Compositions and methods for treating, ameliorating, and / or preventing diseases or disorders caused by or associated with dnase1 and / or dnase1l3 deficiency

Abstract

To provide novel DNAse1 and / or DNAse1L3 constructs with improved in vivo half-lives, enzymatic stability, and / or developability properties.SOLUTION: Provided is a construct comprising the amino acid sequence: DNAse1-X1-LINKER-Fc-X2 (I) (where, DNAse 1 is a human DNAse1 polypeptide; X1 is a covalent bond, or X1 is the peptide comprising a specific amino acid sequence or a fragment thereof; LINKER is a chemical bond or a polypeptide comprising 1-100 amino acids; X2 is null, or X2 is the peptide comprising another specific amino acid sequence or a fragment thereof; Fc is the Fc domain of human IgG1).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 959,932, filed January 11, 2020, all of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Background of the Disclosure Lupus erythematosus (or lupus) is a general term for a group of autoimmune diseases in which the human immune system becomes overactive and attacks healthy tissue. Symptoms of these diseases can affect many different body systems, including joints, skin, kidneys, blood cells, heart, and lungs. The most common and most severe form of lupus is systemic lupus erythematosus (SLE).

[0003] Lupus symptoms vary from person to person and can occur sporadically. Common symptoms are joint pain and swelling and arthritis, primarily in the fingers, hands, wrists, and knees. Other common symptoms include: chest pain while breathing; mouth ulcers; fatigue; weight loss; fever without other cause; a general feeling of discomfort, discomfort, or ill-health (malaise); hair loss; photosensitivity; "butterfly" facial rash, seen in approximately half of people with SLE; and enlarged lymph nodes. Lupus is a highly prevalent condition, and the majority of patients experience life-threatening nephritis.

[0004] Lupus is thought to be influenced by multiple genes and genetic polymorphisms, more than 30 of which are currently associated with the disorder. The pathogenesis of SLE is related to a reduced ability to remove DNA released from apoptotic cells, and the accumulation of that DNA over time triggers an autoimmune response and the formation of anti-DNA autoantibodies. Two key features are associated with SLE: dysregulated activation of both T and B lymphocytes, and the development of anti-DNA autoantibodies, particularly against double-stranded DNA, which are involved in tissue damage. Major histocompatibility complex (MHC) class II genes, certain class III genes [complement components 2 (C2) and 4 (C4A), tumor necrosis factor (TNF), and heat shock protein 70 kD (HSPA1A) alleles], and other non-MHC genes [receptor for the Fc fragment of IgG, low affinity IIa and IIIa (FCGR2A and FCGR3A), interleukin 6 and 10 (IL6 and IL10), and B-cell CLL / lymphoma 2 (BCL2)] may each contribute to susceptibility. However, the primary loci and alleles involved in disease susceptibility are currently unknown.

[0005] Recently, a rare autosomal recessive form of SLE with a null mutation in the DNAse1L3 gene was reported (Al-Mayouf, et al., 2011, Nature Genetics 43(12), 1186-1188). DNAse1L3-associated SLE has a childhood onset and correlates with a high incidence of lupus nephritis. Indeed, DNAse1L3-deficient mice were found to rapidly develop antibodies against double-stranded DNA and chromatin, followed by immune activation, IgG deposition in kidney glomeruli, and glomerulonephritis. DNAse1L3 is one of the three human homologs of DNase I; this enzyme functions as an endonuclease capable of cleaving both single- and double-stranded DNA, is not inhibited by actin, and mediates DNA degradation during apoptosis. On the other hand, DNAse 1 binds with very high (subnanomolar) affinity to actin monomers and with lower affinity to actin polymers; actin-bound DNase I is enzymatically inactive. DNAse 1 cleaves uncomplexed DNA, whereas DNAse 1L3 cleaves chromatin.

[0006] Unfortunately, no stable and bioavailable DNAse1 and / or DNAse1L3 enzyme biologics designed to treat lupus associated with DNAse1 and / or DNAse1L3 deletions, as well as other conditions associated with DNAse1 and / or DNAse1L3 deletions, have been described in the literature to date.

[0007] Thus, there is a need in the art for compositions and methods that can be used to treat diseases or disorders caused by and / or associated with DNAse 1 and / or DNAse 1L3 deficiency, such as, but not limited to, SLE. The present disclosure meets this need. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Al-Mayouf, et al., 2011, Nature Genetics 43(12), 1186-1188 Summary of the Invention

[0009] BRIEF SUMMARY OF THE DISCLOSURE The present disclosure provides certain constructs, such as, but not limited to, DNAse 1-X1-linker-Fc-X2 and DNAse 1 L3-X1-linker-Fc-X2, where DNAse 1, DNAse 1 L3, X1, X2, linker, and Fc are defined elsewhere herein. Additionally, the present disclosure provides certain homodimeric constructs comprising at least one construct of the present disclosure.

[0010] The present disclosure provides methods for treating, ameliorating, and / or preventing multiple forms of lupus associated with DNAse1 and / or DNAse1L3 deficiency in a subject using certain constructs of the present disclosure. Furthermore, the present disclosure provides methods for treating, ameliorating, and / or preventing diseases and / or disorders associated with inefficient NET hydrolysis in a subject using certain constructs of the present disclosure. Furthermore, the present disclosure provides methods for treating, ameliorating, and / or preventing autoimmune disorders associated with DNAse1 and / or DNAse1L3 deficiency in a subject using certain constructs of the present disclosure. Furthermore, the present disclosure provides methods for treating, ameliorating, and / or preventing pathological thrombosis in a subject using certain constructs of the present disclosure. Furthermore, the present disclosure provides methods for treating, ameliorating, and / or preventing myocardial infarction in a subject using certain constructs of the present disclosure. Additionally, the present disclosure provides methods of treating, ameliorating, and / or preventing cancer metastasis in a subject using certain constructs of the present disclosure. [Brief explanation of the drawings]

[0011] The following detailed description of exemplary embodiments of the present disclosure will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present disclosure, exemplary embodiments are shown in the drawings. It should be understood, however, that the present disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Figure 1] Illustrating neutrophil extracellular trap (NET) formation. Scanning electron microscopy of a neutrophil (marked A) casting a net (marked B) that captures Helicobacter pylori bacteria (some of them marked C). Image taken from Kumamoto T, et al., 2006, Eur Heart J. 27(17):2081-7. [Figure 2] 1 illustrates non-limiting DNAse1-Fc constructs of the present disclosure, with certain contemplated point mutations highlighted. [Figure 3] 1 illustrates non-limiting DNAse1L3-Fc constructs of the present disclosure, with certain contemplated point mutations highlighted. [Figure 4] 1 illustrates non-limiting DNAse1-Fc constructs of the present disclosure, with certain contemplated point mutations highlighted. [Figure 5]

[0023] Figure 1 illustrates non-limiting constructs of the present disclosure, highlighting certain contemplated point mutations. In certain embodiments, certain mutations render rDNAse hyperactive and / or actin resistant (i.e., have reduced affinity for actin) and / or increase the half-life of the construct. A non-limiting alignment of the amino acid sequences of mouse DNAse 1 (SEQ ID NO: 42) and mouse DNAse 1L3 (SEQ ID NO: 43) is illustrated. [Figure 6] 1 illustrates non-limiting constructs of the present disclosure, with certain contemplated point mutations highlighted. In certain aspects, the construct lacks at least a portion of the DNAse1L3 nuclear localization domain. [Figure 7]1 depicts a gel showing that certain DNAse1L3 clones cleave chromatin, but this is not the case for certain DNAse1 clones. [Figure 8] 1 illustrates non-limiting constructs of the present disclosure. In certain embodiments, a DNAse1 polypeptide is fused to the C-terminal tail of DNAse1L3. [Figure 9] 1 illustrates certain aspects of the production and purification of DNAse-Fc constructs. [Figure 10A] 10A-10B illustrate the in vivo pharmacokinetics of certain NET-degrading constructs of the present disclosure. [Figure 10B] 10A-10B illustrate the in vivo pharmacokinetics of certain NET-degrading constructs of the present disclosure. [Figure 11] 1 depicts a non-limiting purification gel of certain NET-degrading constructs of the present disclosure. [Figure 12] This figure illustrates the discovery that a non-limiting optimized heterodimer comprising DNAse1 and DNAse1L3 exhibits increased NET degradation compared to the optimized DNAse1 or DNAse1L3 constructs alone. The DNAse degradation activity of various constructs described in this application is shown for DNA alone (top gel) and protein-bound DNA (bottom gel). Purified heterodimers composed of DNAse1 and DNAse1L3 (heterodimers of constructs 1669 and 1689) are shown in lanes 2 and 5; various purified optimized constructs of DNAse1 are shown in lanes 1, 3, 4, and 6; and a purified optimized DNAse1L3 construct is in lane 7. As shown, only the heterodimer digests both plasmid DNA (top gel) and chromatin DNA (bottom gel), as confirmed by the reduced size of the DNA bands in the gel. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description of the Disclosure In one aspect, the present disclosure relates to the discovery that certain constructs can be used to treat, ameliorate, and / or prevent diseases or disorders associated with DNAse1 and / or DNAse1L3 deficiency.

[0013] In certain embodiments, the constructs contemplated herein can be used to treat, ameliorate, and / or prevent multiple forms of lupus (including SLE) associated with DNAse1L3 deficiency.

[0014] In certain embodiments, the constructs contemplated herein can be used to treat, ameliorate, and / or prevent diseases and / or disorders associated with inefficient NET hydrolysis ("NETolysis").

[0015] In certain embodiments, the constructs contemplated herein can be used to treat, ameliorate, and / or prevent autoimmune disorders, such as, but not limited to, lupus (including SLE), thyroid autoimmune disease, and hypocomplementemic urticarial vasculitis syndrome (HUVS).

[0016] In certain embodiments, the constructs contemplated herein can be used to treat, ameliorate, and / or prevent pathological thrombosis, including, but not limited to, microvascular thrombosis, venous thrombosis, and / or arterial thrombosis. In certain embodiments, pathological thrombosis includes neutrophilic thrombosis, including, but not limited to, antineutrophil cytoplasmic autoantibody (ANCA) vasculitis, thrombotic thrombocytopenic purpura (TTP), and Bechet's (or Behcet's) disease or syndrome. In certain embodiments, pathological thrombosis includes thrombosis resulting in stroke.

[0017] In certain embodiments, the constructs contemplated herein can be used to treat, ameliorate, and / or prevent myocardial infarction.

[0018] In certain embodiments, the constructs contemplated herein can be used to treat, ameliorate, and / or prevent cancer spread and progression (e.g., cancer metastasis).

[0019] The present disclosure provides stable and bioavailable constructs comprising DNAse1L3 and / or DNAse1 polypeptides (or fragments, rearrangements, (point) mutations, truncations, and / or any other modifications and / or analogs and / or derivatives thereof) fused to certain proteins. In certain embodiments, the constructs contemplated herein have increased bioavailability and / or developability over DNAse1L3 and / or DNAse1 polypeptides known in the art. In certain embodiments, the constructs contemplated herein have enhanced enzymatic activity over DNAse1L3 and / or DNAse1 polypeptides known in the art. In certain embodiments, the constructs contemplated herein have improved pharmacokinetic behavior over DNAse1L3 and / or DNAse1 polypeptides known in the art. In certain embodiments, the constructs contemplated herein have enhanced stability over DNAse1L3 and / or DNAse1 polypeptides known in the art.

[0020] In certain embodiments, the in vivo half-life of the constructs of the present disclosure is at least about 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, and / or 20 times greater than that of DNAse1 and / or DNAse1L3 polypeptides described in the art. In other embodiments, the constructs of the present disclosure are administered to a subject at a lower dose and / or less frequently than other DNAse1 and / or DNAse1L3 polypeptides in the art. In still other embodiments, the constructs of the present disclosure are administered to a subject monthly, twice monthly, three times monthly, and / or four times monthly. In still other embodiments, less frequent administration of the constructs of the present disclosure results in better patient compliance and / or increased efficacy compared to other DNAse1 and / or DNAse1L3 polypeptides in the art.

[0021] The disclosed constructs can be used to treat diseases or disorders resulting from and / or associated with and / or related to reduced and / or inefficient and / or suboptimal degradation and / or clearance and / or hydrolysis of neutrophil extracellular traps (NETs). Polymorphonuclear leukocytes (PMNs), the most abundant form of white blood cells, circulate in tissues and blood, where they seek out invading microorganisms. When invading microorganisms encounter PMNs, the cells respond with a series of mechanisms to attempt to contain the infection, including phagocytosis, the release of stored antimicrobial compounds in a process called degranulation, and, as a last resort, suicide, causing the PMNs to "rupture" and release webs encapsulating DNA and cytotoxic substances, known as "neutrophil extracellular traps" or NETs. NETs are extracellular, neutrophil-derived webs of DNA that trap invading pathogens. The backbone of NETs is a sticky mesh of chromatin to which various antimicrobial cytotoxic proteins and peptides are attached, which are released along with the chromatin when PMNs degranulate in response to infectious stimuli. The high concentrations of antimicrobial compounds maintained by NETs in close proximity to invading organisms enhance the efficacy of cytotoxic agents, thereby neutralizing invading pathogens, preventing their spread, and eliminating the threat of infection.

[0022] Despite their beneficial role in fighting infection, NETs must be rapidly and efficiently cleared from tissues and the circulation, or else serious pathological consequences will result. In particular, diseases associated with inefficient NET hydrolysis ("NETolysis") include autoimmune disorders such as lupus, pathological thrombosis (e.g., but not limited to, thrombosis leading to stroke) and myocardial infarction, and cancer propagation and progression.

[0023] NETs are typically degraded by blood-based metalloenzymes, several circulating enzyme isoforms that hydrolyze high-energy bonds in DNA, resulting in NETolysis. To do so, different enzyme isoforms recognize DNA either as free nucleic acid or bound to proteins such as chromatin in the protein backbone of NETs. Loss-of-function mutations in these enzymes have been identified in systemic lupus erythematosus (SLE), including hereditary and highly aggressive forms of SLE that appear in pediatric populations. Furthermore, NETs promote cancer progression and metastasis, and inhibition of NETs has been shown to reduce cancer metastasis in mouse models.

[0024] NETs in Tumor Progression and Metastasis: Currently, locoregional control of cancer through complete surgical resection constitutes the primary curative modality for all forms of solid tumors and, in the adjuvant setting, significantly improves disease-free and overall survival for most tumors. Therefore, control of distant metastases after surgery is paramount to patient outcomes. Systemic chemotherapy is currently used to control tumor recurrence, with mixed results. Infectious complications associated with cancer treatment have long been associated with adverse oncological outcomes independent of the morbidity associated with infectious complications, a phenomenon observed across a wide range of malignancies, including lung, breast, colon, and esophageal tumors. In particular, post-procedural infections after surgery or chemotherapy are strongly associated with increased mortality from metastatic disease. Unfortunately, infectious complications are frequent in oncology, approaching 40% in some series. Recently, the association between infection and tumor recurrence has been linked to the presence of NETs in the tumor microenvironment. It appears that infection-induced neutrophil degranulation increases NETs, ​​which bind to tumor cells in the blood and support their metastatic progression.

[0025] Following the discovery that infection-induced NET formation can lead to cancer progression and spread, researchers investigated the ability of cancer cells themselves to induce NET formation and found that highly aggressive cancer cells can induce NET formation in the absence of infection. Cancer cells appear to "hijack" the immune system by inducing neutrophils to extrude NETs, ​​which then enter the circulation and support their attachment to distant sites of metastasis. Supporting these findings, the levels of circulating NETs in cancer patients with advanced esophageal, lung, and GI cancers directly correlated with their cancer stage, i.e., the presence of cancer at sites distal to the primary tumor (i.e., stage I-II cancers had fewer NETs in the blood than stage III-IV cancers). This finding suggests that the number of NETs can be used as an independent biomarker for cancer progression in these aggressive forms of cancer. Preclinical mouse models of lung, GI, and breast cancer, in which inhibition of NETs prevented the anchoring of circulating tumor cells to the liver sinusoids and reduced the appearance of lung metastases, also support the idea that NETs aid in the metastasis of aggressive forms of cancer. These studies demonstrate that NETs promote liver and lung metastasis in mouse models of human tumors and that targeting NETs can inhibit metastatic spread of tumors.

[0026] Current research into the effects of NETs in cancer indicates that circulating NET levels are a prognostically important biomarker for tumor progression and metastasis, that human cancer cells can induce metastasis-promoting NETs to aid their growth and spread, and that inhibition of NET formation can suppress tumor progression and metastasis in mouse models of human GI, lung, and breast cancer. Overall, the research supports the use of NET-based therapies in cancer treatment.

[0027] Autoimmune Diseases with a Focus on Systemic Lupus Erythematosus (SLE): Inadequate clearance of dead and apoptotic cells has long been thought to be directly involved in the pathogenesis of autoimmune diseases, including some forms of SLE. This idea stems from the discovery first reported 40 years ago that patients with SLE have lower serum DNAse 1 activity than healthy subjects, a blood enzyme primarily involved in the removal of DNA from dead and apoptotic cells and the clearance of NETs. Furthermore, low DNAse 1 activity is associated with the active phase of nephropathy in lupus (type III or type IV), implicating DNAse 1 activity in the development of SLE nephropathy.

[0028] Following these clinical findings, researchers engineered transgenic DNAse1 knockout mice lacking DNAse1 activity and found that these mice recapitulated the clinical and biochemical phenotype present in human SLE, including the appearance of double-stranded DNA (dsDNA) antibodies, glomerulonephritis caused by the deposition of autoantibodies on the glomerular surface, and perivascular infiltrates. Remarkably, DNAse1-deficient mice also recapitulated the female-type bias seen in humans with SLE.

[0029] Inactivating mutations in DNAse1 were present in a heterozygous pattern in two Japanese patients with childhood lupus (Yasutomo I et al., 2001, Nat Genet. 2001;28(4):313-4). Importantly, these girls had very low DNAse1 activity and very high titers of anti-nucleosome and anti-double-stranded DNA (dsDNA) antibodies. Following this study, homozygous inactivating mutations in DNAse1L3 were found in several Arab families with children who developed lupus as early as 2 years of age (Al-Mayouf, et al., 2011, Nature Genetics 43(12), 1186-1188), suggesting a highly aggressive form of the disease. Further evidence linking DNAse1 and DNAse1L3 to human autoimmune disease was the identification of functionally defective variants of DNase1 in Spanish patients with SLE, Caucasian patients with thyroid autoimmune disease, and Turkish and Italian patients with hypocomplementemic urticarial vasculitis syndrome, an autoimmune disorder with overlapping clinical features of SLE.

[0030] These findings support the idea that DNAse1 and DNAse1L3 are involved in the pathogenesis of human autoimmune diseases, but subsequent studies have determined that the frequency of these mutations is extremely rare, occurring in essentially less than 1% of patients with autoimmune disease. Finally, the identification of a single nucleotide polymorphism (SNP) in a Caucasian-specific allele of DNAse1L3 was confirmed in SLE, producing a completely inactive enzyme. The allele frequencies were found in three Caucasian populations—Mexicans, Turks, and Germans—with predicted minor allele frequencies of 0.017, 0.052, and 0.077, respectively. Because a homozygous functional defect in DNAse1L3 is required to predispose patients to autoimmune disease, the number of genetically defined cases resulting from this form of DNAse1L3 deletion is also expected to be very low.

[0031] A common DNAse1 polymorphism identified in lupus patients dramatically altered this disease picture. The Q244R polymorphism in exon 8, also identified as rs1053874, is distributed globally with a minor allele frequency of 0.494. The R244 allele was found to be strongly associated with anti-ribonucleoprotein (anti-RNP) antibody production in Korean lupus patients and significantly associated with SLE susceptibility in Argentine patients. Following this association, Japanese researchers determined that the R244 variant has half the enzymatic activity of the Q244 variant, thereby explaining the greater propensity for autoimmune disease in patients with the R244 polymorphism. The same researchers then undertook a systematic evaluation of all nonsynonymous DNAse1 SNPs in the Ensembl database (ensemble.org), ultimately identifying 60 loss-of-function variants of DNAse1, all of which are potentially pathogenic. Although the occurrence of many of these forms is fairly rare, the frequency of the most common variant (rs1053874-Q244R, MAR 0.494) is such that it is expected to be present as homozygotes in approximately 25% of the world's population.

[0032] NETs in pathological thrombosis: Another area of ​​therapeutic intervention in which targeting NETs may prove therapeutic is vascular thrombosis, including but not limited to microvascular thrombosis, venous thrombosis, and arterial thrombosis. In certain embodiments, the present disclosure contemplates neutrophilic thrombosis, including but not limited to antineutrophil cytoplasmic autoantibody (ANCA) vasculitis, thrombotic thrombocytopenic purpura (TTP), and Behcet's disease or syndrome. NETs are prothrombotic and can promote thrombosis in pathological conditions such as cancer and myocardial infarction (Thalin C, et al., 2019, Arterioscler Thromb Vasc Biol. 39(9):1724-38). Serum DNAse 1 activity suddenly increased early in acute myocardial infarction, and the DNAse 1 low-activity polymorphism (rs1053874) described above was significantly elevated in Japanese patients with active myocardial infarction (as opposed to stable angina). These findings directly link reduced DNAse 1 function to myocardial infarction.

[0033] Reference will now be made in detail to certain aspects of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0034] Throughout this document, values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​expressly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were expressly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the recited range. The statement "about X to Y" has the same meaning as "about X to about Y" unless otherwise indicated. Similarly, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z" unless otherwise indicated.

[0035] definition As used herein, each of the following terms has the meaning associated with it in this section. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In general, the nomenclature used herein and laboratory methods in animal pharmacology, pharmaceutical science, separation science, and organic chemistry are those well known and commonly used in the art. It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present teachings remain operable. Any use of section headings is intended to aid in the reading and comprehension of the document and should not be construed as limiting; information relevant to a section heading may be located within or outside of that particular section. All publications, patents, and patent documents mentioned in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference.

[0036] In this application, when an element or component is said to be included in and / or selected from a list of described elements or components, it is understood that the element or component can be any one of the described elements or components, and can be selected from a group consisting of two or more of the described elements or components.

[0037] In the methods described herein, acts may be performed in any order unless a temporal or operational order is expressly recited. Moreover, certain acts may be performed simultaneously unless express claim language recites that they be performed separately. For example, a claimed act of doing X and a claimed act of doing Y may be performed simultaneously in a single operation, and the resulting process would fall within the literal scope of the claimed process.

[0038] In this document, the terms "a," "an," or "the" are used to include one or more than one, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. The phrases "at least one of A and B" or "at least one of A or B" have the same meaning as "A, B, or A and B."

[0039] As used herein, "about" when referring to a measurable value such as amount, duration, and the like, is intended to encompass variations of ±20% or ±10%, in certain embodiments ±5%, in certain embodiments ±1%, and in certain embodiments ±0.1% from the particular value, as such variations are appropriate for performing the disclosed methods.

[0040] A disease or disorder is "alleviated" if the severity of the symptoms of the disease or disorder, the frequency with which the patient experiences such symptoms, or both, is reduced.

[0041] As used herein, the terms "alteration," "deletion," "mutation," or "mutation" refer to a mutation in a cellular gene that affects the function, activity, expression (transcription or translation), or conformation of the polypeptide it encodes, including missense and nonsense mutations, insertions, deletions, frameshifts, and premature terminations.

[0042] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a specific epitope on an antigen. An antibody can be an intact immunoglobulin derived from natural sources or recombinant sources, or an immunoreactive portion of an intact immunoglobulin.

[0043] As used herein, the term "AUC" refers to the area under the plasma drug concentration-time curve (AUC), which correlates with the body's actual exposure to a drug after administration of a dose of the drug. In certain embodiments, AUC is expressed in mg*h / L. AUC can be used to measure the bioavailability of a drug, which is the proportion of unchanged drug absorbed intact and reaching the site of action or systemic circulation after administration by any route.

[0044] AUC can be calculated using the linear trapezoidal method or the logarithmic trapezoidal method. The linear trapezoidal method calculates AUC using linear interpolation between data points. This method is required by the OGD and the FDA and is the standard for bioequivalence studies. For a given time interval (t1-t2), AUC can be calculated as follows: TIFF2025134821000001.tif9128 where C1 and C2 are the average concentrations over the time intervals (t1 and t2).

[0045] The logarithmic trapezoidal method calculates the AUC using logarithmic interpolation between data points. This method is more accurate when concentrations are declining because drug elimination is exponential (which makes drug elimination linear on a logarithmic scale). For a given time interval (t1-t2), the AUC can be calculated as follows: TIFF2025134821000002.tif12128 (assuming C1>C2).

[0046] As used herein, the term "bioavailability" refers to the extent and rate at which an active ingredient (protein, drug, or metabolite) enters the systemic circulation and thereby accesses the site of action or enters the systemic circulation after administration by any route. The bioavailability of an active ingredient is largely determined by the characteristics of the dosage form, which in part depends on its design and manufacture. Differences in bioavailability between formulations of a given drug or protein can have clinical significance; therefore, it is essential to know whether drug formulations are equivalent. The most reliable measure of drug or protein bioavailability is the area under the plasma concentration-time curve (AUC). AUC is directly proportional to the total amount of unchanged drug or therapeutic protein that reaches the systemic circulation. Drugs or therapeutic proteins can be considered bioequivalent in the extent and rate of absorption if their plasma concentration curves are essentially superimposable. For intravenously administered drugs, bioavailability is defined as 1. For drugs administered by other routes of administration, bioavailability is often less than 1. Incomplete bioavailability can be due to several factors, which can be subdivided into the categories of dosage form effects, membrane effects, and administration site effects. Half-life and AUC provide information about the bioavailability of a drug or biologic.

[0047] As used herein, the term " conservative mutation " or " conservative substitution " refers to the replacement of an amino acid residue with another biologically similar residue.Conservative mutation or substitution is unlikely to change the shape of peptide chain.Examples of conservative mutation or substitution include the replacement of a hydrophobic residue, such as isoleucine, valine, leucine or methionine, with another hydrophobic residue, or the replacement of a polar residue with another polar residue, such as the replacement of arginine with lysine, the replacement of glutamic acid with aspartic acid, or the replacement of glutamine with asparagine.

[0048] As used herein, a "construct" of the present disclosure refers to a fusion polypeptide comprising DNAse1 and / or DNAse1L3 polypeptides, or any fragments, rearrangements, (point) mutations, truncations, or any other modifications and / or analogs and / or derivatives thereof.

[0049] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and the animal's health continues to deteriorate unless the disease is remitted.

[0050] An animal "disorder" is a health condition in which the animal is able to maintain homeostasis, but the animal is no better off than it would be in the absence of the disorder. Left untreated, the disorder does not necessarily cause a further decline in the animal's health.

[0051] As used herein, the terms "effective amount," "pharmaceutically effective amount," and "therapeutically effective amount" refer to a non-toxic but sufficient amount of an agent to produce a desired biological result. The result may be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. The appropriate therapeutic amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

[0052] As used herein, the term "DNAse 1" refers to deoxyribonuclease-1 (UniProtKB=P24855). The sequence of human DNAse 1 is provided herein (SEQ ID NO: 1). In certain embodiments, the signal peptide of DNAse 1 corresponds to residues 1-22 of SEQ ID NO: 1. TIFF2025134821000003.tif83128

[0053] The sequence of mouse DNAse1 is provided herein (SEQ ID NO:29): TIFF2025134821000004.tif29159

[0054] A sequence alignment of human DNAse 1 (SEQ ID NO: 1, hereinafter sequence "1") and mouse DNAse 1 (SEQ ID NO: 29, hereinafter sequence "2") follows: TIFF2025134821000005.tif117159

[0055] As used herein, "human DNAse 1" refers to the human DNAse 1 sequence described herein, or any fragment, rearrangement, (point) mutation, truncation, or any other modification and / or analog and / or derivative thereof. As used herein, the term "enzymatically active" with respect to DNAse 1 is defined as being able to bind to and hydrolyze DNA.

[0056] As used herein, the term "DNAse1L3" refers to deoxyribonuclease gamma (UniProtKB=Q13609). The sequence of human DNAse1L3 is provided herein (SEQ ID NO:2). In certain embodiments, the signal peptide of DNAse1L3 corresponds to residues 1-20 of SEQ ID NO:2. In certain embodiments, the nuclear localization signal of DNAse1L3 corresponds to residues 296-304 of SEQ ID NO:2. In certain embodiments, the nuclear localization signal of DNAse1L3 corresponds to residues 292-304 of SEQ ID NO:2. In certain embodiments, the nuclear localization signal of DNAse1L3 corresponds to residues 291-305 of SEQ ID NO:2. In certain embodiments, the nuclear localization signal of DNAse1L3 corresponds to residues A-B of SEQ ID NO:2, where A ranges from 291-296 and B ranges from 304-305. TIFF2025134821000006.tif96128

[0057] The sequence of mouse DNAse1L3 is provided herein (SEQ ID NO:30): TIFF2025134821000007.tif30159

[0058] A sequence alignment of human DNAse1L3 (SEQ ID NO:2, hereinafter sequence "1") and mouse DNAse1L3 (SEQ ID NO:30, hereinafter sequence "2") follows: TIFF2025134821000008.tif142159

[0059] As used herein, "human DNAse1L3" refers to the human DNAse1L3 sequence described herein, or any fragment, rearrangement, (point) mutation, truncation, or any other modification and / or analog and / or derivative thereof. As used herein, the term "enzymatically active" with respect to DNAse1L3 is defined as being able to bind to and hydrolyze DNA.

[0060] As used herein, the term "DNAse1-Fc" refers to a DNAse1 polypeptide that is recombinantly fused and / or chemically conjugated (including both covalent and non-covalent conjugation) to the FcR binding domain of an IgG molecule (preferably human IgG). In certain embodiments, the C-terminus of DNAse1 is fused or conjugated to the N-terminus of the FcR binding domain. In certain embodiments, the N-terminus of DNAse1 is fused or conjugated to the C-terminus of the FcR binding domain.

[0061] As used herein, the term "DNAse1L3-Fc" refers to a DNAse1L3 polypeptide that is recombinantly fused and / or chemically conjugated (including both covalent and non-covalent conjugation) to the FcR binding domain of an IgG molecule (preferably human IgG). In certain embodiments, the C-terminus of DNAse1L3 is fused or conjugated to the N-terminus of the FcR binding domain. In certain embodiments, the N-terminus of DNAse1L3 is fused or conjugated to the C-terminus of the FcR binding domain.

[0062] As shown in Figure 5 herein, a sequence alignment of mouse DNAse1 (SEQ ID NO:42, hereinafter "Query") and mouse DNAse1L3 (SEQ ID NO:43, hereinafter "Subject") follows: TIFF2025134821000009.tif197155

[0063] As used herein, the term "Fc" refers to the human IgG (immunoglobulin) Fc domain. Subtypes of IgG, such as IgG1, IgG2, IgG3, and IgG4, are contemplated for use as the Fc domain.

[0064] As used herein, the term "Fc region" refers to the portion of an IgG molecule that corresponds to the crystallizable fragment obtained by papain digestion of the IgG molecule. The Fc region comprises the C-terminal halves of the two heavy chains of an IgG molecule, which are linked by disulfide bonds. It lacks antigen-binding activity but contains carbohydrate moieties and binding sites for complement and Fc receptors, such as the FcRn receptor. The Fc fragment contains the complete second constant domain CH2 (residues 231-340 of human IgG1 according to the Kabat numbering system) and the third constant domain CH3 (residues 341-447). The term "IgG hinge-Fc region" or "hinge-Fc fragment" refers to the region of an IgG molecule consisting of the Fc region (residues 231-447) and the hinge region (residues 216-230) extending from the N-terminus of the Fc region. The term "constant domain" refers to the portion of an immunoglobulin molecule that contains the antigen-binding site and has a more conserved amino acid sequence than the other portions of the immunoglobulin, i.e., the variable domain. The constant domain includes the CH1, CH2, and CH3 domains of the heavy chain and the CHL domain of the light chain.

[0065] As used herein, the term "Fc receptor" refers to a protein found on the surface of certain cells (e.g., B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, human platelets, and mast cells, among others) that contribute to the protective functions of the immune system. Fc receptors bind to antibodies bound to infected cells or invading pathogens. Immunoglobulin Fc receptors (FcRs) are expressed on all hematopoietic cells and play a key role in antibody-mediated immune responses. Binding of immune complexes to FcRs activates effector cells, leading to phagocytosis, endocytosis of IgG-opsonized particles, release of inflammatory mediators, and antibody-dependent cellular cytotoxicity (ADCC). Fc receptors have been described for all classes of immunoglobulins: FcγR and neonatal FcR (FcRn) for IgG, FcεR for IgE, FcαR for IgA, FcδR for IgD, and FcμR for IgM. All known Fc receptors structurally belong to the immunoglobulin superfamily, except for FcRn and FcεRII, which are structurally related to class I major histocompatibility complexes and C-type lectins, respectively (Fc Receptors, Neil A. Fangera, et al., in Encyclopedia of Immunology (2012)). nd Edition), 1998).

[0066] As used herein, the term "FcRn receptor" refers to the neonatal Fc receptor (FcRn), also known as the Brambell receptor, which in humans is a protein encoded by the FCGRT gene. FcRn specifically binds to the Fc domain of antibodies. FcRn extends the half-life of IgG and serum albumin in endothelial cells by reducing lysosomal degradation. IgG, serum albumin, and other serum proteins are continuously internalized through pinocytosis. Generally, serum proteins are transported from endosomes to lysosomes, where they are degraded. FcRn-mediated transcytosis of IgG across epithelial cells is possible because FcRn binds to IgG at acidic pH (<6.5) but not at neutral or higher pH. IgG and serum albumin bind to FcRn at slightly acidic pH (<6.5) and are recycled to the cell surface, where they are released at the neutral pH of blood (>7.0). In this way, IgG and serum albumin avoid lysosomal degradation.

[0067] The Fc portion of an IgG molecule is located in the constant region of the heavy chain, particularly the CH2 domain. The Fc region binds to the Fc receptor (FcRn), a surface receptor on B cells, and also binds to proteins of the complement system. Binding of the Fc region of an IgG molecule to FcRn activates cells bearing the receptor, thereby activating the immune system. Fc residues important for mouse Fc-mouse FcRn and human Fc-human FcRn interactions have been identified (Dall'Acqua et al., 2002, J. Immunol. 169(9):5171-80). The FcRn-binding domain includes the CH2 domain of an IgG molecule (or its FcRn-binding portion).

[0068] As used herein, the term "fragment," when applied to nucleic acids, refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid can be at least about 15, 50-100, 100-500, 500-1000, 1000-1500 nucleotides, 1500-2500, or 2500 nucleotides (and any integer value therebetween). As used herein, the term "fragment," when applied to a protein or peptide, refers to a subsequence of a larger protein or peptide and can be at least about 20, 50, 100, 200, 300, or 400 amino acids in length (and any integer value therebetween).

[0069] The term "functional equivalent" or "functional derivative," in the context of a functional derivative of an amino acid sequence, refers to a molecule that retains substantially similar biological activity (either functional or structural) to the sequence of the DNAse1-Fc and / or DNAse1E3-Fc constructs set forth herein. Functional derivatives or equivalents may be naturally occurring derivatives or synthetically prepared. Functionally equivalent polypeptides of the present disclosure may also be polypeptides identified using one or more techniques of structural and / or sequence alignment known in the art.

[0070] Exemplary functional derivatives include amino acid sequences with one or more amino acid substitutions, deletions, or additions, provided that the biological activity of the protein is preserved. The substituting amino acid desirably has chemical-physical properties similar to those of the amino acid being replaced. Desirable similar chemical-physical properties include similar charge, bulkiness, hydrophobicity, hydrophilicity, etc. Typically, greater than 30% identity between two polypeptides is considered to be indicative of functional equivalence. Preferably, functionally equivalent polypeptides of the present disclosure have greater than 80% sequence identity with the DNAse1-Fc and / or DNAse1L3-Fc constructs. More preferred polypeptides have greater than 85%, 90%, 95%, 98%, or 99% identity, respectively. Methods for determining whether a functional equivalent or functional derivative has the same or similar, or higher biological activity than the DNAse1-Fc and / or DNAse1L3-Fc constructs can be determined by using enzymatic assays known in the art.

[0071] "Gene transfer" and "gene delivery" refer to a method or system for precisely inserting specific nucleic acid sequences into target cells.

[0072] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell only when an inducer corresponding to the promoter is substantially present in the cell.

[0073] As used herein, the term "in vivo half-life" for proteins and / or polypeptides contemplated within the present disclosure (e.g., DNAse 1 and / or DNAse 1L3 constructs comprising an FcRn binding site) refers to the time required for half of an administered dose to be removed from the animal's circulation and / or other tissues. When a clearance curve of a fusion protein is constructed as a function of time, the curve is typically biphasic, with a rapid α-phase (which represents equilibration of the administered molecule between the intravascular and extravascular spaces and is determined in part by the size of the molecule) and a longer β-phase (which represents catabolism of the molecule in the intravascular space). In certain embodiments, the term "in vivo half-life" actually corresponds to the half-life of the molecule in the β-phase.

[0074] "Instructional materials," as that term is used herein, include publications, recordings, diagrams, or any other medium of expression that can be used to communicate the usefulness of the nucleic acids, peptides, and / or compounds of the present disclosure in kits for identifying or alleviating or treating the various diseases or disorders described herein.

[0075] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or polypeptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0076] An "isolated nucleic acid" refers to a nucleic acid segment or fragment separated from sequences that naturally flank it, i.e., a DNA fragment removed from the sequences that normally flank the fragment, i.e., the sequences that flank the fragment in the genome in which it naturally occurs. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, i.e., the RNA or DNA or proteins that naturally accompany it in the cell. Thus, the term includes recombinant DNA that is incorporated, for example, into a vector, an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or exists as a separate molecule (i.e., as cDNA or a fragment of a genomic or cDNA generated by PCR or restriction enzyme digestion) independent of other sequences. It also includes recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0077] An "oligonucleotide" or "polynucleotide" is a nucleic acid ranging in length from at least 2, and in certain embodiments at least 8, 15, or 25 nucleotides, but may be up to 50, 100, 1000, or 5000 nucleotides in length, or a compound that specifically hybridizes to a polynucleotide.

[0078] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to link two protein coding regions, are in the same reading frame.

[0079] As used herein, the terms "patient," "individual," or "subject" refer to a human.

[0080] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful in the present disclosure and a pharmaceutically acceptable carrier. Pharmaceutical compositions facilitate the administration of compounds to patients. There are multiple techniques for administering compounds in the art, including but not limited to subcutaneous, intravenous, oral, aerosol, inhalation, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical administration.

[0081] As used herein, the term "pharmaceutically acceptable" refers to a material, e.g., a carrier or diluent, that does not interfere with the biological activity or properties of the compound and is relatively non-toxic, i.e., the material can be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any one of the components of the composition in which it is contained.

[0082] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickener, solvent, or encapsulating material, involved in carrying or transporting a compound useful within the present disclosure into or to a patient so that it can perform its intended function. Each carrier should be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the present disclosure, and not toxic to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, absorption delaying agents, and the like, that are compatible with the activity of the compound useful within the present disclosure and are physiologically acceptable to the patient. "Pharmaceutically acceptable carriers" can further include pharmaceutically acceptable salts of compounds useful within the present disclosure. Other additional ingredients that may be included in pharmaceutical compositions used in the practice of the present disclosure are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0083] As used herein, the phrase "pharmaceutically acceptable salts" refers to salts of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.

[0084] As used herein, the term "polypeptide" refers to a polymer of amino acid residues, naturally occurring and related structural variants, and non-naturally occurring synthetic analogues thereof, linked through peptide bonds.

[0085] As used herein, the term "prevent" or "prevention" means that a disorder or disease will not occur if none has occurred, or that a disorder or disease will not occur if the disorder or disease has already occurred. The ability to prevent some or all of the symptoms associated with a disorder or disease is also contemplated.

[0086] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate the specific transcription of a polynucleotide sequence.

[0087] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be the core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0088] As used herein, the term "recombinant polypeptide" is defined as a polypeptide produced by using recombinant DNA methods.

[0089] As used herein, the term "recombinant DNA" is defined as DNA that is produced by joining fragments of DNA from different sources.

[0090] As used herein, "sample" or "biological sample" refers to biological material isolated from a subject. A biological sample can include any biological material suitable for detecting mRNA, polypeptides, or other markers of a physiological or pathological process in a subject, and can include fluids, tissues, cellular and / or non-cellular material obtained from an individual.

[0091] As used herein, the term "signal peptide" refers to a sequence of amino acid residues (e.g., ranging from 10 to 30 residues in length) that is attached to the amino terminus of a nascent protein of interest during protein translation. The signal peptide is recognized by the signal recognition particle (SRP) and cleaved by a signal peptidase after transport in the endoplasmic reticulum (Lodish, et al., 2000, Molecular Cell Biology, 4 th edition).

[0092] As used herein, "substantially purified" refers to being essentially free of other components. For example, a substantially purified polypeptide is one that has been separated from other components with which it is normally associated in its natural state. Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity, and 100% purity.

[0093] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0094] As used herein, the phrases "under transcriptional control" or "operably linked" mean that the promoter is in the correct location and orientation relative to the polynucleotide so as to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0095] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell has been transfected with, transformed with, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0096] As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., a compound useful within the present disclosure (alone or in combination with another pharmaceutical agent), to a patient, or to a tissue or cell line isolated from a patient having a disease or disorder or symptoms of a disease or disorder (e.g., for diagnostic or ex vivo applications), for the purpose of curing, healing, alleviating, mitigating, altering, curing, ameliorating, improving, or affecting a disease or disorder or symptoms of a disease or disorder. Such treatments can be specifically adapted or modified based on knowledge obtained from the field of pharmacogenomics.

[0097] A "variant," as the term is used herein, refers to a nucleic acid or peptide sequence that differs in sequence from a reference nucleic acid or peptide sequence, respectively, but retains essential properties of the reference molecule. Sequence changes in nucleic acid variants may not change the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Sequence changes in peptide variants are typically limited or conservative, so that the sequences of the reference peptide and variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions, in any combination. Nucleic acid or peptide variants may be naturally occurring, such as allelic variants, or may be variants that are not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides can be generated by mutagenesis techniques or by direct synthesis.

[0098] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0099] As used herein, the term "virus" is defined as a particle consisting of nucleic acid (RNA or DNA) encapsulated in a protein coat with or without an outer lipid envelope, and which is capable of transfecting the nucleic acid into cells.

[0100] As used herein, the term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is the most frequently observed gene in a population, and is therefore arbitrarily designated as the "normal" or "wild-type" form of the gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product that exhibits altered sequence and / or functional properties (i.e., altered characteristics) when compared with a wild-type gene or gene product. Naturally occurring mutants can be isolated; they are identified by the fact that they have altered characteristics (including altered nucleic acid sequences) when compared with a wild-type gene or gene product.

[0101] Ranges: Throughout this disclosure, various aspects of the present disclosure may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within the range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0102] Constructs and Polypeptides In one aspect, the present disclosure provides DNAse1-Fc and / or DNAse1L3-Fc constructs. The present disclosure contemplates that the constructs contemplated herein can have one or more of the mutations described herein.

[0103] Additionally, the present disclosure provides a homodimeric construct comprising two independently selected DNAse 1 constructs of the present disclosure. Additionally, the present disclosure provides a homodimeric construct comprising two independently selected DNAse 1 L3 constructs of the present disclosure. Additionally, the present disclosure provides a heterodimeric construct comprising a DNAse 1 construct of the present disclosure and a DNAse 1 L3 construct of the present disclosure.

[0104] The present disclosure provides the constructs described herein, as well as any glycosylation variants (alternative glycoforms), and constructs modified by site-directed mutagenesis or any type of protein chemistry engineering to improve solubility and / or enzymatic activity and / or in vivo half-life.

[0105] In certain embodiments, the construct comprises the amino acid sequence: DNAse1-X1-Linker-Fc-X2 (I) Contains, where: DNAse 1 is the human DNAse 1 polypeptide described elsewhere herein; X1 is a covalent bond or X1 is an amino acid sequence TIFF2025134821000010.tif4128 peptide or a fragment thereof; the linker is a chemical bond or a polypeptide comprising 1 to 100 amino acids; X2 is null or X2 is an amino acid sequence TIFF2025134821000011.tif4128 peptide or a fragment thereof; Fc is the Fc domain of human IgG1 as described elsewhere herein.

[0106] In certain embodiments, (I) describes the construct from left to right, from its N-terminus to its C-terminus, where the N-terminus of the Fc is linked to the C-terminus of DNAse 1. In certain embodiments, (I) describes the construct from left to right, from its C-terminus to its N-terminus, where the C-terminus of the Fc is linked to the N-terminus of DNAse 1.

[0107] In certain embodiments, the polypeptide has the amino acid sequence: DNAse1L3-X1-linker-Fc-X2 (II) Contains, where: DNAse1L3 is the human polypeptide DNAse1L3 described elsewhere herein; X1 is a covalent bond or X1 is an amino acid sequence TIFF2025134821000012.tif4128 peptide or a fragment thereof; the linker is a covalent bond or a polypeptide comprising 1 to 100 amino acids; X2 is null or X2 is an amino acid sequence TIFF2025134821000013.tif4128 peptide or a fragment thereof; Fc is the Fc domain of human IgG1 as described elsewhere herein.

[0108] In certain embodiments, (II) describes the construct from left to right, from its N-terminus to its C-terminus, where the N-terminus of the Fc is linked to the C-terminus of DNAse1L3. In certain embodiments, (II) describes the construct from left to right, from its C-terminus to its N-terminus, where the C-terminus of the Fc is linked to the N-terminus of DNAse1L3.

[0109] Fc: In one particular embodiment, the Fc domain of human IgG1 has the following sequence: SEQ ID NO:4 hIgG Fc domain, Fc (human) TIFF2025134821000014.tif25158

[0110] In one particular embodiment, the Fc domain of a mouse IgG1 has the following sequence: SEQ ID NO:31 hIgG Fc domain, Fc (mouse) TIFF2025134821000015.tif25158

[0111] In certain embodiments, Cys6 (C6) for SEQ ID NO:4 is mutated to another amino acid, such as, but not limited to, G or S. In certain embodiments, Cys9 (C9) for SEQ ID NO:4 is mutated to another amino acid, such as, but not limited to, Gly or Ser. In non-limiting embodiments, any one of such mutations in the C6 / C9 residues involved in the interchain disulfide bond in the heavy chain of the Fc domain converts a dimeric enzyme fusion into a monomeric fusion, thus allowing greater access to chromatin and microparticle DNA.

[0112] In certain embodiments, the hIgG Fc domain has at least one of the following mutations with respect to SEQ ID NO:4: M32Y, S34T, and T36E. In non-limiting embodiments, any such mutation enhances endosomal recycling of the corresponding construct. In certain embodiments, the hIgG Fc domain has the following mutations with respect to SEQ ID NO:4: M32Y, S34T, and T36E.

[0113] A non-limiting list of mutations contemplated in the Fc domain of a construct of the present disclosure includes C6S, C9S, M32Y, S34T, and / or T36E with respect to SEQ ID NO:4. In certain embodiments, the Fc domain of a construct comprises a C6S mutation with respect to SEQ ID NO:4. In certain embodiments, the Fc domain of a construct comprises a C9S mutation with respect to SEQ ID NO:4. In certain embodiments, the Fc domain of a construct comprises an M32Y mutation with respect to SEQ ID NO:4. In certain embodiments, the Fc domain of a construct comprises an S34T mutation with respect to SEQ ID NO:4. In certain embodiments, the Fc domain of a construct comprises a T36E mutation with respect to SEQ ID NO:4.

[0114] Linker: In certain embodiments, the linker is a chemical bond or is absent. In certain embodiments, the linker is a polypeptide comprising 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, and / or 1 to 5 amino acids. In certain embodiments, the linker comprises Gly and / or Ser amino acids.

[0115] In certain embodiments, the linker comprises GS. In certain embodiments, the linker comprises GSC. In certain embodiments, the linker comprises GGGGSGGGGS (SEQ ID NO:5). In certain embodiments, the linker comprises SSTMVRS (SEQ ID NO:40). In certain embodiments, the linker comprises SSTMVGS (SEQ ID NO:41).

[0116] In certain embodiments, the linker is TIFF2025134821000016.tif4128, wherein each occurrence of X is C, G, or S and each occurrence of Z is C, G, or S. In certain non-limiting embodiments, at least one of X and Z is not C, preventing disulfide bridge formation. In certain embodiments, SEQ ID NO:6 corresponds to the hinge region of human IgG1.

[0117] X1 and X2: In certain embodiments, X1 is a covalent bond. In certain embodiments, X1 is the amino acid sequence TIFF2025134821000017.tif4128 or a fragment thereof.

[0118] In certain embodiments, X2 is a covalent bond. In certain embodiments, X2 is the amino acid sequence TIFF2025134821000018.tif4128 or a fragment thereof.

[0119] DNAse1: An exemplary construct of the present disclosure comprises the amino acid sequence of SEQ ID NO:7, where the bolded sequence corresponds to the DNAse 1 polypeptide, the underlined sequence corresponds to the Fc, and the italicized sequence corresponds to the linker. TIFF2025134821000019.tif70158

[0120] In certain embodiments, the construct has one or more of the following mutations in the Fc: C290S, C293S, M316Y, S318T, and / or T320E relative to SEQ ID NO:7.

[0121] An exemplary construct of the present disclosure comprises the amino acid sequence of SEQ ID NO:8, where the bolded sequence corresponds to the DNAse 1 polypeptide, the underlined sequence corresponds to the Fc, and the italicized sequence corresponds to the linker. TIFF2025134821000020.tif69158

[0122] In certain embodiments, the construct lacks at least a portion of the signal peptide of DNAse 1 corresponding to residues 1-22 of SEQ ID NO: 1. In certain embodiments, the construct lacks the signal peptide of DNAse 1 corresponding to residues 1-22 of SEQ ID NO: 1.

[0123] A non-limiting list of contemplated mutations in the DNAse 1 domain of the constructs of the present disclosure with respect to SEQ ID NO:1 includes, but is not limited to, Q31R, E35R, Y46H, Y46S, V88N, N96K, D109N, V111T, A136F, R148S, E149N, M186I, L208P, D220N, D250N, A252T, G262N, D265N, and L267T.

[0124] In certain embodiments, the DNAse 1 domain of the construct comprises the mutation Q31R with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation E35R with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation Y46H with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation Y46S with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation V88N with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation N96K with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation D109N with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation V111T with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation A136F with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation R148S with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation E149N with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation M186I with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation L208P with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation D220N with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation D250N with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation A252T with respect to SEQ ID NO:1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation G262N with respect to SEQ ID NO:1.In certain embodiments, the DNAse 1 domain of the construct comprises the mutation D265N with respect to SEQ ID NO: 1. In certain embodiments, the DNAse 1 domain of the construct comprises the mutation L267T with respect to SEQ ID NO: 1.

[0125] In one specific, non-limiting embodiment, the mutation A136F with respect to SEQ ID NO:1 reduces actin binding of the construct.

[0126] In certain non-limiting embodiments, the mutations E35R, Y46H, Y46S, R148S, E149N, M186I, L208P, and / or D220N increase the enzymatic activity of the construct.

[0127] In certain non-limiting embodiments, the mutations V88N, D109N, V111T, G262N, D265N, and / or L267T alter the overall glycosylation status of the construct.

[0128] Non-limiting examples of constructs of the present disclosure include the following amino acid sequences, where the bolded sequence corresponds to the DNAse 1 polypeptide, the underlined sequence corresponds to Fc, the italicized sequence corresponds to the linker, and the italicized / underlined sequence corresponds to X1 / X2. Certain mutations are indicated as double underlined. TIFF2025134821000021.tif242158TIFF2025134821000022.tif221158In the sequence, X and Z are independently Cys, Gly, or Ser.

[0129] In certain non-limiting embodiments, where at least one of X and Z is not Cys (C), disulfide bridge formation is prevented. TIFF2025134821000023.tif228158

[0130] DNAse1L3: An exemplary construct of the present disclosure comprises the amino acid sequence of SEQ ID NO:18, where the bolded sequence corresponds to the DNAse1L3 polypeptide, the underlined sequence corresponds to the Fc, and the italicized sequence corresponds to the linker. TIFF2025134821000024.tif69158

[0131] In certain embodiments, the construct has one or more of the following mutations in the Fc: C313S, C316S, M339Y, S341T, and / or T342E relative to SEQ ID NO:18.

[0132] An exemplary construct of the present disclosure comprises the amino acid sequence of SEQ ID NO:19, where the bolded sequence corresponds to the DNAse1L3 polypeptide, the underlined sequence corresponds to the Fc, and the italicized sequence corresponds to the linker. TIFF2025134821000025.tif69158

[0133] In certain embodiments, the construct lacks at least a portion of the signal peptide of DNAse1L3 corresponding to residues 1-20 of SEQ ID NO:2. In certain embodiments, the construct lacks the signal peptide of DNAse1L3 corresponding to residues 1-20 of SEQ ID NO:2.

[0134] In certain embodiments, the construct lacks at least a portion of the nuclear localization sequence (NLS) of the DNAse1L3 polypeptide. In certain embodiments, the construct lacks residues 291-305 of SEQ ID NO:2. In certain embodiments, the construct lacks residues 292-304 of SEQ ID NO:2. In certain embodiments, the construct lacks residues 296-304 of SEQ ID NO:2. In certain embodiments, the construct lacks residues A-B of SEQ ID NO:2, where A is in the range of 291-296 and B is in the range of 304-305.

[0135] With respect to SEQ ID NO:18, a non-limiting list of contemplated mutations in the Fc domain of the constructs of the present disclosure include C313S, C316S, M339Y, S341T, and / or T342E.

[0136] With respect to SEQ ID NO:2, a non-limiting list of contemplated mutations in the DNAse1 L3 domain of the constructs of the present disclosure includes E33R, M42T, V44H, V88T, N96K, A127N, V129T, K147S, D148N, L207P, D219N, and / or V254T.

[0137] In certain embodiments, the DNAseIL3 domain of the construct comprises the mutation E33R with respect to SEQ ID NO:2. In certain embodiments, the DNAseIL3 domain of the construct comprises the mutation M42T with respect to SEQ ID NO:2. In certain embodiments, the DNAseIL3 domain of the construct comprises the mutation V44H with respect to SEQ ID NO:2. In certain embodiments, the DNAseIL3 domain of the construct comprises the mutation V88T with respect to SEQ ID NO:2. In certain embodiments, the DNAseIL3 domain of the construct comprises the mutation N96K with respect to SEQ ID NO:2. In certain embodiments, the DNAseIL3 domain of the construct comprises the mutation A127N with respect to SEQ ID NO:2. In certain embodiments, the DNAseIL3 domain of the construct comprises the mutation V129T with respect to SEQ ID NO:2. In certain embodiments, the DNAseIL3 domain of the construct comprises the mutation K147S with respect to SEQ ID NO:2. In certain embodiments, the DNAseIL3 domain of the construct comprises a mutation D148N with respect to SEQ ID NO:2. In certain embodiments, the DNAseIL3 domain of the construct comprises a mutation L207P with respect to SEQ ID NO:2. In certain embodiments, the DNAseIL3 domain of the construct comprises a mutation D219N with respect to SEQ ID NO:2. In certain embodiments, the DNAseIL3 domain of the construct comprises a mutation V254T with respect to SEQ ID NO:2.

[0138] In one specific, non-limiting embodiment, the mutation A136F with respect to SEQ ID NO:1 reduces actin binding of the construct.

[0139] In certain non-limiting embodiments, the mutations E33R, V44H, N96K, K147S, D148N, L207P, and / or D219N relative to SEQ ID NO:1 increase the enzymatic activity of the construct.

[0140] In certain non-limiting embodiments, the mutation V254T alters the global glycosylation status of the construct.

[0141] Non-limiting examples of constructs of the present disclosure include the following amino acid sequences, where the bolded sequence corresponds to the DNAse1L3 polypeptide, the underlined sequence corresponds to Fc, the italicized sequence corresponds to the linker, and the italicized / underlined sequence corresponds to X1 / X2. Certain mutations are indicated as double underlined. TIFF2025134821000026.tif141158TIFF2025134821000027.tif242158TIFF2025134821000028.tif4157In the sequence, each occurrence of X and Z is independently Cys, Gly, or Ser.

[0142] In certain non-limiting embodiments, where at least one of X and Z is not Cys (C), disulfide bridge formation is prevented. In the TIFF2025134821000029.tif69158 sequence, X and Z are independently C, G, or S.

[0143] In certain non-limiting embodiments, where at least one of X and Z is not Cys (C), disulfide bridge formation is prevented. TIFF2025134821000030.tif105158TIFF2025134821000031.tif242158TIFF2025134821 000032.tif242158TIFF2025134821000033.tif242158TIFF2025134821000034.tif19158

[0144] In certain embodiments, the present disclosure contemplates constructs expressed from mammalian cell lines, such as, but not limited to, CHO cell lines, stably transfected with human ST6 beta-galactosamide alpha-2,6-sialyltransferase (ST6GAL1). In certain embodiments, such expression enhances sialylation of the construct. The present disclosure further provides constructs that are grown in cell culture supplemented with sialic acid and / or N-acetylmannosamine (1,3,4-O-Bu3ManNAc). In certain embodiments, such growth enhances sialic acid capping of the construct.

[0145] In certain embodiments, enhancing protein sialylation by expressing the biologic in CHO cells stably transfected with human alpha-2,6-sialyltransferase increases the bioavailability (C) of the construct when administered subcutaneously. max ) was substantially improved. In other embodiments, engineering the Fc domain to increase pH-dependent FcRn-mediated cellular recycling led to improved in vivo biological half-life. In yet other embodiments, combining CHO cells stably transfected with human α-2,6-sialyltransferase with growing the cells in N-acetylmannosamine led to a dramatic increase in half-life and / or biological exposure (AUC). In yet other embodiments, combining two or more methods described herein into a single construct led to a dramatic increase in half-life and / or biological exposure (AUC).

[0146] In certain embodiments, the constructs of the present disclosure are more highly glycosylated than other DNAse1 and / or DNAse1L3 constructs in the art. In other embodiments, the constructs of the present disclosure have a higher affinity for the neonatal orphan receptor (FcRn) than other DNAse1 and / or DNAse1L3 constructs in the art. In still other embodiments, the constructs of the present disclosure have a longer in vivo half-life than other DNAse1 and / or DNAse1L3 constructs in the art. In still other embodiments, the in vivo half-life of the constructs of the present disclosure is at least about 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 times longer than DNAse1 and / or DNAse1L3 constructs described in the art. In still other embodiments, the constructs of the present disclosure are administered to a subject at a lower dose and / or less frequently than other DNAse 1 and / or DNAse 1L3 constructs in the art. In still other embodiments, the constructs of the present disclosure are administered to a subject once a month, twice a month, three times a month, and / or four times a month. In still other embodiments, less frequent administration of the constructs of the present disclosure results in better patient compliance and / or increased efficacy when compared to other DNAse 1 and / or DNAse 1L3 constructs in the art.

[0147] In certain embodiments, the construct is soluble. In other embodiments, the construct is a recombinant polypeptide.

[0148] In certain embodiments, the construct comprises a signal peptide that results in the secretion of a precursor of the DNAse1 and / or DNAse1L3 polypeptide, which undergoes proteolytic processing to yield a processed construct comprising the DNAse1 and / or DNAse1L3 polypeptide.

[0149] In certain embodiments, the DNAse 1 and / or DNAse 1L3 polypeptides are C-terminally fused to the Fc domain of human immunoglobulin 1 (IgG1), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In other embodiments, the DNAse 1 and / or DNAse 1L3 polypeptides are N-terminally fused to the Fc domain of human immunoglobulin 1 (IgG1), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In yet other embodiments, the presence of the IgFc domain improves half-life, solubility, reduces immunogenicity, and enhances activity of the DNAse 1 and / or DNAse 1L3 polypeptides.

[0150] In certain embodiments, the DNAse1 and / or DNAse1L3 polypeptide is fused at the C-terminus to human serum albumin. Human serum albumin may be conjugated to the DNAse1 and / or DNAse1L3 protein via a chemical linker, including but not limited to, a naturally occurring or engineered disulfide bond, and / or by genetic fusion to DNAse1 and / or DNAse1L3, and / or fragments and / or variants thereof.

[0151] In certain embodiments, the construct is further pegylated (ie, fused with poly(ethylene glycol) chains).

[0152] In certain aspects, the constructs are formulated as liquid formulations. In other aspects, the present disclosure provides a dry product form of a pharmaceutical composition comprising a therapeutic amount of a construct of the present disclosure, the dry product being reconstitutable into a solution of the construct in liquid form.

[0153] The present disclosure provides kits comprising at least one construct of the present disclosure and / or a salt or solvate thereof, and instructions for using the construct within the methods of the present disclosure.

[0154] It will be understood that DNAse1 and / or DNAse1L3 polypeptides according to the present disclosure include not only the native human proteins, but also any fragments, derivatives, fusions, conjugates, or mutants thereof. In this disclosure, as used herein, the phrase "DNAse1 and / or DNAse1L3 polypeptides, mutants and / or mutant fragments thereof" also includes any compound or polypeptide (e.g., without limitation, fusion proteins) comprising DNAse1 and / or DNAse1L3 polypeptides, mutants and / or mutant fragments thereof. Fusion proteins according to the present disclosure are considered biological equivalents of DNAse1 and / or DNAse1L3, but in certain embodiments may provide longer half-life or greater potency due to increased in vivo biological exposure, as determined by "area under the curve" (AUC) or prolonged half-life in pharmacokinetic studies.

[0155] Vectors and cells The present disclosure further provides an autonomously replicating or integrating mammalian cell vector comprising a recombinant nucleic acid encoding a polypeptide of the present disclosure. In certain embodiments, the vector comprises a plasmid or a virus. In other embodiments, the vector comprises a mammalian cell expression vector. In still other embodiments, the vector further comprises at least one nucleic acid sequence that directs and / or controls expression of the polypeptide. In still other embodiments, the recombinant nucleic acid encodes a construct comprising a DNAse1 and / or DNAse1L3 polypeptide and a signal peptide, which is proteolytically processed upon secretion from the cell to yield the DNAse1 and / or DNAse1L3 construct of the present disclosure.

[0156] In yet another aspect, the present disclosure provides an isolated host cell comprising a vector of the present disclosure. In certain embodiments, the cell is a non-human cell. In other embodiments, the cell is mammalian. In still other embodiments, the vector of the present disclosure comprises a recombinant nucleic acid encoding a construct comprising a DNAse1 and / or DNAse1L3 polypeptide and a signal peptide. In still other embodiments, the polypeptide is proteolytically processed upon secretion from the cell to yield the DNAse1 and / or DNAse1L3 construct of the present disclosure.

[0157] Production and purification of DNAse1 and / or DNAse1L3 fusion proteins In certain embodiments, soluble DNAse1 and / or DNAse1L3 constructs comprising an IgG Fc domain or an enzymatically / biologically active fragment thereof are effective in treating, reducing, and / or preventing the progression of the diseases or disorders contemplated herein.

[0158] To generate soluble recombinant DNAse1 and / or DNAse1L3 constructs for in vitro use, DNAse1 and / or DNAse1L3 polypeptides can be fused to the Fc domain of IgG (referred to as "DNAse1-Fc" or "DNAse1L3-Fc"), and the fusion constructs can be expressed in stable CHO cell lines. The constructs can also be expressed in Hek293 cells, baculovirus-insect cell systems, CHO cells, or yeast Pichia expression systems using appropriate vectors. The constructs can be produced in either adherent or suspension cells. To establish stable cell lines, the nucleic acid sequences encoding the DNAse1 and / or DNAse1L3 constructs are cloned into vectors suitable for large-scale protein production.

[0159] Many expression systems are known that can be used to produce DNAse 1 and / or DNAse 1L3 constructs, including bacteria (e.g., E. coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae, Kluyveromyces lactis, and Pichia pastoris), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. The desired protein can be produced by conventional methods, for example, from a coding sequence inserted into a host chromosome or on a free plasmid.

[0160] Yeast can be transformed with the coding sequence for the desired protein by any one of the conventional methods, for example, electroporation. Methods for transforming yeast by electroporation are disclosed in Becker & Guarente, 1990, Methods Enzymol. 194:182. Successfully transformed cells, i.e., cells containing the DNA construct of the present disclosure, can be identified by well-known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce the desired polypeptide. Cells can be harvested and lysed, and their DNA content can be examined for the presence of the DNA using methods such as those described by Southern, 1975, J. Mol. Biol. 98:503 and / or Berent, et al., 1985, Biotech 3:208. Alternatively, antibodies can be used to detect the presence of the protein in the supernatant.

[0161] Useful yeast plasmid vectors include pRS403-406 and pRS413-416, which are publicly available from Strat:1.gene Cloning Systems, La Jolla, CA, USA. Plasmids pRS403, pRS404, pRS405, and pRS406 are Yeast Integrating plasmids (Y1p) and incorporate the yeast selectable markers I-11S3, TRP1, LEU2, and IJRA3. Plasmids pRS413-416 are Yeast Centromeric Plasmids (YCp).

[0162] Various methods have been developed for the functional linking of DNA to vectors via complementary cohesive ends.For example, complementary homopolymer tracts can be added to the DNA segment to be inserted into vector DNA.Then, the vector and DNA segment are linked by hydrogen bonds between complementary homopolymer tails to form recombinant DNA molecules.

[0163] Synthetic linkers containing one or more restriction sites provide an alternative method for joining DNA segments to vectors. DNA segments generated by endonuclease restriction digestion are treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding 3' single-stranded ends with their 3'-5' exonuclease activity and fill in recessed 3' ends with their polymerization activity.

[0164] Thus, the combination of these activities produces blunt-ended DNA segments.Then, in the presence of an enzyme that can catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase, the blunt-ended segments are incubated with a large molar excess of linker molecules.The product of this reaction is therefore a DNA segment that carries a polymer linker sequence at its end.Then, these DNA segments are cut with appropriate restriction enzymes and ligated into an expression vector that has been cut with an enzyme that produces ends that are compatible with the ends of the DNA segments.

[0165] Stably transfected single-cell clones are then established and screened for high-expression clones of the desired fusion protein. Screening of single-cell clones for DNAse1 and / or DNAse1L3 protein expression can be achieved in a high-throughput format in 96-well plates. Once high-expression clones are identified through screening, protein production can be achieved in shake flasks or bioreactors.

[0166] Purification of the DNAse1 and / or DNAse1L3 constructs can be achieved using a combination of standard purification techniques known in the art.

[0167] Gene therapy Nucleic acids encoding polypeptides useful within the present disclosure can be used in gene therapy protocols for the treatment of diseases or disorders contemplated herein. The improved constructs encoding the polypeptides can be inserted into appropriate gene therapy vectors, which can be administered to patients to treat or prevent the disease or disorder of interest.

[0168] Vectors, such as viral vectors, have been used in the prior art to introduce genes into a variety of different target cells. Typically, the vector is exposed to the target cells so that a sufficient proportion of cells can be transformed to produce a useful therapeutic or prophylactic effect from the expression of a desired polypeptide (e.g., a receptor). The transfected nucleic acid can be permanently integrated into the genome of each target cell, resulting in a long-lasting effect, or alternatively, the treatment may need to be repeated periodically. In certain embodiments, the (viral) vector transfects the genetic material encoding the polypeptide of the present disclosure into hepatocytes in vivo.

[0169] A variety of vectors, both viral and plasmid vectors, are known in the art (see, for example, U.S. Pat. No. 5,252,479 and WO 93 / 07282). In particular, several viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpesviruses, including HSV and EBV, and retroviruses. Many gene therapy protocols in the prior art have used neutralized murine retroviruses. Several recently issued patents relate to methods and compositions for performing gene therapy (see, for example, U.S. Pat. Nos. 6,168,916; 6,135,976; 5,965,541, and 6,129,705). Each of the aforementioned patents is incorporated herein by reference in its entirety.

[0170] AAV-mediated gene therapy: AAV, a parvovirus belonging to the Dependovirus genus, has several features that make it particularly well suited for gene therapy applications. For example, AAV can infect a wide range of host cells, including non-dividing cells. Furthermore, AAV can infect cells from a variety of species. Importantly, AAV has not been associated with any human or animal disease and does not appear to alter the physiological properties of host cells upon integration. Finally, AAV is stable under a wide range of physical and chemical conditions, which meets the requirements for production, storage, and transportation.

[0171] The AAV genome, a linear, single-stranded DNA molecule comprising approximately 4,700 nucleotides (the AAV-2 genome consists of 4,681 nucleotides, and the AAV-4 genome consists of 4,767 nucleotides), generally contains an internal unique segment flanked at each end by inverted terminal repeats (ITRs). The ITRs are approximately 145 nucleotides in length (AAV-1 has an ITR of 143 nucleotides) and have multiple functions, including serving as origins of replication and as packaging signals for the viral genome.

[0172] The internal, non-repeated portion of the genome contains two large open reading frames (ORFs), known as the AAV replication (rep) and capsid (cap) regions. These ORFs encode the replication and capsid gene products that enable replication, assembly, and packaging of complete AAV virions. More specifically, at least four families of viral proteins are expressed from the AAV rep region: Rep 78, Rep 68, Rep 52, and Rep 40, all of which are named for their apparent molecular weight. The AAV cap region encodes at least three proteins: VP1, VP2, and VP3.

[0173] AAV is a helper-dependent virus; that is, it requires coinfection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) to form functionally complete AAV virions. In the absence of coinfection with a helper virus, AAV establishes a latent state in which the viral genome integrates into the host cell chromosome or exists in an episomal form, but no infectious virions are produced. Subsequent infection with a helper virus "rescues" the integrated genome, allowing it to replicate and be packaged into viral capsids, thereby reconstituting infectious virions. While AAV can infect cells from different species, the helper virus must be of the same species as the host cell. Thus, for example, human AAV replicates in canine cells coinfected with canine adenovirus.

[0174] To produce infectious recombinant AAV (rAAV) containing heterologous nucleic acid sequences, AAV vectors containing heterologous nucleic acid sequences but lacking AAV helper function genes, rep and cap, can be transfected into suitable host cell lines.Then, AAV-helper function genes can be provided on separate vectors.In addition, instead of providing replication-competent helper viruses (such as adenovirus, herpesvirus, or vaccinia), only the helper virus genes (i.e., accessory function genes) required for AAV production can be provided on vectors.

[0175] In summary, AAV helper function genes (i.e., rep and cap) and accessory function genes can be provided on one or more vectors. The products of the helper and accessory function genes can then be expressed in host cells, where they are believed to act in trans on the rAAV vector containing the heterologous nucleic acid sequence. The rAAV vector containing the heterologous nucleic acid sequence is then believed to replicate and package as if it were a wild-type (WT) AAV genome, forming recombinant virions. When a patient's cells are infected with the resulting rAAV virions, the heterologous nucleic acid sequence enters the patient's cells and is expressed therein. Because the patient's cells lack the rep and cap genes and accessory function genes, the rAAV cannot further replicate and package its genome. Furthermore, without a source of the rep and cap genes, wtAAV cannot be formed in the patient's cells.

[0176] There are 11 known AAV serotypes, AAV-1 through AAV-11 (Mori, et al., 2004, Virology 330(2):375-83). AAV-2 is the most prevalent serotype in the human population; one study estimated that at least 80% of the general population has been infected with wild-type AAV-2 (Berns and Linden, 1995, Bioessays 17:237-245). AAV-3 and AAV-5 are also prevalent in the human population, with infection rates of up to 60% (Georg-Fries, et al., 1984, Virology 134:64-71). Although AAV-1 and AAV-4 are simian isolates, both serotypes can transduce human cells (Chiorini, et al., 1997, J Virol 71:6823-6833; Chou, et al., 2000, Mol Ther 2:619-623). Of the six known serotypes, AAV-2 is the best characterized. For example, AAV-2 has been used in a wide range of in vivo transduction experiments and has been shown to transduce many different tissue types, including mouse (U.S. Pat. No. 5,858,351; U.S. Pat. No. 6,093,392), dog muscle; mouse liver (Couto, et al., 1999, Proc. Natl. Acad. Sci. USA 96:12725-12730; Couto, et al., 1997, J. Virol. 73:5438-5447; Nakai, et al., 1999, J. Virol. 73:5438-5447; and Snyder, et al., 1997, Nat. Genet. 16:270-276); mouse heart (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806); rabbit lungs (Flotte, et al., 1993, Proc. Natl. Acad. Sci. USA 90:10613-10617); and rodent photoreceptor cells (Flannery et al., 1997, Proc. Natl. Acad. Sci. USA 94:6916-6921).

[0177] The broad tissue tropism of AAV-2 can be exploited to deliver tissue-specific transgenes. For example, AAV-2 vectors have been used to deliver the following genes: the cystic fibrosis transmembrane conductance regulator gene to rabbit lung (Flotte, et al., 1993, Proc. Natl. Acad. Sci. USA 90:10613-10617); the factor NIII gene (Burton, et al., 1999, Proc. Natl. Acad. Sci. USA 96:12725-12730) and the factor IX gene (Nakai, et al., 1999, J. Virol. 73:5438-5447; Snyder, et al., 1997, Nat. Genet. 1999) to mouse liver, dog, and mouse muscle (U.S. Patent No. 6,093,392). 16:270-276; U.S. Patent No. 6,093,392); the erythropoietin gene into mouse muscle (U.S. Patent No. 5,858,351); the vascular endothelial growth factor (VEGF) gene into mouse heart (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806); and the aromatic 1-amino acid decarboxylase gene into monkey neurons. Expression of certain transgenes delivered by rAAV has therapeutic effects in experimental animals; for example, expression of factor IX was reported to restore phenotypic normality in a canine model of hemophilia B (U.S. Patent No. 6,093,392). Furthermore, expression of NEGF delivered by rAAV to the myocardium of mice resulted in the formation of new blood vessels (Su, et al., 2000, Proc. Natl. Acad. Sci. USA 97:13801-13806), and expression of AADC delivered by rAAV to the brain of Parkinson's disease-like monkeys resulted in the restoration of dopaminergic function.

[0178] The delivery of protein of interest to mammalian cells is achieved by first producing the AAV vector that comprises the DNA that encodes protein of interest, and then administering this vector to mammalian cells.Therefore, the present disclosure should be interpreted as including the AAV vector that comprises the DNA that encodes polypeptide of interest.Once equipped with the present disclosure, the generation of the AAV vector that comprises the DNA that encodes this / these polypeptides will be clear to those skilled in the art.

[0179] In certain embodiments, the rAAV vector of the present disclosure comprises several essential DNA elements. In certain embodiments, these DNA elements include at least two copies of the AAV ITR sequence, a promoter / enhancer element, a transcription termination signal, and any necessary 5' or 3' untranslated region adjacent to the DNA encoding the protein of interest or its biologically active fragment. The rAAV vector of the present disclosure can also comprise a portion of the intron of the protein of interest. Optionally, the rAAV vector of the present disclosure also comprises DNA encoding a mutant polypeptide of interest.

[0180] In certain embodiments, the vector comprises a promoter / regulatory sequence, including a promiscuous promoter, capable of driving high levels of heterologous gene expression in many different cell types. Such promoters include, but are not limited to, the cytomegalovirus (CMV) immediate-early promoter / enhancer sequence, the Rous sarcoma virus promoter / enhancer sequence, and the like. In certain embodiments, the promoter / regulatory sequence in the rAAV vectors of the present disclosure is a CMV immediate-early promoter / enhancer. However, the promoter sequence used to drive expression of the heterologous gene may also be an inducible promoter, such as, but not limited to, a steroid-inducible promoter, or a tissue-specific promoter, such as, but not limited to, the skeletal alpha-actin promoter, which is specific to muscle tissue, and the muscle creatine kinase promoter / enhancer.

[0181] In certain embodiments, the rAAV vectors of the present disclosure comprise a transcription termination signal. While any transcription termination signal can be included in the vectors of the present disclosure, in certain embodiments, the transcription termination signal is an SV40 transcription termination signal.

[0182] In certain embodiments, the rAAV vector of the present disclosure comprises an isolated DNA encoding a polypeptide of interest or a biologically active fragment of a polypeptide of interest.The present disclosure should be interpreted as including any mammalian sequence of a polypeptide of interest, whether known or unknown.Therefore, the present disclosure should be interpreted as including a gene from a mammal other than human, whose polypeptide functions in a manner substantially similar to that of a human polypeptide.Preferably, the nucleotide sequence comprising the gene encoding the polypeptide of interest is about 50% homologous to the gene encoding the polypeptide of interest, more preferably about 70% homologous, even more preferably about 80% homologous, and most preferably about 90% homologous.

[0183] Additionally, the present disclosure should be construed to include naturally occurring variants or recombinantly derived mutants of the wild-type protein sequence, which variants or mutants cause the polypeptides encoded thereby to be either as therapeutically effective as the full-length polypeptides in the gene therapy methods of the present disclosure, or even more therapeutically effective than the full-length polypeptides.

[0184] The present disclosure should also be construed as including DNA encoding variants that retain the biological activity of the polypeptide. Such variants include proteins or polypeptides that have been modified or can be modified using recombinant DNA technology so that the protein or polypeptide has additional properties that enhance its suitability for use in the methods described herein, including, but not limited to, variants that enhance the stability of the protein in plasma and enhance the specific activity of the protein. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences, or by modifications that do not affect the sequence, or both. For example, conservative amino acid changes can be made that change the primary sequence of the protein or peptide but do not usually change its function.

[0185] The present disclosure is not limited to the particular rAAV vectors exemplified in the experimental examples; rather, the present disclosure should be construed to include any suitable AAV vector, including, but not limited to, vectors based on AAV-1, AAV-3, AAV-4, and AAV-6.

[0186] The present disclosure also includes a method for treating a mammal with a disease or disorder with an amount effective to produce a therapeutic effect. The method comprises administering to the mammal an rAAV vector encoding a polypeptide of interest. Preferably, the mammal is a human.

[0187] Typically, the number of viral vector genomes / mammal administered in a single injection is approximately 1 x 10 8 ~Approx. 5×10 16 Preferably, the number of viral vector genomes administered in a single injection per mammal is in the range of about 1 x 10 10 ~Approx. 1×10 15 more preferably, the number of viral vector genomes administered in a single injection / mammal is about 5 x 10 10 ~Approx. 5×10 15 most preferably, the number of viral vector genomes administered to a mammal in a single injection is about 5 x 10 11~Approx. 5×10 14 There are individuals.

[0188] When the methods of the present disclosure involve multiple site simultaneous injections, or several multiple site injections, including injections at different sites over the course of several hours (e.g., less than about 1 hour to about 2 or 3 hours), the total number of viral vector genomes administered can be the same as, or a fraction of, or a multiple of, that described for single site injection methods.

[0189] For administration of the rAAV vectors of the present disclosure in a single-site injection, in certain embodiments, a composition comprising the virus is injected directly into an organ of the subject (for example, but not limited to, the liver of the subject).

[0190] For administration to mammals, the rAAV vector can be suspended in a pharmaceutically acceptable carrier, such as HEPES-buffered saline at a pH of about 7.8. Other useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol, and other pharmaceutically acceptable salt solutions, such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0191] The rAAV vectors of the disclosure can also be provided in the form of a kit, including, for example, a lyophilized preparation of the vector in a dry salt formulation, sterile water for suspension of the vector / salt composition, and instructions for suspension of the vector and its administration to a mammal.

[0192] method The present disclosure includes methods of treating, ameliorating, and / or preventing multiple forms of lupus (including SLE) associated with DNAse1L3 deficiencies.

[0193] The present disclosure includes methods of treating, ameliorating, and / or preventing diseases and / or disorders associated with inefficient NET hydrolysis ("NETolysis").

[0194] The present disclosure includes methods of treating, ameliorating, and / or preventing autoimmune disorders. In certain embodiments, the autoimmune disorders include lupus (including SLE), autoimmune thyroid disease, and / or hypocomplementemic urticarial vasculitis syndrome (HUVS).

[0195] The present disclosure includes methods of treating, ameliorating, and / or preventing pathological thrombosis, including, but not limited to, microvascular thrombosis, venous thrombosis, and / or arterial thrombosis. In certain embodiments, the pathological thrombosis includes neutrophilic thrombosis, including, but not limited to, antineutrophil cytoplasmic autoantibody (ANCA) vasculitis, thrombotic thrombocytopenic purpura (TTP), and Behcet's disease or syndrome. In certain embodiments, the pathological thrombosis includes thrombosis resulting in stroke.

[0196] The present disclosure includes methods of treating, ameliorating, and / or preventing myocardial infarction.

[0197] The present disclosure includes methods of treating, ameliorating, and / or preventing cancer spread and progression (eg, cancer metastasis).

[0198] In certain embodiments, the methods comprise administering a construct of the present disclosure to a subject suffering from, suspected of suffering from, and / or susceptible to developing any disease or disorder contemplated herein.

[0199] In certain embodiments, the constructs of the present disclosure are secreted products of DNAse1 and / or DNAse1L3 precursor constructs (themselves contemplated within the present disclosure) expressed in mammalian cells. In other embodiments, the DNAse1 and / or DNAse1L3 precursor constructs include a signal peptide sequence and a DNAse1 and / or DNAse1L3 polypeptide, and the DNAse1 and / or DNAse1L3 precursor constructs undergo proteolytic processing to result in a processed construct comprising a DNAse1 and / or DNAse1L3 polypeptide. In yet other embodiments, in the DNAse1 and / or DNAse1L3 precursor constructs, the signal peptide sequence is conjugated to the N-terminus of the DNAse1 and / or DNAse1L3 polypeptide. Upon proteolysis, the signal sequence is cleaved from the DNAse1 and / or DNAse1L3 precursor construct, resulting in a construct comprising a DNAse1 and / or DNAse1L3 polypeptide.

[0200] In certain embodiments, the construct is administered to the subject acutely or chronically, while in other embodiments, the construct is administered to the subject locally, regionally, parenterally, or systemically.

[0201] In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

[0202] In certain embodiments, the construct and / or its precursor construct is administered by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical. In other embodiments, the construct and / or its precursor construct is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

[0203] In certain embodiments, the construct and / or precursor construct thereof is administered to a subject acutely or chronically. In other embodiments, the construct and / or precursor construct thereof is administered to a subject locally, regionally, or systemically. In yet other embodiments, the construct and / or precursor construct thereof is delivered on an encoded vector, the vector encoding a protein, which is transcribed and translated from the vector upon administration of the vector to a subject.

[0204] Armed with the present disclosure, including the methods detailed herein, those skilled in the art will recognize that the present disclosure is not limited to the treatment of disease or disorder once established.In particular, the symptoms of disease or disorder do not need to be manifested to the point that they cause harm to the subject; in fact, disease or disorder does not need to be detected in the subject before treatment is administered.That is, serious symptoms from disease or disorder do not need to occur before the present disclosure can provide benefits.

[0205] Thus, the present disclosure includes methods for preventing disease and disorders in a subject, in that a polypeptide or construct of the present disclosure, as described more fully herein, can be administered to a subject prior to the onset of the disease or disorder, thereby preventing the occurrence of the disease or disorder. In particular, when the symptoms of the disease or disorder have not yet manifested to the point of causing harm to the subject; in fact, the disease or disorder does not need to be detected in a subject before treatment is administered. That is, significant symptoms from the disease or disorder do not need to occur before the present disclosure can provide benefits. Thus, the present disclosure includes methods for preventing or delaying the onset of a disease or disorder in a subject and / or reducing the progression or growth of a disease or disorder in a subject, in that a polypeptide of the present disclosure can be administered to a subject prior to the detection of the disease or disorder. In certain embodiments, a polypeptide of the present disclosure is administered to a subject with a strong family history of the disease or disorder, thereby preventing or delaying the onset or progression of the disease or disorder.

[0206] Armed with the present disclosure herein, one of skill in the art will therefore recognize that preventing a disease or disorder in a subject encompasses administering to the subject a polypeptide of the present disclosure as a preventative measure against the disease or disorder.

[0207] Pharmaceutical Compositions and Formulations The present disclosure provides pharmaceutical compositions comprising the polypeptides of the present disclosure within the methods described herein.

[0208] Such pharmaceutical compositions are in a form suitable for administration to a subject, and / or may further comprise one or more pharmaceutically acceptable carriers, one or more additional components, and / or some combination thereof. Various components of the pharmaceutical composition can be present in the form of a physiologically acceptable salt, for example, in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0209] In one embodiment, pharmaceutical compositions useful for practicing the methods of the present disclosure can be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day, hi other embodiments, pharmaceutical compositions useful for practicing the present disclosure can be administered to deliver a dose of 1 ng / kg / day to 500 mg / kg / day.

[0210] The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical compositions of the present disclosure will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition can contain from about 0.1% to about 100% (w / w) of the active ingredient.

[0211] Pharmaceutical compositions useful within the methods of the present disclosure may be suitably developed for inhaled, oral, rectal, vaginal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ocular, intrathecal, intravenous, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed red blood cells containing the active ingredient, and immunologically-based formulations. The route of administration will be readily apparent to one of skill in the art and will depend on any number of factors, including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, etc.

[0212] The preparation of pharmaceutical compositions described herein can be prepared by any method known or developed in the field of pharmacology.Generally, such preparation method comprises the step of combining active ingredient with carrier or one or more other accessory components, and then, if necessary or desirable, shaping or packaging the product into desired single or multiple dose units.

[0213] As used herein, a "unit dose" is a discrete amount of pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, or a convenient fraction of such a dosage, for example, half or one-third of such a dosage. The unit dosage form may be for a single daily dose, or for one of multiple daily doses (for example, about 1 to 4 times or more per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0214] Administration / Dosage The administration regimen can affect what constitutes an effective amount. For example, several divided doses as well as staggered doses can be administered daily or consecutively, or the dose can be continuously infused or injected as a bolus. Furthermore, the dosage of the therapeutic formulation can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0215] The compositions of the present disclosure can be administered to patients, such as mammals, for example, humans, at dosages and for periods effective to treat diseases or disorders in patients using known procedures. The effective amount of therapeutic compound required to achieve a therapeutic effect can vary depending on factors such as the activity of the specific compound used; the time of administration; the excretion rate of the compound; the duration of treatment; other drugs, compounds, or materials used in combination with the compound; the state of the disease or disorder of the patient being treated, age, sex, weight, condition, overall health, and previous medical history, as well as similar factors well known in the medical field. The dosage regimen can be adjusted to provide an optimal therapeutic response. The dosage is determined based on the biological activity of the therapeutic compound, which depends on the half-life and the area under the plasma time curve of the therapeutic compound. The polypeptide according to the present disclosure can be administered at appropriate time intervals, such as every 2 days, every 4 days, weekly, or monthly. The therapeutic dosage of the polypeptide of the present disclosure can also be determined based on the half-life or the rate at which the therapeutic polypeptide is excreted from the body. Polypeptides according to the present disclosure are administered at appropriate time intervals, either every two days, every four days, weekly, or monthly, to achieve a constant level of DNAse 1 and / or DNAse 1L3 enzymatic activity.

[0216] For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the present disclosure is about 0.01 to 50 mg / kg body weight / day. In some embodiments, the effective dose range for a therapeutic compound of the present disclosure is about 50 ng to 500 ng / kg body weight, preferably 100 ng to 300 ng / kg body weight. Those skilled in the art will be able to study the relevant factors and determine the effective amount of a therapeutic compound without undue experimentation.

[0217] The compound can be administered to patients as frequently as several times a day, or less frequently, for example, once a day, once a week, once every two weeks, once a month, or even less frequently, for example, once every few months, or even once a year or less.It is understood that the amount of compound administered per day can be administered, for example, every day, every other day, every two days, every three days, every four days, or every five days, including, but not limited to, every other day.For example, in every other day administration, a 5 mg dose per day is administered starting on Monday, followed by the first 5 mg dose per day on Wednesday, followed by the second 5 mg dose per day on Friday, etc.The frequency of administration is readily apparent to those skilled in the art and depends on a number of factors, for example, but not limited to, the type and severity of the disease being treated, and the type and age of the patient.

[0218] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0219] A physician, such as a physician, having ordinary skill in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, the physician or veterinarian can start the dose of the compound of the present disclosure used in the pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0220] In certain embodiments, the compositions of the present disclosure are administered to patients in dosages ranging from 1 to 5 or more times per day. In other embodiments, the compositions of the present disclosure are administered to patients in dosage ranges including, but not limited to, daily, every two days, once every three days to once a week and once every two weeks. The frequency of administration of the various combination compositions of the present disclosure will vary from subject to subject, depending on many factors, including, but not limited to, age, disease or disorder being treated, gender, overall health, and other factors. Therefore, the present disclosure should not be construed as limited to any particular dosing regime, and the exact dosage and composition administered to any patient will be determined by the attending physician, taking into account all other factors related to the patient.

[0221] In certain embodiments, the present disclosure relates to packaged pharmaceutical compositions comprising a container holding a therapeutically effective amount of a compound of the present disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

[0222] Administration route Routes of administration of any one of the compositions of the present disclosure include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, ​​vaginal (e.g., transvaginal and peri-vaginal), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and topical administration.

[0223] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration, etc. The formulations and compositions contemplated as useful in the present disclosure are not limited to the specific formulations and compositions described herein.

[0224] Parenteral administration As used herein, "parenteral administration" of a pharmaceutical composition includes any administration route characterized by physically creating a break in the tissue of a subject and administering the pharmaceutical composition through the break in the tissue.Thus, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injecting the composition, applying the composition through a surgical incision, applying the composition through a non-surgical wound that penetrates the tissue, etc.In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialysis infusion techniques.

[0225] Further dosage forms Additional dosage forms of the present disclosure include those described in U.S. Patent Nos. 6,340,475, 6,488,962, 6,451,808, 5,972,389, 5,582,837, and 5,007,790. Additional dosage forms of the present disclosure also include those described in U.S. Patent Application Nos. 20030147952, 20030104062, 20030104053, 20030044466, 20030039688, and 20020051820. Additional dosage forms of the present disclosure also include those described in PCT Application Nos. WO 03 / 35041, WO 03 / 35040, WO 03 / 35029, WO 03 / 35177, WO 03 / 35039, WO 02 / 96404, WO 02 / 32416, WO 01 / 97783, WO 01 / 56544, WO 01 / 32217, WO 98 / 55107, WO 98 / 11879, WO 97 / 47285, WO 93 / 18755, and WO 90 / 11757.

[0226] Controlled-Release Formulations and Drug Delivery Systems The controlled or sustained release formulation of pharmaceutical compositions of the present disclosure can be prepared using conventional technology.In some cases, the dosage form used can be provided as a sustained or controlled release of one or more active ingredients therein, for example, by using hydropropylmethylcellulose, other polymer matrices, gel, permeable membrane, osmotic system, multi-layer coating, microparticle, liposome or microsphere or their combinations, to provide desired release profile with various ratios.The single unit dosage form suitable for oral administration, for example, tablet, capsule, gel capsule and caplet, that is suitable for controlled release, is included in the present disclosure.

[0227] In certain aspects, the formulations of the present disclosure may be, but are not limited to, short-term, fast-release, and controlled, eg, sustained-release, delayed-release, and pulsed-release formulations.

[0228] The term "sustained release" is used in its conventional sense to refer to a drug formulation that provides gradual release of a drug over a long period of time, and can, although not necessarily, result in a substantially constant blood level of the drug over a long period of time.The period can be as long as one month or more, and should be longer than the release of the same amount of drug administered in a bolus form.For sustained release, the compound can be formulated with a suitable polymer or hydrophobic material that provides the compound with sustained release properties.Therefore, the compound for use in the method of the present disclosure can be administered, for example, in the form of microparticles by injection, or in the form of wafers or disks by implantation.In certain embodiments of the present disclosure, the compound of the present disclosure is administered to patients using a sustained release formulation, alone or in combination with another pharmaceutical agent.

[0229] The term "delayed release" is used herein in its conventional sense to refer to a drug formulation that provides an initial release of drug after some delay following drug administration, which may include, but is not necessarily, a delay of from about 10 minutes up to about 12 hours. The term "pulse release" is used herein in its conventional sense to refer to a drug formulation that provides release of drug in a manner that provides a pulsed plasma profile of drug after drug administration. The term "immediate release" is used herein in its conventional sense to refer to a drug formulation that provides release of drug immediately following drug administration.

[0230] As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any or all whole or partial increments thereof.

[0231] As used herein, rapid elimination refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration, and any and all whole or partial increments thereof.

[0232] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents are considered to be within the scope of this disclosure and encompassed by the claims appended hereto. For example, modifications of the reaction and preparation conditions, using art-recognized substitutes and no more than routine experimentation, are understood to be within the scope of this application.

[0233] Whenever values ​​and ranges are provided herein, it is understood that all values ​​and ranges subsumed within those values ​​and ranges are intended to be encompassed within the scope of the disclosure. Furthermore, all values ​​that fall within these ranges, as well as the upper or lower limits of a range of values, are also contemplated by the application.

[0234] The following examples further illustrate aspects of the present disclosure, but are in no way a limitation of the teachings or disclosure described herein. [Example]

[0235] The present disclosure will now be described in connection with the following examples, which are provided for illustrative purposes only, and the present disclosure is not limited to these examples, but rather encompasses all variations that become apparent as a result of the teachings provided herein.

[0236] Methods and Materials Unless otherwise stated, expression of constructs in CHO cells or modified CHO cells with and without supplementation, enzymatic assays, AUC assays, half-life assays can be performed using protocols described elsewhere herein or known in the art.

[0237] Area under the curve assay The area under the plasma concentration versus time curve, also known as the area under the curve (AUC), can be used as a means to assess the volume of distribution (V), total elimination clearance (CL), and bioavailability (F) for extravascular drug delivery. The area under the plasma concentration versus time curve for each expressed and purified DNAse1-Fc and / or DNAse1L3-Fc construct can be performed using the standard formula for determining half-life and bioavailability after a single subcutaneous injection of a biologic, as described in Equation 1.

[0238] Determining half-life Drug half-life (t 1 / 2 ) is the time it takes for the plasma concentration or amount of a drug or biologic in the body to decrease by 50%. The half-life value for each expressed and purified construct can be performed according to protocols described in the prior art and / or herein, e.g., Equation 1, which allows for the determination of half-life and bioavailability after a single subcutaneous injection of a biologic.

[0239] Drug half-life can be calculated using Equation 1, which correlates the relationship between systemic fractional concentration and time of a drug administered to a subcutaneous depot in a single injection. By plotting the data as fraction of drug absorbed (F) versus time (t), the data can be fitted to an equation for total systemic absorption of a drug administered to a subcutaneous depot at time t = 0, yielding elimination (k e ) and absorption (k a ) constants can be determined. TIFF2025134821000035.tif12128

[0240] Example Figure 1 illustrates neutrophil extracellular trap (NET) formation. Scanning electron microscopy of a neutrophil (marked A) casting a net (marked B) that captures Helicobacter pylori bacteria (some of which are marked C). Image taken from Kumamoto T, et al., 2006, Eur Heart J. 27(17):2081-7.

[0241] FIG. 2 illustrates non-limiting DNAse1-Fc constructs of the present disclosure, highlighting certain contemplated point mutations.

[0242] FIG. 3 illustrates non-limiting DNAse1L3-Fc constructs of the present disclosure, highlighting certain contemplated point mutations.

[0243] FIG. 4 illustrates non-limiting DNAse1-Fc constructs of the present disclosure, highlighting certain contemplated point mutations.

[0244] Figure 5 illustrates non-limiting constructs of the present disclosure, highlighting certain contemplated point mutations. In certain embodiments, certain mutations render rDNAse hyperactive and / or actin resistant (i.e., have reduced affinity for actin) and / or increase the half-life of the construct.

[0245] 6 illustrates non-limiting constructs of the present disclosure, with certain contemplated point mutations highlighted. In certain embodiments, the construct lacks at least a portion of the DNAse1L3 nuclear localization domain.

[0246] FIG. 7 depicts a gel showing that certain DNAse1L3 clones cleave chromatin, but this is not the case for certain DNAse1 clones.

[0247] Figure 8 illustrates non-limiting constructs of the present disclosure. In certain embodiments, the DNAse1 polypeptide is fused to the C-terminal tail of DNAse1L3.

[0248] FIG. 9 illustrates certain aspects of the production and purification of DNAse-Fc constructs.

[0249] 10A-10B illustrate the in vivo pharmacokinetics of certain NET-degrading constructs of the present disclosure.

[0250] FIG. 11 depicts a non-limiting purification gel of certain NET-degrading constructs of the present disclosure.

[0251] List of aspects: The following exemplary aspects are provided, the numbering of which should not be construed as designating a level of importance. Aspect 1 provides the following: Amino acid sequence: DNAse1-X1-Linker-Fc-X2 (I) A construct comprising: where: DNAse1 is a human DNAse1 polypeptide; X1 is a covalent bond or X1 is an amino acid sequence TIFF2025134821000036.tif4128 peptide or a fragment thereof; the linker is a chemical bond or a polypeptide containing 1 to 100 amino acids; X2 is null or X2 is a peptide of amino acid sequence SEQ ID NO:3 or a fragment thereof; Fc is the Fc domain of human IgG1; construct. Aspect 1 provides the following: Amino acid sequence: DNAse1L3-X1-linker-Fc-X2 (II) A construct comprising: where: DNAse1L3 is a human DNAse1L3 polypeptide; X1 is a covalent bond or X1 is an amino acid sequence TIFF2025134821000037.tif4128 peptide or a fragment thereof; the linker is a covalent bond or a polypeptide comprising 1 to 100 amino acids; X2 is null or X2 is a peptide of amino acid sequence SEQ ID NO:3 or a fragment thereof; Fc is the Fc domain of human IgG1; construct. Aspect 3 provides the following: The construct according to any one of aspects 1 to 2, wherein the Fc comprises the amino acid sequence of SEQ ID NO:4. Aspect 4 provides the following: The construct of embodiment 3, wherein at least one of C6 and C9 with respect to SEQ ID NO:4 is independently mutated to G or S. Aspect 5 provides the following: 5. The construct of any of embodiments 3-4, wherein each one of C6 and C9 with respect to SEQ ID NO:4 is independently mutated to G or S. Aspect 6 provides the following: 6. The construct of any of aspects 3 to 5, comprising at least one of the following mutations with respect to SEQ ID NO:4: M32Y, S34T, T36E. Aspect 7 provides the following: 7. The construct of any of aspects 3 to 6, comprising one of the following mutations with respect to SEQ ID NO:4: M32Y, S34T, T36E. Aspect 8 provides the following: A construct according to any one of aspects 1 to 7, wherein the linker is a chemical bond or is absent. Aspect 9 provides the following: 8. The construct of any of aspects 1 to 7, wherein the linker is a polypeptide comprising 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, and / or 1 to 5 amino acids. Aspect 10 provides the following: The construct according to any of embodiments 1 to 7 and 9, wherein the linker comprises GS and / or GSC. Aspect 11 provides the following: 11. The construct of any of aspects 1 to 7 and 9 to 10, wherein the linker comprises GGGGSGGGGS (SEQ ID NO: 5), SSTMVRS (SEQ ID NO: 40), and / or SSTMVGS (SEQ ID NO: 41). Aspect 12 provides the following: The linker TIFF2025134821000038.tif4128, wherein each occurrence of X is C, G, or S, and each occurrence of Z is C, G, or S. Aspect 13 provides the following: 13. The construct according to any one of aspects 1 to 12, wherein X1 is a covalent bond. Aspect 14 provides the following: X1 is the amino acid sequence 13. The construct according to any one of aspects 1 to 12, which is a peptide of TIFF2025134821000039.tif4128 or a fragment thereof. Aspect 15 provides the following: The construct according to any one of aspects 1 to 14, wherein X2 is a covalent bond. Aspect 16 provides the following: X2 is the amino acid sequence 15. The construct according to any one of embodiments 1 to 14, which is a peptide of TIFF2025134821000040.tif4128 or a fragment thereof. Aspect 17 provides the following: 17. The construct of any of embodiments 1 and 3 to 16, wherein the DNAse 1 lacks at least a portion of residues 1 to 22 corresponding to SEQ ID NO:1. Aspect 18 provides the following: 18. The construct of any of embodiments 1 and 3 to 17, wherein the DNAse 1 lacks residues 1 to 22 corresponding to SEQ ID NO: 1. Aspect 19 provides the following: 19. The construct of any of embodiments 1 and 3-18, wherein the DNAse 1 comprises at least one of the following mutations with respect to SEQ ID NO: 1: Q31R, E35R, Y46H, Y46S, V88N, N96K, D109N, V111T, A136F, R148S, E149N, M186I, L208P, D220N, D250N, A252T, G262N, D265N, and L267T. Aspect 20 provides the following: 20. The construct of any of embodiments 1 and 3-19, wherein the Fc comprises at least one of the following mutations with respect to SEQ ID NO:4: C6G, C6S, C9G, C9S, M32Y, S34T, and T36E. Aspect 21 provides the following: 21. The construct according to any one of embodiments 1 and 3 to 20, which is selected from the group consisting of SEQ ID NOs: 7 to 17 and 32 to 35. Aspect 22 provides the following: 17. The construct of any of aspects 2 to 16, wherein the DNAse1L3 lacks at least one of the following: residues 291 to 305 of SEQ ID NO:2; residues 292 to 304 of SEQ ID NO:2; residues 296 to 304 of SEQ ID NO:2; or residues A to B of SEQ ID NO:2, wherein A is in the range of 291 to 296 and B is in the range of 304 to 305. Aspect 23 provides the following: 23. The construct of any of aspects 2 to 16 and 22, wherein the DNAse1L3 comprises at least one of the following mutations with respect to SEQ ID NO:2: E33R, M42T, V44H, V88T, N96K, A127N, V129T, K147S, D148N, L207P, D219N, and V254T. Aspect 24 provides the following: 24. The construct of any of embodiments 2 to 16 and 22 to 23, wherein the Fc comprises at least one of the following mutations with respect to SEQ ID NO:4: C6G, C6S, C9G, C9S, M32Y, S34T, and T36E. Embodiment 25 provides the following: 25. The construct according to any one of aspects 2 to 16 and 22 to 24, which is selected from the group consisting of SEQ ID NOs: 18 to 28 and 36 to 39. Embodiment 26 provides the following: 26. The construct of any one of embodiments 1 to 25, which is expressed in a mammalian cell. Aspect 27 provides the following: 27. The construct of embodiment 26, wherein said mammalian cells are stably transfected with human ST6 beta-galactosamide alpha-2,6-sialyltransferase (also known as ST6GAL1). Embodiment 28 provides the following: 27. The construct of embodiment 26, wherein said mammalian cells are grown in a cell culture supplemented with sialic acid and / or N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc). Aspect 29 provides the following: 29. The construct of any of embodiments 1 to 28, which is soluble. Aspect 30 provides the following: A homodimeric construct comprising two independently selected constructs according to any one of embodiments 1, 3 to 20, and 26 to 29. Aspect 31 provides the following: A homodimeric construct comprising two independently selected constructs according to any one of aspects 2 to 16 and 22 to 29. Aspect 32 provides the following: A heterodimer construct comprising the construct according to any one of embodiments 1, 3 to 20, and 26 to 29, and the construct according to any one of embodiments 2 to 16 and 22 to 29. Aspect 33 provides the following: A method for treating, ameliorating, and / or preventing multiple forms of lupus associated with DNAse1 and / or DNAse1L3 deficiency in a subject, comprising administering to the subject a therapeutically effective amount of a construct described in any of embodiments 1 to 29. Embodiment 34 provides the following: 34. The method of embodiment 33, wherein said lupus comprises systemic lupus erythematosus (SLE). Embodiment 35 provides the following: 30. A method of treating, ameliorating, and / or preventing a disease and / or disorder associated with inefficient NETolysis in a subject, comprising administering to the subject a therapeutically effective amount of a construct of any of embodiments 1 to 29. Embodiment 36 provides the following: A method for treating, ameliorating, and / or preventing an autoimmune disorder associated with DNAse1 and / or DNAse1L3 deficiency in a subject, comprising administering to the subject a therapeutically effective amount of a construct described in any of embodiments 1 to 29. Aspect 37 provides the following: 37. The method of embodiment 36, wherein said autoimmune disorder comprises lupus, autoimmune thyroid disease, and / or hypocomplementemic urticarial vasculitis syndrome (HUVS). Embodiment 38 provides the following: 30. A method of treating, ameliorating, and / or preventing pathological thrombosis in a subject, comprising administering to the subject a therapeutically effective amount of a construct according to any one of embodiments 1 to 29. Aspect 39 provides the following: The method of embodiment 38, wherein said pathological thrombosis comprises microvascular thrombosis, venous thrombosis, and / or arterial thrombosis. Embodiment 40 provides the following: A method according to any of aspects 38-39, wherein said pathological thrombosis results in or predisposes said subject to stroke. Aspect 41 provides the following: A method according to any one of aspects 38 to 40, wherein the pathological thrombosis comprises neutrophilic thrombosis. Aspect 42 provides the following: 42. The method of embodiment 41, wherein said neutrophilic thrombosis comprises at least one of antineutrophil cytoplasmic autoantibody (ANCA) vasculitis, thrombotic thrombocytopenic purpura (TTP), and Behcet's (or Behcet's) disease or syndrome. Aspect 43 provides the following: 30. A method of treating, ameliorating, and / or preventing myocardial infarction in a subject, comprising administering to the subject a therapeutically effective amount of the construct of any of embodiments 1 to 29. Embodiment 44 provides the following: 30. A method of treating, ameliorating, and / or preventing cancer metastasis in a subject, the method comprising administering to the subject a therapeutically effective amount of the construct of any of embodiments 1 to 29. Embodiment 45 provides the following: A method according to any one of aspects 33 to 44, wherein in the DNAse1 and / or DNAse1L3 precursor construct, a signal peptide sequence is conjugated to the N-terminus of the DNAse1 and / or DNAse1L3 polypeptide. Embodiment 46 provides the following: the construct is a secreted product of a DNAse1 and / or DNAse1L3 precursor construct expressed in a mammalian cell; the DNAse1 and / or DNAse1L3 precursor construct comprises a signal peptide sequence and a DNAse1 and / or DNAse1L3 polypeptide; the DNAse1 and / or DNAse1L3 precursor construct undergoes proteolytic processing to yield said DNAse1 and / or DNAse1L3 construct; A method according to any one of aspects 33 to 45. Aspect 47 provides the following: 47. The method of embodiment 46, wherein said mammalian cells are stably transfected with human ST6 beta-galactosamide alpha-2,6-sialyltransferase (also known as ST6GAL1). Embodiment 48 provides the following: 47. The method of embodiment 46, wherein said mammalian cells are grown in a cell culture supplemented with sialic acid and / or N-acetylmannosamine (also known as 1,3,4-O-Bu3ManNAc). Aspect 49 provides the following: A method according to any one of aspects 33 to 48, wherein the construct is administered to the subject acutely or chronically. Embodiment 50 provides the following: Aspect 49. The method of any of aspects 33-48, wherein the construct is administered to the subject locally, regionally, parenterally, and / or systemically. Embodiment 51 provides the following: 51. The method of any of aspects 33 to 50, wherein the construct and / or precursor construct thereof is delivered to the subject on an encoding vector, the vector encoding the construct or precursor construct, which is transcribed and translated from the vector upon administration of the vector to the subject. Embodiment 52 provides the following: 52. The method of any of aspects 33-51, wherein the construct is administered to the subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical. Embodiment 53 provides the following: Aspect 53. The method of any of aspects 33-52, wherein the construct is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier. Embodiment 54 provides the following: The construct comprises: a homodimeric construct comprising two independently selected constructs according to any one of embodiments 1, 3 to 20, and 26 to 29; a homodimeric construct comprising two independently selected constructs according to any of aspects 2 to 16 and 22 to 29, and / or A heterodimer construct comprising a construct according to any one of aspects 1, 3 to 20, and 26 to 29, and a construct according to any one of aspects 2 to 16 and 22 to 29. 54. The method of any one of aspects 33 to 53, comprising: Embodiment 55 provides the following: The method according to any one of aspects 33 to 54, wherein the subject is a mammal. Embodiment 56 provides the following: 56. The method of embodiment 55, wherein the mammal is a human.

[0252] The disclosures of any and all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present disclosure has been disclosed in connection with specific embodiments, it is clear that other embodiments and modifications of the present disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the present disclosure. It is intended that the appended claims be construed to include all such embodiments and equivalent modifications.

[0253] Sequence information SEQUENCE LISTING <110> Yale University <120> Compositions And Methods For Treating, Ameliorating, and / or Preventing Diseases or Disorders Caused By or Associated With DNAse1 and / or DNAse1L3 Deficiency <150> US 62 / 959,932 <151> 2020-01-11 <160> 43 <170> PatentIn version 3.5 <210> 1 <211> 282 <212> PRT <213> Homo sapiens <400> 1 Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys 275 280 <210> 2 <211> 305 <212> PRT <213> Homo sapiens <400> 2 Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45 Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60 Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Ser 305 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 3 Arg Ala Phe Thr Asn Asn Arg Lys Ser Val Ser Leu Lys Lys Arg Lys 1 5 10 15 Lys Gly Asn Arg Ser 20 <210> 4 <211> 227 <212> PRT <213> Homo sapiens <400> 4 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 5 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 6 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <220> <221> MOD_RES <222> (13)..(13) <223> Cys, Gly, or Ser <220> <221> MOD_RES <222> (16)..(16) <223> Cys, Gly, or Ser <400> 6 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Xaa Pro Arg Xaa 1 5 10 15 Pro Ala Pro Glu Leu Leu Gly Gly Pro 20 25 <210> 7 <211> 511 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 7 Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys Gly Ser Asp Lys Thr His 275 280 285 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 290 295 300 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 305 310 315 320 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 325 330 335 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 340 345 350 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 355 360 365 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 370 375 380 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 385 390 395 400 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 405 410 415 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 420 425 430 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 435 440 445 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 450 455 460 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 465 470 475 480 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 485 490 495 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 510 <210> 8 <211> 511 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 8 Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys Gly Ser Asp Lys Thr His 275 280 285 Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 290 295 300 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu 305 310 315 320 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 325 330 335 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 340 345 350 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 355 360 365 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 370 375 380 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 385 390 395 400 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 405 410 415 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 420 425 430 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 435 440 445 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 450 455 460 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 465 470 475 480 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 485 490 495 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 510 <210> 9 <211> 511 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 9 Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30 Phe Gly Arg Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Asn Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asn Gln Thr Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Phe Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Asn Leu Ser Asn Gln Thr Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys Gly Ser Asp Lys Thr His 275 280 285 Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 290 295 300 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu 305 310 315 320 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 325 330 335 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 340 345 350 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 355 360 365 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 370 375 380 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 385 390 395 400 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 405 410 415 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 420 425 430 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 435 440 445 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 450 455 460 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 465 470 475 480 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 485 490 495 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 510 <210> 10 <211> 553 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 10 Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys Arg Ala Phe Thr Asn Asn 275 280 285 Arg Lys Ser Val Ser Leu Lys Lys Arg Lys Lys Gly Asn Arg Ser Gly 290 295 300 Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu 305 310 315 320 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 325 330 335 Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser 340 345 350 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 355 360 365 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 370 375 380 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 385 390 395 400 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 405 410 415 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 420 425 430 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 435 440 445 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 450 455 460 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 465 470 475 480 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 485 490 495 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 500 505 510 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 515 520 525 Ser Pro Gly Lys Arg Ala Phe Thr Asn Asn Arg Lys Ser Val Ser Leu 530 535 540 Lys Lys Arg Lys Lys Gly Asn Arg Ser 545 550 <210> 11 <211> 532 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 11 Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys Gly Ser Asp Lys Thr His 275 280 285 Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 290 295 300 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu 305 310 315 320 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 325 330 335 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 340 345 350 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 355 360 365 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 370 375 380 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 385 390 395 400 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 405 410 415 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 420 425 430 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 435 440 445 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 450 455 460 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 465 470 475 480 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 485 490 495 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Arg 500 505 510 Ala Phe Thr Asn Asn Arg Lys Ser Val Ser Leu Lys Lys Arg Lys Lys 515 520 525 Gly Asn Arg Ser 530 <210> 12 <211> 532 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 12 Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys Arg Ala Phe Thr Asn Asn 275 280 285 Arg Lys Ser Val Ser Leu Lys Lys Arg Lys Lys Gly Asn Arg Ser Gly 290 295 300 Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu 305 310 315 320 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 325 330 335 Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser 340 345 350 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 355 360 365 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 370 375 380 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 385 390 395 400 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 405 410 415 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 420 425 430 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 435 440 445 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 450 455 460 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 465 470 475 480 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 485 490 495 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 500 505 510 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 515 520 525 Ser Pro Gly Lys 530 <210> 13 <211> 519 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 13 Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys Gly Gly Gly Gly Ser Gly 275 280 285 Gly Gly Gly Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro 290 295 300 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 305 310 315 320 Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val 325 330 335 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 340 345 350 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 355 360 365 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 370 375 380 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 385 390 395 400 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 405 410 415 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 420 425 430 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 435 440 445 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 450 455 460 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 465 470 475 480 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 485 490 495 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 500 505 510 Leu Ser Leu Ser Pro Gly Lys 515 <210> 14 <211> 534 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <220> <221> MOD_RES <222> (295)..(295) <223> Cys, Gly, or Ser <220> <221> MOD_RES <222> (298)..(298) <223> Cys, Gly, or Ser <400> 14 Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys Glu Leu Lys Thr Pro Leu 275 280 285 Gly Asp Thr Thr His Thr Xaa Pro Arg Xaa Pro Ala Pro Glu Leu Leu 290 295 300 Gly Gly Pro Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu 305 310 315 320 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 325 330 335 Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp 340 345 350 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 355 360 365 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 370 375 380 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 385 390 395 400 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 405 410 415 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 420 425 430 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 435 440 445 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 450 455 460 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 465 470 475 480 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 485 490 495 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 500 505 510 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 515 520 525 Ser Leu Ser Pro Gly Lys 530 <210> 15 <211> 512 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 15 Met Arg Gly Met Lys Leu Leu Gly Leu Ala Leu Ala Leu Ala Leu Ala Leu Ala Leu Ala Leu 1 5 10 15 Leu Gln Gly Val Ser Leu Lys Ile Ile Ile Ile Phe Asn I Arg Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Is Containing Arg Tyr Asp and Is Containing Arg Asp 50 55 60 His Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Lys 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Phe Ile Val Arg Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Gln Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys Gly Ser Cys Asp Lys Thr 275 280 285 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 290 295 300 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg 305 310 315 320 Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 325 330 335 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 340 345 350 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 355 360 365 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 370 375 380 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 385 390 395 400 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 405 410 415 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 420 425 430 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 435 440 445 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 450 455 460 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 465 470 475 480 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 485 490 495 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 510 <210> 16 <211> 512 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 16 Met Arg Gly Met Lys Leu Leu Gly Ala Leu Leu Ala Leu Ala Ala Leu 1 5 10 15 Leu Gln Gly Ala Val Ser Leu Lys Ile Ala Ala Phe Asn Ile Arg Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Ile Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp 50 55 60 Ser His Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Lys 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Phe Ile Val Arg Phe Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Gln Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asn Gln Thr Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys Gly Ser Cys Asp Lys Thr 275 280 285 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 290 295 300 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg 305 310 315 320 Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 325 330 335 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 340 345 350 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 355 360 365 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 370 375 380 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 385 390 395 400 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 405 410 415 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 420 425 430 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 435 440 445 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 450 455 460 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 465 470 475 480 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 485 490 495 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 510 <210> 17 <211> 512 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 17 Met Arg Gly Met Lys Leu Leu Gly Leu Ala Leu Ala Leu Ala Leu Ala Leu Ala Leu Ala Leu 1 5 10 15 Leu Gln Gly Val Ser Leu Lys Ile Ile Ile Ile Phe Asn I Arg Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val 35 40 45 Gln Is Containing Arg Tyr Asp and Is Containing Arg Asp 50 55 60 His Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp 65 70 75 80 Ala Pro Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Lys 85 90 95 Ser Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Asn Arg Glu Pro Phe Ile Val Arg Phe Ser Arg Phe 130 135 140 Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly 145 150 155 160 Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Gln Glu Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Trp Thr Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Ala Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Met Leu Leu Gln Gly Ala Val Val Pro Asn Ser Thr Leu Pro Phe Asn 245 250 255 Phe Gln Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Met Leu Lys Gly Ser Cys Asp Lys Thr 275 280 285 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 290 295 300 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg 305 310 315 320 Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 325 330 335 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 340 345 350 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 355 360 365 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 370 375 380 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 385 390 395 400 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 405 410 415 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 420 425 430 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 435 440 445 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 450 455 460 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 465 470 475 480 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 485 490 495 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 510 <210> 18 <211> 534 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 18 Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45 Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60 Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Lys 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Ser Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 305 310 315 320 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 325 330 335 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 340 345 350 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 355 360 365 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 370 375 380 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 385 390 395 400 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 405 410 415 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 420 425 430 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 435 440 445 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 450 455 460 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 465 470 475 480 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 485 490 495 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 500 505 510 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 515 520 525 Ser Leu Ser Pro Gly Lys 530 <210> 19 <211> 534 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 19 Met Ser Arg Glu Lew Wing Pro Lew Lew Lew Lew Lew Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45 Arg Cys Asp Ile Leu Is Met Glu Lys Asp Ser Asn 50 55 60 Asn Arg With Cys Pro With Glue Lys With Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly With Thr Tyr Asn Tyr Val Served With Arg Leu Gly Arg Lys 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Ser Gly Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu 305 310 315 320 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 325 330 335 Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp 340 345 350 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 355 360 365 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 370 375 380 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 385 390 395 400 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 405 410 415 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 420 425 430 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 435 440 445 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 450 455 460 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 465 470 475 480 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 485 490 495 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 500 505 510 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 515 520 525 Ser Leu Ser Pro Gly Lys 530 <210> 20 <211> 555 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 20 Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45 Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60 Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Lys 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Ser Gly Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu 305 310 315 320 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 325 330 335 Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp 340 345 350 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 355 360 365 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 370 375 380 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 385 390 395 400 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 405 410 415 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 420 425 430 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 435 440 445 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 450 455 460 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 465 470 475 480 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 485 490 495 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 500 505 510 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 515 520 525 Ser Leu Ser Pro Gly Lys Arg Ala Phe Thr Asn Asn Arg Lys Ser Val 530 535 540 Ser Leu Lys Arg Lys Gly Asn Arg Ser 545 550 555 <210> 21 <211> 576 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 21 Met Ser Arg Glu Lew Wing Pro Lew Lew Lew Lew Lew Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45 Arg Cys Asp Ile Leu Is Met Glu Lys Asp Ser Asn 50 55 60 Asn Arg With Cys Pro With Glue Lys With Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly With Thr Tyr Asn Tyr Val Served With Arg Leu Gly Arg Lys 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Ser Arg Ala Phe Thr Asn Asn Arg Light Ser Val Ser Leu Light Light Arg 305 310 315 320 Lys Lys Gly Asn Arg Ser Gly Ser Asp Lys Thr His Thr Ser Pro Pro 325 330 335 Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 340 345 350 Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr 355 360 365 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 370 375 380 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 385 390 395 400 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 405 410 415 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 420 425 430 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 435 440 445 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 450 455 460 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 465 470 475 480 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 485 490 495 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 500 505 510 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 515 520 525 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 530 535 540 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Arg Ala Phe Thr Asn 545 550 555 560 Asn Arg Lys Ser Val Ser Leu Lys Lys Arg Lys Lys Gly Asn Arg Ser 565 570 575 <210> 22 <211> 555 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 22 Met Ser Arg Glu Lew Wing Pro Lew Lew Lew Lew Lew Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45 Arg Cys Asp Ile Leu Is Met Glu Lys Asp Ser Asn 50 55 60 Asn Arg With Cys Pro With Glue Lys With Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly With Thr Tyr Asn Tyr Val Served With Arg Leu Gly Arg Lys 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Ser Arg Ala Phe Thr Asn Asn Arg Lys Ser Val Ser Leu Lys Lys Arg 305 310 315 320 Lys Lys Gly Asn Arg Ser Gly Ser Asp Lys Thr His Thr Ser Pro Pro 325 330 335 Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 340 345 350 Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr 355 360 365 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 370 375 380 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 385 390 395 400 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 405 410 415 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 420 425 430 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 435 440 445 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu 450 455 460 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 465 470 475 480 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 485 490 495 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 500 505 510 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 515 520 525 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 530 535 540 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 545 550 555 <210> 23 <211> 542 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 23 Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45 Arg Cys Asp Ile Leu Is Met Glu Lys Asp Ser Asn 50 55 60 Asn Arg With Cys Pro With Glue Lys With Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly With Thr Tyr Asn Tyr Val Served With Arg Leu Gly Arg Lys 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Will Lys Glu Ile Asp Glu Leu Will Glu Will Tyr Thr Asp Will Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr 305 310 315 320 Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 325 330 335 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro 340 345 350 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 355 360 365 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 370 375 380 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 385 390 395 400 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 405 410 415 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 420 425 430 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 435 440 445 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 450 455 460 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 465 470 475 480 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 485 490 495 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 500 505 510 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 515 520 525 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535 540 <210> 24 <211> 557 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <220> <221> MOD_RES <222> (318)..(318) <223> Cys, Gly, or Ser <220> <221> MOD_RES <222> (321)..(321) <223> Cys, Gly, or Ser <220> <221> MOD_RES <222> (336)..(336) <223> Cys, Gly, or Ser <400> 24 Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45 Arg Cys Asp Ile Leu Is Met Glu Lys Asp Ser Asn 50 55 60 Asn Arg With Cys Pro With Glue Lys With Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly With Thr Tyr Asn Tyr Val Served With Arg Leu Gly Arg Lys 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Will Lys Glu Ile Asp Glu Leu Will Glu Will Tyr Thr Asp Will Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Ser Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Xaa Pro Arg 305 310 315 320 Xaa Pro Ala Pro Glu Phe Leu Gly Gly Pro Asp Lys Thr His Thr Xaa 325 330 335 Pro Pro Glx Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 340 345 350 Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu 355 360 365 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 370 375 380 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 385 390 395 400 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 405 410 415 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 420 425 430 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 435 440 445 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 450 455 460 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 465 470 475 480 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 485 490 495 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 500 505 510 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 515 520 525 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 530 535 540 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 545 550 555 <210> 25 <211> 519 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <220> <221> MOD_RES <222> (298)..(298) <223> Cys, Gly, or Ser <220> <221> MOD_RES <222> (301)..(301) <223> Cys, Gly, or Ser <400> 25 Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45 Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60 Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Lys 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Gly Ser Asp Lys Thr His Thr Xaa Pro Pro Glx Pro Ala Pro 290 295 300 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 305 310 315 320 Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val 325 330 335 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 340 345 350 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 355 360 365 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 370 375 380 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 385 390 395 400 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 405 410 415 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 420 425 430 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 435 440 445 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 450 455 460 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 465 470 475 480 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 485 490 495 How Does Glu Ala Leu Discover Tyr Thr Gln Lys Ser? 500 505 510 Leu Ser Leu Ser Pro Gly Lys 515 <210> 26 <211> 535 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 26 Met Ser Arg Glu Lew Wing Pro Lew Lew Lew Lew Lew Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Arg Ser Lys Gln Glu Asp Lys Asn Ala Thr Asp Val Ile Val Lys Val 35 40 45 Arg Cys Asp Ile Leu Is Met Glu Lys Asp Ser Asn 50 55 60 Asn Arg With Cys Pro With Glue Lys With Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly With Thr Tyr Asn Tyr Val Served With Arg Leu Gly Arg Lys 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 305 310 315 320 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 325 330 335 Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val 340 345 350 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 355 360 365 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 370 375 380 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 385 390 395 400 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 405 410 415 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 420 425 430 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 435 440 445 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 450 455 460 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 465 470 475 480 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 485 490 495 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 500 505 510 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 515 520 525 Leu Ser Leu Ser Pro Gly Lys 530 535 <210> 27 <211> 535 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 27 Met Ser Arg Glu Lew Wing Pro Lew Lew Lew Lew Lew Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Arg Ser Lys Gln Glu Asp Lys Asn Ala Thr Asp Val Ile Val Lys Val 35 40 45 Arg Cys Asp Ile Leu Is Met Glu Lys Asp Ser Asn 50 55 60 Asn Arg With Cys Pro With Glue Lys With Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly With Thr Tyr Asn Tyr Val Served With Arg Leu Gly Arg Lys 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Asn Asp 115 120 125 Thr Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 305 310 315 320 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 325 330 335 Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val 340 345 350 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 355 360 365 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 370 375 380 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 385 390 395 400 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 405 410 415 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 420 425 430 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 435 440 445 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 450 455 460 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 465 470 475 480 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 485 490 495 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 500 505 510 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 515 520 525 Leu Ser Leu Ser Pro Gly Lys 530 535 <210> 28 <211> 535 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 28 Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Arg Ser Lys Gln Glu Asp Lys Asn Ala Thr Asp Val Ile Val Lys Val 35 40 45 Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60 Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly Ile Thr Tyr Asn Tyr Thr Ile Ser Ser Arg Leu Gly Arg Lys 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Ser Gly Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 305 310 315 320 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 325 330 335 Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val 340 345 350 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 355 360 365 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 370 375 380 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 385 390 395 400 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 405 410 415 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 420 425 430 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 435 440 445 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 450 455 460 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 465 470 475 480 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 485 490 495 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 500 505 510 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 515 520 525 Leu Ser Leu Ser Pro Gly Lys 530 535 <210> 29 <211> 283 <212> PRT <213> Mus musculus <400> 29 Met Arg Tyr Thr Gly Leu Met Gly Thr Leu Leu Thr Leu Val Asn Leu 1 5 10 15 Leu Gln Leu Ala Gly Thr Leu Arg Ile Ala Ala Phe Asn Ile Arg Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Ser Val Tyr Phe Val 35 40 45 Lys Ile Leu Ser Arg Tyr Asp Ile Ala Val Ile Gln Glu Val Arg Asp 50 55 60 Ser His Leu Val Ala Val Gly Lys Leu Leu Asp Glu Leu Asn Arg Asp 65 70 75 80 Lys Pro Asp Thr Tyr Arg Tyr Val Val Ser Glu Pro Leu Gly Arg Lys 85 90 95 Ser Tyr Lys Glu Gln Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ile Leu Asp Ser Tyr Gln Tyr Asp Asp Gly Cys Glu Cys Gly Asn Asp 115 120 125 Thr Phe Ser Arg Glu Pro Ala Ile Val Lys Phe Phe Ser Pro Tyr Thr 130 135 140 Glu Val Gln Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Thr Glu 145 150 155 160 Ala Val Ser Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Trp 165 170 175 Gln Lys Trp Gly Leu Glu Asp Ile Met Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Thr Ser Ser Gln Trp Ser Ser Ile Arg Leu Arg 195 200 205 Thr Ser Pro Ile Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr 210 215 220 Val Thr Ser Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Ala 225 230 235 240 Leu Leu Gln Ala Ala Val Val Pro Asn Ser Ala Val Pro Phe Asp Phe 245 250 255 Gln Ala Glu Tyr Gly Leu Ser Asn Gln Leu Ala Glu Ala Ile Ser Asp 260 265 270 His Tyr Pro Val Glu Val Thr Leu Arg Lys Ile 275 280 <210> 30 <211> 310 <212> PRT <213> Mus musculus <400> 30 Met Ser Leu His Pro Ala Ser Pro Arg Leu Ala Ser Leu Leu Phe 1 5 10 15 His Asp Thr Leu Ala Leu Arg Leu Cys Ser Phe Asn 20 25 30 Val Arg Ser Phe Gly Ala Ser Lys Lys Glu Asn His Glu Ala Met Asp 35 40 45 Val Lys Ile Ile Lys Arg Cys Asp Leu Ile Leu Met Glu 50 55 60 Ile Lys Asp Ser Ser Asn Asn Ile Cys Pro Met Leu Met Glu Lys Leu 65 70 75 80 Asn Gly Asn Ser Arg Arg Ser Thr Thr Tyr Asn Tyr Val Ile Ser Ser 85 90 95 Arg Leu Gly Arg Asn Thr Tyr Lys Glu Gln Tyr Ala Phe Val Tyr Lys 100 105 110 Glu Lys Leu Val Ser Val Lys Thr Lys Tyr His Tyr His Asp Tyr Gln 115 120 125 Asp Gly Asp Thr Asp Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe 130 135 140 His Ser Pro Phe Thr Ala Val Lys Asp Phe Val Ile Val Pro Leu His 145 150 155 160 Thr Thr Pro Glu Thr Ser Val Lys Glu Ile Asp Glu Leu Val Asp Val 165 170 175 Tyr Thr Asp Val Arg Ser Gln Trp Lys Thr Glu Asn Phe Ile Phe Met 180 185 190 Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Gln 195 200 205 Asn Ile Arg Leu Arg Thr Asp Pro Lys Phe Val Trp Leu Ile Gly Asp 210 215 220 Gln Glu Asp Thr Thr Val Lys Lys Ser Thr Ser Cys Ala Tyr Asp Arg 225 230 235 240 Ile Val Leu Cys Gly Gln Glu Ile Val Asn Ser Val Val Pro Arg Ser 245 250 255 Ser Gly Val Phe Asp Phe Gln Lys Ala Tyr Asp Leu Ser Glu Glu Glu 260 265 270 Ala Leu Asp Val Ser Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser 275 280 285 Ser Arg Ala Phe Thr Asn Asn Arg Lys Ser Val Ser Leu Lys Lys Arg 290 295 300 Lys Lys Gly Asn Arg Ser 305 310 <210> 31 <211> 222 <212> PRT <213> Mus musculus <400> 31 Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe 1 5 10 15 Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Tyr Ile Thr Leu Glu Pro 20 25 30 Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val 35 40 45 Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr 50 55 60 Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu 65 70 75 80 Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys 85 90 95 Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser 100 105 110 Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro 115 120 125 Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile 130 135 140 Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly 145 150 155 160 Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp 165 170 175 Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp 180 185 190 Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His 195 200 205 Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 210 215 220 <210> 32 <211> 512 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 32 Met Arg Tyr Thr Gly Leu Met Gly Thr Leu Leu Thr Leu Val Asn Leu 1 5 10 15 Leu Gln Leu Ala Gly Thr Leu Arg Ile Ala Ala Phe Asn Ile Arg Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Ser Val Tyr Phe Val 35 40 45 Lys Ile Leu Ser Arg Tyr Asp Ile Ala Val Ile Gln Glu Val Arg Asp 50 55 60 Ser His Leu Val Ala Val Gly Lys Leu Leu Asp Glu Leu Asn Arg Asp 65 70 75 80 Lys Pro Asp Thr Tyr Arg Tyr Val Val Ser Glu Pro Leu Gly Arg Lys 85 90 95 Ser Tyr Lys Glu Gln Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ile Leu Asp Ser Tyr Gln Tyr Asp Asp Gly Cys Glu Cys Gly Asn Asp 115 120 125 Thr Phe Ser Arg Glu Pro Ala Ile Val Lys Phe Phe Ser Pro Tyr Thr 130 135 140 Glu Val Gln Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Thr Glu 145 150 155 160 Ala Val Ser Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Trp 165 170 175 Gln Lys Trp Gly Leu Glu Asp Ile Met Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Thr Ser Ser Gln Trp Ser Ser Ile Arg Leu Arg 195 200 205 Thr Ser Pro Ile Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr 210 215 220 Val Thr Ser Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Ala 225 230 235 240 Leu Leu Gln Ala Ala Val Val Pro Asn Ser Ala Val Pro Phe Asp Phe 245 250 255 Gln Ala Glu Tyr Gly Leu Ser Asn Gln Leu Ala Glu Ala Ile Ser Asp 260 265 270 His Tyr Pro Val Glu Val Thr Leu Arg Lys Ile Ser Ser Thr Met Val 275 280 285 Arg Ser Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser 290 295 300 Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Tyr Ile Thr Leu 305 310 315 320 Glu Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro 325 330 335 Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala 340 345 350 Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val 355 360 365 Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe 370 375 380 Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr 385 390 395 400 Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile 405 410 415 Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys 420 425 430 Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp 435 440 445 Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp 450 455 460 Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser 465 470 475 480 Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly 485 490 495 Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 500 505 510 <210> 33 <211> 513 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 33 Met Arg Tyr Thr Gly Leu Met Gly Thr Leu Leu Thr Leu Val Asn Leu 1 5 10 15 Leu Gln Leu Ala Gly Thr Leu Arg Ile Ala Ala Phe Asn Ile Arg Thr 20 25 30 Phe Gly Glu Thr Lys Met Ser Asn Ala Thr Leu Ser Val Tyr Phe Val 35 40 45 Lys Ile Leu Ser Arg Tyr Asp Ile Ala Val Ile Gln Glu Val Arg Asp 50 55 60 Ser His Leu Val Ala Val Gly Lys Leu Leu Asp Glu Leu Asn Arg Asp 65 70 75 80 Lys Pro Asp Thr Tyr Arg Tyr Val Val Ser Glu Pro Leu Gly Arg Lys 85 90 95 Ser Tyr Lys Glu Gln Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ile Leu Asp Ser Tyr Gln Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Ser Arg Glu Pro Phe Ile Val Lys Phe Phe Ser Pro Tyr 130 135 140 Thr Glu Val Gln Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Thr 145 150 155 160 Glu Ala Val Ser Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Trp Gln Lys Trp Gly Leu Glu Asp Ile Met Phe Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Thr Ser Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Arg Thr Ser Pro Ile Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Val Thr Ser Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Ala Leu Leu Gln Ala Ala Val Val Pro Asn Ser Ala Val Pro Phe Asp 245 250 255 Phe Gln Ala Glu Tyr Gly Leu Ser Asn Gln Leu Ala Glu Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Thr Leu Arg Lys Ile Ser Ser Thr Met 275 280 285 Val Gly Ser Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser 290 295 300 Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Tyr Ile Thr 305 310 315 320 Leu Glu Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp 325 330 335 Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr 340 345 350 Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser 355 360 365 Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu 370 375 380 Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys 385 390 395 400 Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr 405 410 415 Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr 420 425 430 Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln 435 440 445 Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met 450 455 460 Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys 465 470 475 480 Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu 485 490 495 Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly 500 505 510 Lys <210> 34 <211> 513 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 34 Met Arg Tyr Thr Gly Leu Met Gly Thr Leu Leu Thr Leu Val Asn Leu 1 5 10 15 Leu Gln Leu Ala Gly Thr Leu Arg Ile Ala Ala Phe Asn Ile Arg Thr 20 25 30 Phe Gly Arg Thr Lys Met Ser Asn Ala Thr Leu Ser Val Tyr Phe Val 35 40 45 Lys Ile Leu Ser Arg Tyr Asp Ile Ala Val Ile Gln Glu Val Arg Asp 50 55 60 Ser His Leu Val Ala Val Gly Lys Leu Leu Asp Glu Leu Asn Arg Asp 65 70 75 80 Lys Pro Asp Thr Tyr Arg Tyr Asn Val Ser Glu Pro Leu Gly Arg Lys 85 90 95 Ser Tyr Lys Glu Gln Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ile Leu Asp Ser Tyr Gln Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Ser Arg Glu Pro Ala Ile Val Lys Phe Phe Ser Pro Tyr 130 135 140 Thr Glu Val Gln Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Thr 145 150 155 160 Glu Ala Val Ser Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Trp Gln Lys Trp Gly Leu Glu Asp Ile Met Phe Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Thr Ser Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Arg Thr Ser Pro Ile Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Val Thr Ser Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Ala Leu Leu Gln Ala Ala Val Val Pro Asn Ser Ala Val Pro Phe Asp 245 250 255 Phe Gln Ala Glu Tyr Gly Leu Ser Asn Gln Leu Ala Glu Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Thr Leu Arg Lys Ile Ser Ser Thr Met 275 280 285 Val Gly Ser Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser 290 295 300 Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Tyr Ile Thr 305 310 315 320 Leu Glu Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp 325 330 335 Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr 340 345 350 Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser 355 360 365 Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu 370 375 380 Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys 385 390 395 400 Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr 405 410 415 Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr 420 425 430 Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln 435 440 445 Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met 450 455 460 Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys 465 470 475 480 Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu 485 490 495 Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly 500 505 510 Lys <210> 35 <211> 513 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 35 Met Arg Tyr Thr Gly Leu Met Gly Thr Leu Leu Thr Leu Val Asn Leu 1 5 10 15 Leu Gln Leu Ala Gly Thr Leu Arg Ile Ala Ala Phe Asn Ile Arg Thr 20 25 30 Phe Gly Arg Thr Lys Met Ser Asn Ala Thr Leu Ser Val Tyr Phe Val 35 40 45 Lys Ile Leu Ser Arg Tyr Asp Ile Ala Val Ile Gln Glu Val Arg Asp 50 55 60 Ser His Leu Val Ala Val Gly Lys Leu Leu Asp Glu Leu Asn Arg Asp 65 70 75 80 Lys Pro Asp Thr Tyr Arg Tyr Asn Val Ser Glu Pro Leu Gly Arg Lys 85 90 95 Ser Tyr Lys Glu Gln Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser 100 105 110 Ile Leu Asp Ser Tyr Gln Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn 115 120 125 Asp Thr Phe Ser Arg Glu Pro Ala Ile Val Lys Phe Phe Ser Pro Tyr 130 135 140 Thr Glu Val Gln Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Thr 145 150 155 160 Glu Ala Val Ser Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val 165 170 175 Trp Gln Lys Trp Gly Leu Glu Asp Ile Met Phe Met Gly Asp Phe Asn 180 185 190 Ala Gly Cys Ser Tyr Val Thr Ser Ser Gln Trp Ser Ser Ile Arg Leu 195 200 205 Arg Thr Ser Pro Ile Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr 210 215 220 Thr Val Thr Ser Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly 225 230 235 240 Ala Leu Leu Gln Ala Ala Val Val Pro Asn Ser Ala Val Pro Phe Asp 245 250 255 Phe Gln Ala Glu Tyr Gly Leu Ser Asn Gln Thr Ala Glu Ala Ile Ser 260 265 270 Asp His Tyr Pro Val Glu Val Thr Leu Arg Lys Ile Ser Ser Thr Met 275 280 285 Val Gly Ser Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser 290 295 300 Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Tyr Ile Thr 305 310 315 320 Leu Glu Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp 325 330 335 Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr 340 345 350 Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser 355 360 365 Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu 370 375 380 Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys 385 390 395 400 Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr 405 410 415 Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr 420 425 430 Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln 435 440 445 Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met 450 455 460 Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys 465 470 475 480 Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu 485 490 495 Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly 500 505 510 Lys <210> 36 <211> 539 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 36 Met Ser Leu His Pro Ala Ser Pro Arg Leu Ala Ser Leu Leu Leu Phe 1 5 10 15 Ile Leu Ala Leu His Asp Thr Leu Ala Leu Arg Leu Cys Ser Phe Asn 20 25 30 Val Arg Ser Phe Gly Ala Ser Lys Lys Glu Asn His Glu Ala Met Asp 35 40 45 Ile Ile Val Lys Ile Ile Lys Arg Cys Asp Leu Ile Leu Leu Met Glu 50 55 60 Ile Lys Asp Ser Ser Asn Asn Ile Cys Pro Met Leu Met Glu Lys Leu 65 70 75 80 Asn Gly Asn Ser Arg Arg Ser Thr Thr Tyr Asn Tyr Val Ile Ser Ser 85 90 95 Arg Leu Gly Arg Asn Thr Tyr Lys Glu Gln Tyr Ala Phe Val Tyr Lys 100 105 110 Glu Lys Leu Val Ser Val Lys Thr Lys Tyr His Tyr His Asp Tyr Gln 115 120 125 Asp Gly Asp Thr Asp Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe 130 135 140 His Ser Pro Phe Thr Ala Val Lys Asp Phe Val Ile Val Pro Leu His 145 150 155 160 Thr Thr Pro Glu Thr Ser Val Lys Glu Ile Asp Glu Leu Val Asp Val 165 170 175 Tyr Thr Asp Val Arg Ser Gln Trp Lys Thr Glu Asn Phe Ile Phe Met 180 185 190 Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Gln 195 200 205 Asn Ile Arg Leu Arg Thr Asp Pro Lys Phe Val Trp Leu Ile Gly Asp 210 215 220 Gln Glu Asp Thr Thr Val Lys Lys Ser Thr Ser Cys Ala Tyr Asp Arg 225 230 235 240 Ile Val Leu Cys Gly Gln Glu Ile Val Asn Ser Val Val Pro Arg Ser 245 250 255 Ser Gly Val Phe Asp Phe Gln Lys Ala Tyr Asp Leu Ser Glu Glu Glu 260 265 270 Ala Leu Asp Val Ser Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser 275 280 285 Ser Arg Ala Phe Thr Asn Asn Arg Lys Ser Val Ser Leu Lys Lys Arg 290 295 300 Lys Lys Gly Asn Arg Ser Ser Ser Thr Met Val Gly Ser Gly Cys Lys 305 310 315 320 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 325 330 335 Pro Lys Pro Lys Asp Val Leu Tyr Ile Thr Leu Glu Pro Lys Val Thr 340 345 350 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 355 360 365 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 370 375 380 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 385 390 395 400 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 405 410 415 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 420 425 430 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 435 440 445 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 450 455 460 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 465 470 475 480 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 485 490 495 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 500 505 510 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 515 520 525 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 530 535 <210> 37 <211> 519 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 37 Met Ser Leu His Pro Ala Ser Pro Arg Leu Ala Ser Leu Leu Phe 1 5 10 15 His Asp Thr Leu Ala Leu Arg Leu Cys Ser Phe Asn 20 25 30 Val Arg Ser Phe Gly Ala Ser Lys Lys Glu Asn His Glu Ala Met Asp 35 40 45 Val Lys Ile Ile Lys Arg Cys Asp Leu Ile Leu Met Glu 50 55 60 Ile Lys Asp Ser Ser Asn Asn Ile Cys Pro Met Leu Met Glu Lys Leu 65 70 75 80 Asn Gly Asn Ser Arg Arg Ser Thr Thr Tyr Asn Tyr Val Ile Ser Ser 85 90 95 Arg Leu Gly Arg Asn Thr Tyr Lys Glu Gln Tyr Ala Phe Val Tyr Lys 100 105 110 Glu Lys Leu Val Ser Val Lys Thr Lys Tyr His Tyr His Asp Tyr Gln 115 120 125 Asp Gly Asp Thr Asp Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe 130 135 140 His Ser Pro Phe Thr Ala Val Lys Asp Phe Val Ile Val Pro Leu His 145 150 155 160 Thr Thr Pro Glu Thr Ser Val Lys Glu Ile Asp Glu Leu Val Asp Val 165 170 175 Tyr Thr Asp Val Arg Ser Gln Trp Lys Thr Glu Asn Phe Ile Phe Met 180 185 190 Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Gln 195 200 205 Asn Ile Arg Leu Arg Thr Asp Pro Lys Phe Val Trp Leu Ile Gly Asp 210 215 220 Gln Glu Asp Thr Thr Val Lys Lys Ser Thr Ser Cys Ala Tyr Asp Arg 225 230 235 240 Ile Val Leu Cys Gly Gln Glu Ile Val Asn Ser Val Val Pro Arg Ser 245 250 255 Ser Gly Val Phe Asp Phe Gln Lys Ala Tyr Asp Leu Ser Glu Glu Glu 260 265 270 Ala Leu Asp Val Ser Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser 275 280 285 Ser Arg Ala Phe Thr Asn Asn Arg Ser Gly Cys Lys Pro Cys Ile Cys 290 295 300 Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys 305 310 315 320 Asp Val Leu Tyr Ile Thr Leu Glu Pro Lys Val Thr Cys Val Val Val 325 330 335 Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp 340 345 350 Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe 355 360 365 Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp 370 375 380 Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe 385 390 395 400 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys 405 410 415 Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys 420 425 430 Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp 435 440 445 Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys 450 455 460 Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser 465 470 475 480 Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr 485 490 495 Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser 500 505 510 Leu Ser His Ser Pro Gly Lys 515 <210> 38 <211> 539 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 38 Met Ser Leu His Pro Ala Ser Pro Arg Leu Ala Ser Leu Leu Leu Phe 1 5 10 15 Ile Leu Ala Leu His Asp Thr Leu Ala Leu Arg Leu Cys Ser Phe Asn 20 25 30 Val Arg Ser Phe Gly Arg Ser Lys Lys Glu Asn His Glu Ala Met Asp 35 40 45 Ile Ile Val Lys Ile Ile Lys Arg Cys Asp Leu Ile Leu Leu Met Glu 50 55 60 Ile Lys Asp Ser Ser Asn Asn Ile Cys Pro Met Leu Met Glu Lys Leu 65 70 75 80 Asn Gly Asn Ser Arg Arg Ser Thr Thr Tyr Asn Tyr Val Ile Ser Ser 85 90 95 Arg Leu Gly Arg Lys Thr Tyr Lys Glu Gln Tyr Ala Phe Val Tyr Lys 100 105 110 Glu Lys Leu Val Ser Val Lys Thr Lys Tyr His Tyr His Asp Tyr Gln 115 120 125 Asp Gly Asp Thr Asp Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe 130 135 140 His Ser Pro Phe Thr Ala Val Lys Asp Phe Val Ile Val Pro Leu His 145 150 155 160 Thr Thr Pro Glu Thr Ser Val Lys Glu Ile Asp Glu Leu Val Asp Val 165 170 175 Tyr Thr Asp Val Arg Ser Gln Trp Lys Thr Glu Asn Phe Ile Phe Met 180 185 190 Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Gln 195 200 205 Asn Ile Arg Leu Arg Thr Asp Pro Lys Phe Val Trp Leu Ile Gly Asp 210 215 220 Gln Glu Asp Thr Thr Val Lys Lys Ser Thr Ser Cys Ala Tyr Asp Arg 225 230 235 240 Ile Val Leu Cys Gly Gln Glu Ile Val Asn Ser Val Val Pro Arg Ser 245 250 255 Ser Gly Val Phe Asp Phe Gln Lys Ala Tyr Asp Leu Ser Glu Glu Glu 260 265 270 Ala Leu Asp Val Ser Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser 275 280 285 Ser Arg Ala Phe Thr Asn Asn Arg Lys Ser Val Ser Leu Lys Lys Arg 290 295 300 Lys Lys Gly Asn Arg Ser Ser Ser Thr Met Val Gly Ser Gly Cys Lys 305 310 315 320 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 325 330 335 Pro Lys Pro Lys Asp Val Leu Tyr Ile Thr Leu Glu Pro Lys Val Thr 340 345 350 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 355 360 365 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 370 375 380 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 385 390 395 400 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 405 410 415 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 420 425 430 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 435 440 445 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 450 455 460 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 465 470 475 480 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 485 490 495 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 500 505 510 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 515 520 525 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 530 535 <210> 39 <211> 539 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 39 Met Ser Leu His Pro Ala Ser Pro Arg Leu Ala Ser Leu Leu Phe 1 5 10 15 His Asp Thr Leu Ala Leu Arg Leu Cys Ser Phe Asn 20 25 30 Val Arg Ser Phe Gly Arg Ser Lys Lys Glu Asn His Glu Ala Met Asp 35 40 45 Val Lys Ile Ile Lys Arg Cys Asp Leu Ile Leu Met Glu 50 55 60 Ile Lys Asp Ser Ser Asn Asn Ile Cys Pro Met Leu Met Glu Lys Leu 65 70 75 80 Asn Gly Asn Ser Arg Arg Ser Thr Thr Tyr Asn Tyr Val Ile Ser Ser 85 90 95 Arg Leu Gly Arg Lys Thr Tyr Lys Glu Gln Tyr Ala Phe Val Tyr Lys 100 105 110 Glu Lys Leu Val Ser Val Lys Thr Lys Tyr His Tyr His Asp Tyr Gln 115 120 125 Asp Gly Asp Thr Asp Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe 130 135 140 His Ser Pro Phe Thr Ala Val Lys Asp Phe Val Ile Val Pro Leu His 145 150 155 160 Thr Thr Pro Glu Thr Ser Val Lys Glu Ile Asp Glu Leu Val Asp Val 165 170 175 Tyr Thr Asp Val Arg Ser Gln Trp Lys Thr Glu Asn Phe Ile Phe Met 180 185 190 Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Gln 195 200 205 Asn Ile Arg Leu Arg Thr Asp Pro Lys Phe Val Trp Leu Ile Gly Asp 210 215 220 Gln Glu Asp Thr Thr Val Lys Lys Ser Thr Ser Cys Ala Tyr Asp Arg 225 230 235 240 Ile Val Leu Cys Gly Gln Glu Ile Val Asn Ser Val Val Pro Arg Ser 245 250 255 Asn Gly Thr Phe Asp Phe Gln Lys Ala Tyr Asp Leu Ser Glu Glu Glu 260 265 270 Ala Leu Asp Val Ser Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser 275 280 285 Ser Arg Ala Phe Thr Asn Asn Arg Lys Ser Val Ser Leu Lys Lys Arg 290 295 300 Lys Lys Gly Asn Arg Ser Ser Ser Thr Met Val Gly Ser Gly Cys Lys 305 310 315 320 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 325 330 335 Pro Lys Pro Lys Asp Val Leu Tyr Ile Thr Leu Glu Pro Lys Val Thr 340 345 350 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 355 360 365 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 370 375 380 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 385 390 395 400 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 405 410 415 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 420 425 430 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 435 440 445 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 450 455 460 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 465 470 475 480 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 485 490 495 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 500 505 510 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 515 520 525 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 530 535 <210> 40 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 40 Ser Ser Thr Met Val Arg Ser 1 5 <210> 41 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 41 Ser Ser Thr Met Val Gly Ser 1 5 <210> 42 <211> 507 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 42 Met Gly Thr Leu Leu Thr Leu Val Asn Leu Leu Gln Leu Ala Gly Thr 1 5 10 15 Leu Arg Ile Ala Ala Phe Asn Ile Arg Thr Phe Gly Glu Thr Lys Met 20 25 30 Ser Asn Ala Thr Leu Ser Val Tyr Phe Val Lys Ile Leu Ser Arg Tyr 35 40 45 Asp Ile Ala Val Ile Gln Glu Val Arg Asp Ser His Leu Val Ala Val 50 55 60 Gly Lys Leu Leu Asp Glu Leu Asn Arg Asp Lys Pro Asp Thr Tyr Arg 65 70 75 80 Tyr Val Val Ser Glu Pro Leu Gly Arg Lys Ser Tyr Lys Glu Gln Tyr 85 90 95 Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ile Leu Asp Ser Tyr Gln 100 105 110 Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Ser Arg Glu 115 120 125 Pro Ala Ile Val Lys Phe Phe Ser Pro Tyr Thr Glu Val Gln Glu Phe 130 135 140 Ala Ile Val Pro Leu His Ala Ala Pro Thr Glu Ala Val Ser Glu Ile 145 150 155 160 Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Trp Gln Lys Trp Gly Leu 165 170 175 Glu Asp Ile Met Phe Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val 180 185 190 Thr Ser Ser Gln Trp Ser Ser Ile Arg Leu Arg Thr Ser Pro Ile Phe 195 200 205 Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Val Thr Ser Thr His 210 215 220 Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Ala Leu Leu Gln Ala Ala 225 230 235 240 Val Val Pro Asn Ser Ala Val Pro Phe Asp Phe Gln Ala Glu Tyr Gly 245 250 255 Leu Ser Asn Gln Leu Ala Glu Ala Ile Ser Asp His Tyr Pro Val Glu 260 265 270 Val Thr Leu Arg Lys Ile Ser Ser Thr Met Val Gly Ser Gly Cys Lys 275 280 285 Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 290 295 300 Pro Lys Pro Lys Asp Val Leu Tyr Ile Thr Leu Glu Pro Lys Val Thr 305 310 315 320 Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser 325 330 335 Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg 340 345 350 Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile 355 360 365 Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn 370 375 380 Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 385 390 395 400 Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu 405 410 415 Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe 420 425 430 Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala 435 440 445 Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr 450 455 460 Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly 465 470 475 480 Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His 485 490 495 Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 500 505 <210> 43 <211> 530 <212> PRT <213> Artificial Sequence <220> <223> Chemically synthesized <400> 43 Leu Ala Ser Leu Leu Leu Phe Ile Leu Ala Leu His Asp Thr Leu Ala 1 5 10 15 Leu Arg Leu Cys Ser Phe Asn Val Arg Ser Phe Gly Arg Ser Lys Lys 20 25 30 Glu Asn His Glu Ala Met Asp Ile Ile Val Lys Ile Ile Lys Arg Cys 35 40 45 Asp Leu Ile Leu Leu Met Glu Ile Lys Asp Ser Ser Asn Asn Ile Cys 50 55 60 Pro Met Leu Met Glu Lys Leu Asn Gly Asn Ser Arg Arg Ser Thr Thr 65 70 75 80 Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Lys Thr Tyr Lys Glu 85 90 95 Gln Tyr Ala Phe Val Tyr Lys Glu Lys Leu Val Ser Val Lys Thr Lys 100 105 110 Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Thr Asp Val Phe Ser Arg 115 120 125 Glu Pro Phe Val Val Trp Phe His Ser Pro Phe Thr Ala Val Lys Asp 130 135 140 Phe Val Ile Val Pro Leu His Thr Thr Pro Glu Thr Ser Val Lys Glu 145 150 155 160 Ile Asp Glu Leu Val Asp Val Tyr Thr Asp Val Arg Ser Gln Trp Lys 165 170 175 Thr Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr 180 185 190 Val Pro Lys Lys Ala Trp Gln Asn Ile Arg Leu Arg Thr Asp Pro Lys 195 200 205 Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr Val Lys Lys Ser 210 215 220 Thr Ser Cys Ala Tyr Asp Arg Ile Val Leu Cys Gly Gln Glu Ile Val 225 230 235 240 Asn Ser Val Val Pro Arg Ser Ser Gly Val Phe Asp Phe Gln Lys Ala 245 250 255 Tyr Asp Leu Ser Glu Glu Glu Ala Leu Asp Val Ser Asp His Phe Pro 260 265 270 Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr Asn Asn Arg Lys 275 280 285 Ser Val Ser Leu Lys Lys Arg Lys Lys Gly Asn Arg Ser Ser Ser Thr 290 295 300 Met Val Gly Ser Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val 305 310 315 320 Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Tyr Ile 325 330 335 Thr Leu Glu Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp 340 345 350 Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His 355 360 365 Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg 370 375 380 Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys 385 390 395 400 Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu 405 410 415 Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr 420 425 430 Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu 435 440 445 Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp 450 455 460 Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile 465 470 475 480 Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln 485 490 495 Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His 500 505 510 Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro 515 520 525 Gly Lys 530

Claims

1. Amino acid sequence: DNAse1-X1-Linker-Fc-X2 (I) A construct comprising: Where: DNAse1 is a human DNAse1 polypeptide; X1 is a covalent bond or X1 is an amino acid sequence or a fragment thereof; the linker is a chemical bond or a polypeptide comprising 1 to 100 amino acids; X2 is null or X2 is a peptide of amino acid sequence SEQ ID NO:3 or a fragment thereof; Fc is the Fc domain of human IgG1; construct.

2. Amino acid sequence: DNAse1L3-X1-linker-Fc-X2 (II) A construct comprising: Where: DNAse1L3 is a human DNAse1L3 polypeptide; X1 is a covalent bond or X1 is an amino acid sequence or a fragment thereof; the linker is a covalent bond or a polypeptide comprising 1 to 100 amino acids; X2 is null or X2 is a peptide of amino acid sequence SEQ ID NO:3 or a fragment thereof; Fc is the Fc domain of human IgG1; construct.

3. The construct of any one of claims 1 to 2, wherein the Fc comprises the amino acid sequence of SEQ ID NO:

4.

4. 4. The construct of claim 3, wherein at least one of C6 and C9 with respect to SEQ ID NO:4 is independently mutated to G or S.

5. 4. The construct of claim 3, wherein each one of C6 and C9 with respect to SEQ ID NO:4 is independently mutated to G or S.

6. 4. The construct of claim 3, comprising at least one of the following mutations with respect to SEQ ID NO:4: M32Y, S34T, T36E.

7. 4. The construct of claim 3, comprising one of the following mutations relative to SEQ ID NO:4: M32Y, S34T, T36E.

8. The construct of any one of claims 1 to 2, wherein the linker is a chemical bond or is absent.

9. 3. The construct of any one of claims 1 to 2, wherein the linker is a polypeptide comprising 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, and / or 1 to 5 amino acids.

10. The construct of any one of claims 1 to 2, wherein the linker comprises GS and / or GSC.

11. 3. The construct of claim 1, wherein the linker comprises GGGGSGGGGS (SEQ ID NO:5), SSTMVRS (SEQ ID NO:40), and / or SSTMVGS (SEQ ID NO:41).

12. The linker wherein each occurrence X is C, G, or S, and each occurrence Z is C, G, or S.

13. The construct of any one of claims 1 to 2, wherein X1 is a covalent bond.

14. X1 is the amino acid sequence The construct of any one of claims 1 to 2, which is a peptide of or a fragment thereof.

15. 3. The construct of any one of claims 1 to 2, wherein X2 is a covalent bond.

16. X2 is the amino acid sequence The construct of any one of claims 1 to 2, which is a peptide of or a fragment thereof.

17. The construct of claim 1, wherein the DNAse 1 lacks at least a portion of residues 1 to 22 corresponding to SEQ ID NO:

1.

18. The construct of claim 1, wherein the DNAse 1 lacks residues 1 to 22 corresponding to SEQ ID NO:

1.

19. 2. The construct of claim 1, wherein the DNAse 1 comprises at least one of the following mutations with respect to SEQ ID NO: 1: Q31R, E35R, Y46H, Y46S, V88N, N96K, D109N, V111T, A136F, R148S, E149N, M186I, L208P, D220N, D250N, A252T, G262N, D265N, and L267T.

20. 2. The construct of claim 1, wherein the Fc comprises at least one of the following mutations with respect to SEQ ID NO:4: C6G, C6S, C9G, C9S, M32Y, S34T, and T36E.

21. 2. The construct of claim 1, selected from the group consisting of SEQ ID NOs: 7-17 and 32-35.

22. 3. The construct of claim 2, wherein the DNAse1L3 lacks at least one of the following: residues 291-305 of SEQ ID NO:2; residues 296-304 of SEQ ID NO:2; residues 292-304 of SEQ ID NO:2; or residues A-B of SEQ ID NO:2, wherein A is in the range of 291-296 and B is in the range of 304-305.

23. The construct of claim 2, wherein the DNAse1L3 comprises at least one of the following mutations relative to SEQ ID NO:2: E33R, M42T, V44H, V88T, N96K, A127N, V129T, K147S, D148N, L207P, D219N, and V254T.

24. 3. The construct of claim 2, wherein the Fc comprises at least one of the following mutations with respect to SEQ ID NO:4: C6G, C6S, C9G, C9S, M32Y, S34T, and T36E.

25. 3. The construct of claim 2, selected from the group consisting of SEQ ID NOs: 18-28 and 36-39.

26. 3. The construct of any one of claims 1 to 2, which is expressed in a mammalian cell.

27. 27. The construct of claim 26, wherein the mammalian cell is stably transfected with human ST6 beta-galactosamide alpha-2,6-sialyltransferase (also known as ST6GAL1).

28. The mammalian cells are capable of expressing sialic acid and / or N-acetylmannosamine (1,3,4-O-Bu 3 27. The construct of claim 26, wherein the construct is grown in a cell culture supplemented with ManNAc (also known as ManNAc).

29. 3. The construct of any one of claims 1 to 2, which is soluble.

30. 10. A homodimeric construct comprising two independently selected constructs of claim 1.

31. A homodimeric construct comprising two independently selected constructs of claim 2.

32. A heterodimeric construct comprising the construct of claim 1 and the construct of claim 2.

33. A method for treating, ameliorating, and / or preventing multiple forms of lupus associated with DNAse1 and / or DNAse1L3 deficiency in a subject, comprising administering to the subject a therapeutically effective amount of a construct described in any one of claims 1 to 2.

34. 34. The method of claim 33, wherein the lupus comprises systemic lupus erythematosus (SLE).

35. 10. A method of treating, ameliorating, and / or preventing diseases and / or disorders associated with inefficient NETolysis in a subject, comprising administering to the subject a therapeutically effective amount of a construct according to any one of claims 1-2.

36. A method for treating, ameliorating, and / or preventing an autoimmune disorder associated with DNAse1 and / or DNAse1L3 deficiency in a subject, comprising administering to the subject a therapeutically effective amount of a construct described in any one of claims 1 to 2.

37. 37. The method of claim 36, wherein the autoimmune disorder comprises lupus, autoimmune thyroid disease, and / or hypocomplementemic urticarial vasculitis syndrome (HUVS).

38. 10. A method for treating, ameliorating, and / or preventing pathological thrombosis in a subject, comprising administering to the subject a therapeutically effective amount of a construct according to any one of claims 1-2.

39. 39. The method of claim 38, wherein the pathological thrombosis comprises microvascular thrombosis, venous thrombosis, and / or arterial thrombosis.

40. 39. The method of claim 38, wherein the pathological thrombosis results in or predisposes the subject to stroke.

41. 39. The method of claim 38, wherein the pathological thrombosis comprises neutrophilic thrombosis.

42. 42. The method of claim 41, wherein the neutrophilic thrombosis comprises at least one of antineutrophil cytoplasmic autoantibody (ANCA) vasculitis, thrombotic thrombocytopenic purpura (TTP), and Behcet's disease or syndrome.

43. A method for treating, ameliorating, and / or preventing myocardial infarction in a subject, comprising administering to the subject a therapeutically effective amount of a construct according to any one of claims 1-2.

44. A method for treating, ameliorating, and / or preventing cancer metastasis in a subject, comprising administering to the subject a therapeutically effective amount of a construct according to any one of claims 1 to 2.

45. The method of claim 44, wherein in the DNAse1 and / or DNAse1L3 precursor construct, a signal peptide sequence is conjugated to the N-terminus of the DNAse1 and / or DNAse1L3 polypeptide.

46. the construct is a secreted product of a DNAse1 and / or DNAse1L3 precursor construct expressed in a mammalian cell; the DNAse1 and / or DNAse1L3 precursor construct comprises a signal peptide sequence and a DNAse1 and / or DNAse1L3 polypeptide; the DNAse1 and / or DNAse1L3 precursor construct undergoes proteolytic processing to yield said DNAse1 and / or DNAse1L3 construct; 45. The method of any one of claims 33, 35, 36, 38, 43, and 44.

47. 47. The method of claim 46, wherein the mammalian cells are stably transfected with human ST6 beta-galactosamide alpha-2,6-sialyltransferase (also known as ST6GAL1).

48. The mammalian cells are capable of expressing sialic acid and / or N-acetylmannosamine (1,3,4-O-Bu 3 47. The method of claim 46, wherein the cell is grown in a cell culture supplemented with ManNAc (also known as ManNAc).

49. 45. The method of any one of claims 33, 35, 36, 38, 43, and 44, wherein the construct is administered to the subject acutely or chronically.

50. 45. The method of any one of claims 33, 35, 36, 38, 43, and 44, wherein the construct is administered to the subject locally, regionally, parenterally, and / or systemically.

51. 45. The method of any one of claims 33, 35, 36, 38, 43, and 44, wherein the construct and / or precursor construct thereof is delivered to the subject on an encoded vector, the vector encoding the construct or precursor construct, which is transcribed and translated from the vector upon administration of the vector to the subject.

52. 45. The method of any one of claims 33, 35, 36, 38, 43, and 44, wherein the construct is administered to the subject by at least one route selected from the group consisting of subcutaneous, oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical.

53. 45. The method of any one of claims 33, 35, 36, 38, 43, and 44, wherein the construct is administered to the subject as a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier.

54. The construct comprises: A homodimeric construct comprising two independently selected constructs of claim 1. a homodimeric construct comprising two independently selected constructs according to claim 2, and / or A heterodimeric construct comprising the construct of claim 1 and the construct of claim 2.

45. The method of any one of claims 33, 35, 36, 38, 43, and 44, comprising:

55. 45. The method of any one of claims 33, 35, 36, 38, 43, and 44, wherein the subject is a mammal.

56. 56. The method of claim 55, wherein the mammal is a human.

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