein the subject has Acute Respiratory Distress Syndrome (ARDS) or Acute Lung Injury (ALI).

52. The method of any one of claims 48 to 51, wherein the subject has pneumonia.

53. The method of claim 52, wherein the subject has a coronavirus infection.

54. The method of claim 53, wherein the coronavirus is SARS-CoV-2 or COVID-19.

55. A method for treating a subject in need of extracellular chromatin degradation, the method comprising administering a composition comprising isolated T cells to the subject, the isolated T cells being modified in vitro to express a heterologous polynucleotide encoding a D1L3 enzyme operably linked to a promoter, so as to express and secrete the D1L3 enzyme.

56. The method of claim 55, wherein the secreted D1L3 enzyme has at least a partial deletion of the C-terminal basic domain.

57. The method of claim 56, wherein the secreted D1L3 enzyme has a deletion of at least 12 amino acids of the C-terminal basic domain.

58. The method of claim 57, wherein the secreted D1L3 enzyme includes enzymes having deletions of one or more of: K291_S305 del, K292_S305 del, K293_S305 del, with respect to SEQ ID NO:4.

59. The method of claim 55, wherein the heterologous polynucleotide is DNA.

60. The method of claim 55, wherein the heterologous polynucleotide is an mRNA or a modified mRNA (mmRNA).

61. The method of claim 55, wherein the polynucleotide is introduced to the cell using a viral vector.

62. The method of claim 55, wherein the polynucleotide is introduced to the cell using a gene-editing system selected from CRISPR-Cas9, zinc finger nuclease, or TALENS.

63. The method of claim 55, wherein the D1L3 enzyme comprises an amino acid sequence that has at least 90% sequence identity to the enzyme of SEQ ID NO: 4 and SEQ ID NO: 5 lacking the C-terminal basic domain.

64. The method of claim 55, wherein the polynucleotide encodes a D1L3 enzyme having a deletion of one or more amino acids of the C-terminal basic domain.

65. The method of claim 64, wherein the polynucleotide encodes a D1L3 enzyme having a deletion of at least five amino acids of the C-terminal basic domain.

66. The method of claim 55, wherein the polynucleotide encodes a D1L3 enzyme having an inactivation of one or more of the NLS1 and the NLS2.

67. The method of claim 55, wherein the D1L3 enzyme includes one or more amino acid substitutions and/or deletions within the NLS1 and/or NLS2.

68. The method of claim 55, wherein the D1L3 enzyme is fused to a carrier protein.

69. The method of claim 68, wherein the carrier protein is albumin.

70. The method of claim 68, wherein the D1L3 is fused to the carrier protein at the N-terminus of the D1L3 protein through a flexible or cleavable linker.

71. The method of claim 55, wherein the subject is in need of extracellular trap (ET) degradation, neutrophil extracellular trap (NET) degradation.

72. The method of claim 55, wherein the subject is at risk of occlusions, including vascular occlusions, involving extracellular chromatin.

73. The method of claim 55, wherein the extracellular chromatin includes chromatin released by cancer cells or injured endothelial cells.

74. The method of claim 55, wherein the subject has a loss of function mutation in one or both D1L3 genes.

75. The method of claim 55, wherein the subject has a condition selected from chronic neutrophilia, neutrophil aggregation or leukostasis, thrombosis or vascular occlusion, ischemia-reperfusion injury, surgical or traumatic tissue injury, an acute or chronic inflammatory reaction or disease, an autoimmune disease, inflammatory disease of the respiratory tract, renal inflammatory disease, inflammatory disease related to transplanted tissue, and cancer.

76. The method of claim 75, wherein the subject has cancer.

77. The method of claim 76, wherein the cancer is leukemia.

78. The method of claim 76, wherein the cancer is a solid tumor.

79. The method of claim 78, wherein the tumor is metastatic.

80. The method of claim 75, wherein the subject has an inflammatory disease of the respiratory tract.

81. The method of claim 80, wherein the subject has an infection of the lower respiratory tract.

82. The method of claim 81, wherein the subject has a viral infection.

83. The method of claim 75, wherein the subject has Acute Respiratory Distress Syndrome (ARDS) or Acute Lung Injury (ALI).

84. The method of claim 75, wherein the subject has pneumonia.