Treatment of Sjögren's disease with nuclease fusion proteins

Abstract

This provides a useful method for treating symptoms associated with Sjögren's disease. [Solution] A method for treating Sjögren's disease in a human patient who requires treatment for Sjögren's disease by reducing fatigue, comprising the step of administering an effective amount of RNase-Fc fusion protein to the patient, thereby treating Sjögren's disease by reducing fatigue in the patient.

Description

Technical Field

[0001] Cross-reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 788,730 filed on January 4, 2019, the entire content of which is incorporated herein by reference. Background Art

[0002] Primary Sjögren's syndrome (pSS) is an autoimmune disorder estimated to affect between 0.5% and 1% of the general population, with 9 out of 10 patients being female (Ramos-Casals, 2005, Ann Rheum Dis. 64, 347-354; Skopouli, 2000, Semin. Arthritis Rheum. 29:296-304). The majority of female pSS patients are characterized by mild to moderate disease that manifests as fatigue, arthralgia, and ocular and / or oral dryness. The disease is characterized by lymphocyte infiltration of the salivary and lacrimal glands, followed by gland inflammation, damage, and loss of function that cause dry eye and dry mouth. Involvement of major organ systems including the lung, kidney, and liver is a common systemic manifestation of pSS (Malladi, 2012, Arthritis Care Res. 64:911-918). At the biochemical level, pSS involves increased immunoglobulin levels and the production of antinuclear antibodies against ribonucleoprotein complexes such as SSA / Ro and SSB / La (Bave, 2005, Arth. & Rheum. 52:1185-1195; Hall 2015, Arth. & Rheum. 67, 2437-2446).

[0003] Once formed, RNA-containing immune complexes are immediately translocated to immune system cells such as dendritic cells, where the RNA bound to the immune complex can interact with RNA sensors and Toll-like receptors (TLRs), such as TLR7. TLRs are thought to function as key elements of the innate immune system by recognizing pathogen-related molecular components. It is now clear that host nucleic acids can also activate specific family members, including TLR7, TLR8, and TLR9 (Theofilopoulos, 2010, Nat. Rev. Rheum., 6:146-156). Cells expressing these receptors do not distribute them on the cell surface; rather, TLR7 / 8 / 9 are isolated within endosomes (Theofilopoulos, 2010, Nat. Rev. Rheum., 6:146-156). This positioning is hypothesized to minimize interaction with host nucleic acids. However, when present within immune complexes, nucleic acid antigens are actively translocated into cells via receptor-mediated endocytosis. Effector Fc, complement, and B cell receptors all facilitate the entry of nucleic acid-containing ICs into endosomes (Means, 2005, J. Clin. Invest. 115:407-417; Lau, 2005, J. Exp. Med. 202:1171-1177;Brkic 2013, Ann. Rheum. Dis. 72(5):728-735). Upon internalization, the nucleic acid binds to endosomal TLRs and is positioned to activate them. The activated TLRs then promote type 1 IFN production from pDCs, activate PMNs, and promote B cell proliferation and autoantibody production. Thus, nucleic acid-containing antigens contribute to multiple aspects of the pathophysiology of pSS disease. Fatigue is one of the most common extraglandular symptoms of Sjögren's syndrome and is defined by persistent generalized fatigue. An estimated 70% of patients with pSS experience marked fatigue, which is reported to have a negative impact on their quality of life. Serologically, approximately 80% of these patients have anti-Ro / SSA autoantibodies that bind to autoantigens containing small non-coding RNA molecules. Fatigue can be characterized in terms of intensity, duration, and effect on daily functioning. Notably, in a primary care setting, fatigue is strongly associated with depressive states (Segal et al. Arthritis Rhem. 2008 December 15; 59(12): 1780-1778). Therefore, there is a need for means to alleviate fatigue in patients with autoimmune diseases such as Sjögren's syndrome. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Ramos-Casals, 2005, Ann Rheum Dis. 64, 347-354 [Non-Patent Document 2] Skopouli, 2000, Semin. Arthritis Rheum. 29:296-304 [Non-Patent Document 3] Malladi, 2012, Arthritis Care Res. 64:911-918 [Non-Patent Document 4] Bave, 2005, Arth. & Rheum. 52:1185-1195 [Non-Patent Document 5] Hall 2015, Arth. & Rheum. 67, 2437-2446 [Non-Patent Document 6] Theofilopoulos, 2010, Nat. Rev. Rheum., 6:146-156 [Non-Patent Document 7] Means, 2005, J. Clin. Invest. 115:407-417 [Non-Patent Document 8] Lau, 2005, J. Exp. Med. 202:1171-1177 [Non-Patent Document 9] Brkic 2013, Ann. Rheum. Dis. 72(5):728-735 [Non-Patent Document 10] Segal et al. Arthritis Rhem. 2008 December 15; 59(12): 1780-1778 [Overview of the project] [Means for solving the problem]

[0005] This disclosure relates, at least in part, to RNase-containing nuclease fusion proteins, including RNase-Fc fusion proteins, that are useful for the treatment of Sjögren's syndrome in human patients requiring treatment for Sjögren's syndrome. In some embodiments, this disclosure relates to RNase-containing nuclease fusion proteins, including RNase-Fc fusion proteins, that are useful for the treatment, reduction, or remission of fatigue in patients with Sjögren's syndrome. In some embodiments, this disclosure relates to RNase-containing nuclease fusion proteins, including RNase-Fc fusion proteins, that are useful for improving, enhancing, or increasing cognitive abilities, or for reducing, decreasing, or relieving agnosia in patients with Sjögren's syndrome. In some embodiments, this disclosure relates to RNase-containing nuclease fusion proteins, including RNase-Fc fusion proteins, that are useful for reducing, decreasing, or relieving depressive states in patients with Sjögren's syndrome, including fatigue-related depressive states. In some embodiments, the disclosure relates to compositions comprising an RNase-Fc fusion protein and one or more pharmaceutically acceptable carriers and / or diluents, which are useful in methods for treating or preventing Sjögren's syndrome, methods for treating, preventing or reducing fatigue in patients with Sjögren's syndrome, and methods for improving cognitive abilities in patients with Sjögren's syndrome. In some embodiments, the RNase-Fc fusion protein is administered to a human patient by injection (e.g., intravenous injection) in doses of about 5–10 mg / kg, about 2–8 mg / kg, about 3–6 mg / kg, about 3 mg / kg, about 5 mg / kg, or about 10 mg / kg.

[0006] In some embodiments, the RNase-Fc fusion protein is RSLV-132. RSLV-132 is a nuclease fusion protein comprising a homodimer of two polypeptides, each having the amino acid sequence shown as SEQ ID NO: 50. Each polypeptide in the homodimer has the configuration shown in Figure 1 from the N-terminus to the C-terminus of the RNase-Fc, with the wild-type human RNase 1 domain (SEQ ID NO: 2) operably linked without a linker to the N-terminus of a human IgG1 Fc domain containing a serine mutation in one of the three hinge region cysteine ​​residues (residue 220 or C220S, also referred to herein as the "SCC hinge") and two mutations P238S and P331S in the CH2 domain. The sequence of the human IgG1 Fc domain with these mutations is shown as SEQ ID NO: 22.

[0007] In some embodiments, the present disclosure provides a method for treating Sjögren's disease by reducing fatigue in a human patient who requires treatment for Sjögren's disease, comprising the step of administering an effective amount of an RNase-containing nuclease fusion protein, such as an RNase-Fc fusion protein, such as RSLV-132, to the patient, thereby providing a method for treating Sjögren's disease by reducing fatigue in the patient.

[0008] In some embodiments, the present disclosure provides a method for treating Sjögren's disease by reducing fatigue in a human patient who requires treatment for Sjögren's disease, comprising the step of administering to the patient by intravenous injection a dose of approximately 5-10 mg / kg of an RNase-containing nuclease fusion protein, such as an RNase-Fc fusion protein, such as RSLV-132, thereby providing a method for treating Sjögren's disease by reducing fatigue in the patient.

[0009] In some embodiments, the Disclosure provides a method for treating Sjögren's disease by improving cognitive effects in a human patient requiring treatment for Sjögren's disease, comprising the step of administering an effective amount of an RNase-containing nuclease fusion protein, e.g., an RNase-Fc fusion protein, e.g., RSLV-132, to the patient, thereby providing a method for treating Sjögren's disease by improving cognitive effects in the patient. In some embodiments, the cognitive effects in the patient are improved by at least 1 point in the mental components of ProF compared to the mental components of ProF before treatment. In some embodiments, the cognitive effects in the patient are improved by more than 1 point, more than 2 points, or more than 3 points in the mental components of ProF compared to the mental components of ProF before treatment.

[0010] In some embodiments, the present disclosure provides a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, comprising the step of administering to the patient an effective amount of an RNase-Fc fusion protein comprising the amino acid sequence shown in SEQ ID NO: 50, thereby providing a method for treating Sjögren's disease by reducing fatigue in the patient. In some embodiments, the effective amount of the RNase-Fc fusion protein comprising the amino acid sequence shown in SEQ ID NO: 50 is a dose of about 5 mg / kg to about 10 mg / kg. In some embodiments, the RNase-Fc fusion protein is administered in the form of a composition containing a pharmaceutically acceptable carrier. In some embodiments, the composition comprising the RNase-Fc fusion protein comprising the amino acid sequence shown in SEQ ID NO: 50 is formulated for intravenous injection (e.g., in solution).

[0011] In some embodiments, the Disclosure provides a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, comprising the step of administering an effective amount of a pharmaceutical composition to the patient, the composition comprising an RNase-Fc fusion protein comprising the amino acid sequence shown in SEQ ID NO: 50; and one or more pharmaceutically acceptable carriers and / or diluents, thereby providing a method for treating Sjögren's disease by reducing fatigue in a patient. In some embodiments, the composition comprising the RNase-Fc fusion protein comprising the amino acid sequence shown in SEQ ID NO: 50 is formulated for intravenous injection (e.g., in solution).

[0012] In any of the aforementioned embodiments or related embodiments of the methods of the present disclosure, the RNase-Fc fusion protein for use herein comprises human pancreatic RNase 1. In some embodiments, human pancreatic RNase 1 comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the RNase-Fc fusion protein of the present disclosure comprises a wild-type human IgG1 Fc domain or a human IgG1 Fc domain comprising one or more mutations. In some embodiments, the human IgG1 Fc domain comprises one or more mutations that reduce binding to the Fcγ receptor on human cells. In some embodiments, the RNase-Fc fusion protein of the present disclosure has a reduction in effector function, which is selected as needed from the group consisting of opsonization, phagocytosis, complement-dependent cytotoxicity, and antibody-dependent cellular cytotoxicity.

[0013] In some embodiments, the human IgG1 Fc domain includes a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, the human IgG1 Fc domain includes one or more serine substitutions among the three hinge region cysteine ​​residues. In some embodiments, the Fc domain includes SCC mutations (residues 220, 226, and 229) numbered according to the EU index. In some embodiments, the human IgG1 Fc domain includes the amino acid sequence shown in Sequence ID No. 22.

[0014] In any of the foregoing or related embodiments of the methods of the present disclosure, the RNase-Fc fusion protein for use herein comprises human pancreatic RNase 1 linked to a human IgG1 Fc domain comprising the C220S mutation, the P238S mutation and the P331S mutation, with or without a linker, according to EU numbering.

[0015] In any of the foregoing or related embodiments of the methods of the present disclosure, the RNase-Fc fusion protein for use herein comprises the amino acid sequence set forth in SEQ ID NO: 50.

[0016] In any of the foregoing or related embodiments of the methods of the present disclosure, the RNase-Fc fusion protein, for example RSLV-132, or a pharmaceutical composition comprising the RNase-Fc fusion protein for use herein is administered to a patient by intravenous injection. In some aspects, the patient is administered an effective dose of the RNase-Fc fusion protein, for example RSLV-132, or a pharmaceutical composition comprising the RNase-Fc fusion protein once every two weeks.

[0017] In some embodiments, the RNase-Fc fusion protein of this disclosure, e.g., RSLV-132, or a pharmaceutical composition containing an RNase-Fc fusion protein, is administered to a patient at a dose of approximately 5 to 10 mg / kg. In some embodiments, the RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition containing an RNase-Fc fusion protein, is administered to a patient at a dose of approximately 10 mg / kg. In some embodiments, the RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition containing an RNase-Fc fusion protein, is administered to a patient at a dose of approximately 5 mg / kg. In some embodiments, the RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition containing an RNase-Fc fusion protein, is administered to a patient at a dose of approximately 5 to 10 mg / kg every two weeks. In some embodiments, an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition containing an RNase-Fc fusion protein is administered to the patient at a dose of approximately 5-10 mg / kg every two weeks for three months. In some embodiments, an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition containing an RNase-Fc fusion protein is administered to the patient in six bi-weekly infusions over three months. In some embodiments, an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition containing an RNase-Fc fusion protein is administered to the patient weekly for three weeks, and then once every two weeks, to achieve or maintain a therapeutic effect.

[0018] In some aspects, the present disclosure provides a method for treating Sjögren's disease by reducing fatigue in a human patient in need of treatment for Sjögren's syndrome, comprising administering a dosing regimen of at least 3 doses of an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein, wherein each dose is administered to the patient at a dose of about 5 to 10 mg / kg, about 2 to 8 mg / kg, about 3 to 6 mg / kg, about 3 mg / kg, about 5 mg / kg or about 10 mg / kg (e.g., by injection, e.g., intravenous injection). In some aspects, the patient is administered at least 4 doses of the RNase-Fc fusion protein. In some aspects, the patient is administered at least 5 doses of the RNase-Fc fusion protein. In some aspects, the patient is administered at least 6 doses of the RNase-Fc fusion protein. In some aspects, the patient is administered at least 7 doses of the RNase-Fc fusion protein. In some aspects, the patient is administered at least 8 doses of the RNase-Fc fusion protein.

[0019] In some embodiments, the Disclosure provides a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's syndrome, comprising the step of administering a drug regimen of doses of an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein, once weekly for at least two weeks, wherein each dose is administered to the patient (e.g., by injection, e.g., intravenous injection) at doses of approximately 5–10 mg / kg, approximately 2–8 mg / kg, approximately 3–6 mg / kg, approximately 3 mg / kg, approximately 5 mg / kg, or approximately 10 mg / kg. In some embodiments, the patient is administered doses of the RNase-Fc fusion protein weekly for at least three weeks. In some embodiments, the patient is administered doses of the RNase-Fc fusion protein weekly for at least four weeks. In some embodiments, the patient is administered doses of the RNase-Fc fusion protein weekly for at least five weeks. In some embodiments, the patient receives a dose of RNase-Fc fusion protein weekly for at least 6 weeks. In some embodiments, the patient receives a dose of RNase-Fc fusion protein weekly for at least 7 weeks. In some embodiments, the patient receives a dose of RNase-Fc fusion protein weekly for at least 8 weeks.

[0020] In some embodiments, the patient receives a dose of RNase-Fc fusion protein every two weeks for at least two weeks. In some embodiments, the patient receives a dose of RNase-Fc fusion protein every two weeks for at least four weeks. In some embodiments, the patient receives a dose of RNase-Fc fusion protein every two weeks for at least six weeks. In some embodiments, the patient receives a dose of RNase-Fc fusion protein every two weeks for at least eight weeks.

[0021] In some embodiments, the patient receives a dose of RNase-Fc fusion protein weekly for three weeks, followed by a dose every two weeks for at least one month. In some embodiments, the patient receives a dose of RNase-Fc fusion protein weekly for three weeks, followed by a dose every two weeks for at least two months. In some embodiments, the patient receives a dose of RNase-Fc fusion protein weekly for three weeks, followed by a dose every two weeks for at least three months.

[0022] In some embodiments, the present disclosure provides a method for treating Sjögren's disease by reducing fatigue in a human patient who requires treatment for Sjögren's disease, comprising the step of administering to the patient an effective amount of an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein, wherein the treatment is EULAR This invention provides a method to reduce fatigue in a patient by at least 1 point on the SS Patient Reported Index (ESSPRI) score compared to a pre-treatment ESSPRI score. In some embodiments, the treatment reduces the ESSPRI score by at least 1 point compared to a pre-treatment ESSPRI score. In some embodiments, fatigue is reduced to a score between 4.5 and 5.5 on an ESSPRI scale of 1 to 10.

[0023] In some embodiments, the Disclosure provides a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, comprising the step of administering to the patient an effective amount of an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein, wherein the treatment improves the patient's fatigue by at least 1 point on the FACIT Fatigue Scale for Assessment of Functional Treatment of Chronic Disease (FACIT) compared to a pre-treatment FACIT score. In some embodiments, the treatment improves the patient's fatigue by at least 2 points on the FACIT Fatigue Scale. In some embodiments, the treatment increases the FACIT fatigue score by at least 1 point compared to a pre-treatment FACIT fatigue score. In some embodiments, the treatment increases the FACIT fatigue score by at least 2 points compared to a pre-treatment FACIT fatigue score.

[0024] In some embodiments, the Disclosure provides a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, comprising the step of administering to the patient an effective amount of an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein, wherein the treatment reduces fatigue in the patient by at least 1 point on the fatigue profile (ProF) score compared to the pre-treatment ProF score. In some embodiments, the treatment reduces fatigue in the patient by at least 1 point on the mental component of the fatigue profile (ProF) score compared to the pre-treatment mental component ProF score. In some embodiments, the treatment reduces fatigue in the patient by at least 1 point on the physical component of the fatigue profile (ProF) score compared to the pre-treatment physical component ProF score.

[0025] In some embodiments, the Disclosure provides a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, comprising the step of administering to the patient an effective amount of an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein, wherein the treatment improves cognitive function in the patient, as measured by a digit-signature substitution test (DSST), compared to a DSST score before treatment. In some embodiments, the treatment increases the number of matches completed by the patient in 90 seconds on the digit-signature substitution test (DSST). In some embodiments, the treatment reduces the time it takes the patient to complete the DSST test.

[0026] In other embodiments, the present disclosure provides a kit comprising a container containing an injectable solution comprising a pharmaceutical composition comprising an effective amount of the present RNase-Fc fusion protein, e.g., RSLV-132, or an RNase-Fc fusion protein comprising the amino acid sequence shown in SEQ ID NO: 50, or an RNase-Fc fusion protein; and one or more pharmaceutically acceptable carriers and / or diluents, and instructions for use in the treatment of Sjögren's disease by reducing fatigue in a human patient requiring such use, wherein the injectable solution is formulated for intravenous administration.

[0027] In some embodiments, the Disclosure provides an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein for use in a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the treatment comprises the step of administering an effective amount of the RNase-Fc fusion protein to the patient.

[0028] In some embodiments, the Disclosure provides an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein for use in the manufacture of a pharmaceutical for treating Sjögren's disease by reducing fatigue in human patients who require treatment for Sjögren's disease.

[0029] In some embodiments, the Disclosure provides an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein for use in a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the treatment comprises the step of administering to the patient a dose of about 5-10 mg / kg of the RNase-Fc fusion protein by intravenous injection.

[0030] In some embodiments, the Disclosure provides for use in the manufacture of a pharmaceutical for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment of Sjögren's disease, wherein the use comprises the step of administering to the patient a dose of about 5-10 mg / kg of the RNase-Fc fusion protein by intravenous injection.

[0031] In some embodiments, the Disclosure provides an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein for use in a method for treating Sjögren's disease by improving cognitive effects in a human patient requiring treatment for Sjögren's disease, wherein the treatment comprises the step of administering an effective amount of the RNase-Fc fusion protein to the patient.

[0032] In some embodiments, the Disclosure provides for use in the manufacture of a pharmaceutical for treating Sjögren's disease by improving cognitive effects in human patients requiring treatment of Sjögren's disease, the use comprising the step of administering an effective amount of the RNase-Fc fusion protein to the patient.

[0033] In some embodiments, the Disclosure provides an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein for use in a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the treatment comprises the step of administering to the patient an effective amount of the RNase-Fc fusion protein having the amino acid sequence shown in SEQ ID NO: 50.

[0034] In some embodiments, the Disclosure provides an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein for use in the manufacture of a medicament for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the use comprises the step of administering to the patient an effective amount of the RNase-Fc fusion protein comprising the amino acid sequence shown in SEQ ID NO: 50.

[0035] In some embodiments, the Disclosure provides an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein for use in a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the treatment comprises the step of administering an effective amount of the pharmaceutical composition to the patient, the composition comprising an RNase-Fc fusion protein comprising the amino acid sequence shown in SEQ ID NO: 50; and one or more pharmaceutically acceptable carriers and / or diluents.

[0036] In some embodiments, the Disclosure provides an RNase-Fc fusion protein, e.g., RSLV-132, or a pharmaceutical composition comprising an RNase-Fc fusion protein for use in the manufacture of a medicament for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the use comprises the step of administering an effective amount of the pharmaceutical composition to the patient, wherein the composition comprises an RNase-Fc fusion protein comprising the amino acid sequence shown in SEQ ID NO: 50; and one or more pharmaceutically acceptable carriers and / or diluents.

[0037] In some embodiments, the Disclosure provides a method for treating Sjögren's disease in a patient requiring treatment for Sjögren's disease, comprising the step of administering an effective amount of an RNA nuclease agent to the patient, wherein the treatment results in a reduction of one or more inflammation-related genes. In some embodiments, the one or more inflammation-related genes are selected from the group including IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4, and STAT5B.

[0038] In some embodiments, the Disclosure provides a method for treating Sjögren's disease in a patient requiring treatment for Sjögren's disease, comprising the step of administering an effective amount of an RNA nuclease agent to the patient, wherein the treatment results in a reduction of one or more inflammation-related genes. In some embodiments, the one or more inflammation-related genes are selected from the group consisting of IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R, and STAT5B.

[0039] In some embodiments, the present disclosure provides a use of an RNA nuclease agent in the manufacture of a medicament for the treatment of Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in a reduction of one or more inflammation-related genes. In some embodiments, the one or more inflammation-related genes are selected from the group consisting of IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4, and STAT5B.

[0040] In some embodiments, the present disclosure provides a use of an RNA nuclease agent in the manufacture of a medicament for the treatment of Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in a reduction of one or more inflammation-related genes. In some embodiments, the one or more inflammation-related genes are selected from the group consisting of IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R, and STAT5B.

[0041] In some embodiments, the Disclosure provides an RNA nuclease agent for use in a method for treating Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in a reduction of one or more inflammation-related genes. In some embodiments, the one or more inflammation-related genes are selected from the group consisting of IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4, and STAT5B.

[0042] In some embodiments, the Disclosure provides an RNA nuclease agent for use in a method for treating Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in a reduction of one or more inflammation-related genes. In some embodiments, the one or more inflammation-related genes are selected from the group consisting of IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R, and STAT5B.

[0043] In some embodiments, the Disclosure provides a method for treating Sjögren's disease in a patient requiring treatment for Sjögren's disease, comprising the step of administering an effective amount of an RNA nuclease agent to the patient, wherein the treatment results in an increase of one or more inflammation-related genes. In some embodiments, the one or more inflammation-related genes are selected from the group consisting of CXCL10 (IP-10), CD163, RIPK2, and CCR2.

[0044] In some embodiments, the present disclosure provides a use of an RNA nuclease agent in the manufacture of a medicament for the treatment of Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in an increase of one or more inflammation-related genes. In some embodiments, the one or more inflammation-related genes are selected from the group consisting of CXCL10 (IP-10), CD163, RIPK2, and CCR2.

[0045] In some embodiments, the Disclosure provides an RNA nuclease agent for use in a method for treating Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in an increase of one or more inflammation-related genes. In some embodiments, the one or more inflammation-related genes are selected from the group consisting of CXCL10 (IP-10), CD163, RIPK2, and CCR2.

[0046] In some embodiments, the Disclosure provides a method for treating Sjögren's disease in a patient requiring treatment for Sjögren's disease, comprising the step of administering an effective amount of an RNA nuclease agent to the patient, wherein the treatment results in an increase of one or more cytokines and an improvement in fatigue. In some embodiments, the cytokine is CXCL10.

[0047] In some embodiments, the present disclosure provides a use of an RNA nuclease agent in the manufacture of a medicament for the treatment of Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in an increase of one or more cytokines and an improvement in fatigue. In some embodiments, the cytokine is CXCL10.

[0048] In some embodiments, the Disclosure provides an RNA nuclease agent for use in a method for treating Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in an increase of one or more cytokines and an improvement in fatigue. In some embodiments, the cytokine is CXCL10.

[0049] In some embodiments, the Disclosure provides a method for identifying a patient with Sjögren's disease as a candidate for RNA nuclease treatment, comprising the steps of (a) determining an inflammation-related gene expression profile in a sample obtained from the patient, and (b) comparing the inflammation-related gene expression profile determined in step (a) with an inflammation-related gene expression profile in a sample obtained from a suitable control subject, wherein the inflammation-related gene expression profile indicates that the patient is a candidate for RNA nuclease treatment. In some embodiments, the inflammation-related genes are selected from the group consisting of MAP3K8, ACKR3, STAT1, STAT2, TRIM37, and ZNF606.

[0050] A patent or application file must contain at least one color drawing. A copy of this patent or patent application publication containing the color drawing(s) is available from the relevant government agency upon request and payment of the required fees.

[0051] These and other features, aspects, and advantages of the present invention will be better understood with reference to the following description and accompanying drawings. In embodiments of the present invention, for example, the following items are provided. (Item 1) A method for treating Sjögren's disease in a human patient requiring treatment for Sjögren's disease by reducing fatigue, comprising the step of administering an effective amount of RNase-Fc fusion protein to the patient, thereby treating Sjögren's disease in the patient by reducing fatigue. (Item 2) The method according to item 1, wherein the RNase-Fc fusion protein contains human pancreatic RNase 1. (Item 3) The method according to item 2, wherein the human pancreatic RNase 1 comprises the amino acid sequence shown in SEQ ID NO: 2. (Item 4) The method according to item 1, wherein the RNase-Fc fusion protein comprises a wild-type human IgG1 Fc domain or a human IgG1 Fc domain containing one or more mutations. (Item 5) The method according to item 4, wherein the Fc domain containing one or more mutations has reduced binding to the Fcγ receptor on human cells. (Item 6) The method according to item 1, wherein the RNase-Fc fusion protein has a reduction in effector function selected as needed from the group consisting of opsonization, phagocytosis, complement-dependent cell injury, and antibody-dependent cell-mediated cytotoxicity. (Item 7) The method according to item 4, wherein the human IgG1 Fc domain contains the P238S mutation and the P331S mutation according to EU numbering. (Item 8) The method according to item 4, wherein the human IgG1 Fc domain includes a hinge domain, a CH2 domain, and a CH3 domain. (Item 9) The method according to item 4, wherein the human IgG1 Fc domain comprises substitution with one or more serine residues from among the three hinge region cysteine ​​residues. (Item 10) The method according to item 9, wherein the Fc domain contains SCC mutations (residues 220, 226, and 229) numbered according to the EU index. (Item 11) The method according to item 1, wherein the human IgG1 Fc domain comprises the amino acid sequence shown in Sequence ID No. 22. (Item 12) The method according to item 1, wherein the RNase-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO: 50. (Item 13) The method according to item 1, wherein the RNase-Fc fusion protein is administered to the patient at a dose of approximately 5-10 mg / kg. (Item 14) The method according to item 1, wherein the RNase-Fc fusion protein is administered to the patient at a dose of approximately 10 mg / kg. (Item 15) The method according to item 1, wherein the RNase-Fc fusion protein is administered to the patient at a dose of approximately 5 mg / kg. (Item 16) The method according to item 1, wherein the RNase-Fc fusion protein is administered to the patient by intravenous injection. (Item 17) The method according to item 1, wherein the RNase-Fc fusion protein is administered to the patient at a dose of approximately 5-10 mg / kg every two weeks. (Item 18) The method according to item 1, wherein the RNase-Fc fusion protein is administered to the patient at a dose of approximately 5-10 mg / kg every two weeks for three months. (Item 19) The method according to item 1, wherein the RNase-Fc fusion protein is administered to the patient in six bi-weekly infusions over a period of three months. (Item 20) The method according to item 1, wherein the RNase-Fc fusion protein is administered to the patient weekly for three weeks, and then once every two weeks, to achieve or maintain a therapeutic effect. (Item 21) The method according to item 1, wherein the treatment reduces fatigue in the patient by at least 1 point on the EULAR SS Patient Reported Index (ESSPRI) score compared to the ESSPRI score before the treatment. (Item 22) The method according to item 1, wherein the treatment reduces the ESSPRI score by at least 1 point compared to the ESSPRI score before the treatment. (Item 23) The method described in item 21, which reduces fatigue to a score between 4.5 and 5.5 on the ESSPRI scale (1-10). (Item 24) The method according to item 21, wherein the patient is administered an effective dose of the RNase-Fc every two weeks. (Item 25) The method according to Item 1, wherein the treatment improves the patient's fatigue by at least 1 point on the FACIT Fatigue Scale of the Functional Assessment of Chronic Disease Treatment (FACIT) compared to the FACIT score before the treatment. (Item 26) The method according to item 25, wherein the treatment improves the patient's fatigue score by at least 2 points on the FACIT fatigue scale. (Item 27) The method according to item 1, wherein the treatment increases the FACIT fatigue score by at least 1 point compared to the FACIT fatigue score before the treatment. (Item 28) The method according to item 27, wherein the treatment increases the FACIT fatigue score by at least 2 points compared to the FACIT fatigue score before the treatment. (Item 29) The method according to item 1, wherein the treatment reduces the fatigue profile (ProF) score in the patient by at least 1 point compared to the ProF score before the treatment. (Item 30) The method according to item 1, wherein the treatment reduces fatigue in the patient by at least 1 point in the mental component of the fatigue profile (ProF) score compared to the mental component ProF score before the treatment. (Item 31) The method according to item 1, wherein the treatment reduces fatigue in the patient by at least 1 point in the physical components of the fatigue profile (ProF) score compared to the physical components ProF score before the treatment. (Item 32) The method according to item 1, wherein the treatment improves the cognitive function of the patient, as measured by the Digital Substitution Test (DSST), compared to the DSST score before the treatment. (Item 33) The method according to item 1, wherein the procedure increases the number of matches completed by the patient in 90 seconds in a digit substitution test (DSST). (Item 34) The method according to item 1, wherein the procedure reduces the time it takes for the patient to complete the DSST examination. (Item 35) A method for treating Sjögren's disease in a human patient requiring treatment for Sjögren's disease by reducing fatigue, comprising the step of administering to the patient a dose of approximately 5-10 mg / kg of RNase-Fc fusion protein by intravenous injection, thereby treating Sjögren's disease in the patient by reducing fatigue. (Item 36) A method for treating Sjögren's disease in a human patient requiring treatment for Sjögren's disease by improving cognitive effects, comprising the step of administering an effective amount of RNase-Fc fusion protein to the patient, thereby treating Sjögren's disease by improving cognitive effects in the patient. (Item 37) The method according to item 36, wherein the cognitive effect in the patient is improved by at least 1 point in the mental components of ProF compared to the mental components of ProF before treatment. (Item 38) A method for treating Sjögren's disease in a human patient requiring treatment for Sjögren's disease by reducing fatigue, comprising the step of administering to the patient an effective amount of an RNase-Fc fusion protein comprising the amino acid sequence shown in SEQ ID NO: 50, thereby treating Sjögren's disease in the patient by reducing fatigue. (Item 39) A method for treating Sjögren's disease in a human patient requiring treatment for Sjögren's disease by reducing fatigue, comprising the step of administering an effective amount of a pharmaceutical composition to the patient, wherein the composition comprises an RNase-Fc fusion protein having the amino acid sequence shown in SEQ ID NO: 50; and one or more pharmaceutically acceptable carriers and / or diluents, thereby treating Sjögren's disease in the patient by reducing fatigue. (Item 40) The method according to item 35 or 36, wherein the RNase-Fc fusion protein comprises human pancreatic RNase 1. (Item 41) The method according to item 40, wherein the human pancreatic RNase 1 comprises the amino acid sequence shown in SEQ ID NO: 2. (Item 42) The method according to any one of items 35 to 41, wherein the RNase-Fc fusion protein comprises a wild-type human IgG1 Fc domain or a human IgG1 Fc domain containing one or more mutations. (Item 43) The method according to item 42, wherein the Fc domain containing one or more mutations has reduced binding to the Fcγ receptor on human cells. (Item 44) The method according to any one of items 35 to 39, wherein the RNase-Fc fusion protein has a reduction in effector function selected as necessary from the group consisting of opsonization, phagocytosis, complement-dependent cell injury, and antibody-dependent cell-mediated cytotoxicity. (Item 45) The method according to item 42, wherein the human IgG1 Fc domain contains the P238S mutation and the P331S mutation according to EU numbering. (Item 46) The method according to item 42, wherein the human IgG1 Fc domain includes a hinge domain, a CH2 domain, and a CH3 domain. (Item 47) The method according to item 42, wherein the human IgG1 Fc domain comprises substitution with one or more serine residues from among the three hinge region cysteine ​​residues. (Item 48) The method according to item 47, wherein the Fc domain contains SCC mutations (residues 220, 226, and 229) numbered according to the EU index. (Item 49) The method according to item 35 or 36, wherein the human IgG1 Fc domain comprises the amino acid sequence shown in SEQ ID NO: 22. (Item 50) The method according to item 35 or 36, wherein the RNase-Fc fusion protein comprises the amino acid sequence shown in SEQ ID NO: 50. (Item 51) The method according to any one of items 36 to 39, wherein the RNase-Fc fusion protein is administered to the patient at a dose of approximately 5 to 10 mg / kg. (Item 52) The method according to any one of items 35 to 39, wherein the RNase-Fc fusion protein is administered to the patient at a dose of approximately 10 mg / kg. (Item 53) The method according to any one of items 35 to 39, wherein the RNase-Fc fusion protein is administered to the patient at a dose of approximately 5 mg / kg. (Item 54) The method according to any one of items 36 to 39, wherein the RNase-Fc fusion protein is administered to the patient by intravenous injection. (Item 55) The method according to any one of items 35 to 39, wherein the RNase-Fc fusion protein is administered to the patient at a dose of approximately 5 to 10 mg / kg every two weeks. (Item 56) The method according to any one of items 35 to 39, wherein the RNase-Fc fusion protein is administered to the patient at a dose of approximately 5 to 10 mg / kg every two weeks for three months. (Item 57) The method according to any one of items 35 to 39, wherein the RNase-Fc fusion protein is administered to the patient in six bi-weekly infusions over a period of three months. (Item 58) The method according to any one of items 35 to 39, wherein the RNase-Fc fusion protein is administered to the patient weekly for three weeks, and then once every two weeks. (Item 59) The method according to any one of items 35 to 39, wherein the treatment reduces fatigue in the patient by at least 1 point on the EULAR SS Patient Reported Index (ESSPRI) score compared to the ESSPRI score before the treatment. (Item 60) The method according to any one of items 35 to 39, wherein the treatment reduces the ESSPRI score by at least 1 point compared to the ESSPRI score before the treatment. (Item 61) The method described in item 59, which reduces fatigue to a score between 4.5 and 5.5 on the ESSPRI scale (1-10). (Item 62) The method according to item 59, wherein the patient is administered an effective dose of the RNase-Fc every two weeks. (Item 63) The method according to any one of items 35 to 39, wherein the treatment improves the patient's fatigue by at least 1 point on the FACIT Fatigue Scale of the Assessment of Functional Independence of Chronic Diseases (FACIT) compared to the patient's FACIT score before the treatment. (Item 64) The method according to item 63, wherein the treatment improves the patient's fatigue score by at least 2 points on the FACIT fatigue scale. (Item 65) The method according to any one of items 35 to 39, wherein the treatment increases the FACIT fatigue score by at least 1 point compared to the FACIT fatigue score before the treatment. (Item 66) The method according to item 65, wherein the treatment increases the FACIT fatigue score by at least 2 points compared to the FACIT fatigue score before the treatment. (Item 67) The method according to any one of items 35 to 39, wherein the treatment reduces fatigue in the patient by at least 1 point on the fatigue profile (ProF) score compared to the ProF score before the treatment. (Item 68) The method according to any one of items 35 to 39, wherein the treatment reduces fatigue in the patient by at least 1 point in the mental component of the fatigue profile (ProF) score compared to the mental component ProF score before the treatment. (Item 69) The method according to any one of items 35 to 39, wherein the treatment reduces fatigue in the patient by at least 1 point in the physical components of the fatigue profile (ProF) score compared to the physical component ProF score before the treatment. (Item 70) The method according to any one of items 35 to 39, wherein the treatment improves cognitive function in the patient, as measured by the Digital Substitution Test (DSST), compared to the DSST score before the treatment. (Item 71) The method according to any one of items 35 to 39, wherein the procedure increases the number of matches completed by the patient in 90 seconds in a digit substitution test (DSST). (Item 72) The method according to any one of items 35 to 39, wherein the procedure reduces the time it takes for the patient to complete the DSST examination. (Item 73) A kit for use in the treatment of Sjögren's disease by reducing fatigue in human patients who require treatment for Sjögren's disease, comprising a container containing an injectable solution and instructions for use, An effective amount of RNase-Fc fusion protein as shown in SEQ ID NO: 50; and One or more pharmaceutically acceptable carriers and / or diluents A kit comprising the above, wherein the injectable solution is formulated for intravenous administration. (Item 74) An RNase-Fc fusion protein for use in a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the treatment comprises the step of administering an effective amount of the RNase-Fc fusion protein to the patient. (Item 75) Use of RNase-Fc fusion protein for use in the manufacture of pharmaceuticals for treating Sjögren's disease by reducing fatigue in human patients requiring treatment for Sjögren's disease. (Item 76) An RNase-Fc fusion protein for use in a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the treatment comprises the step of administering to the patient a dose of approximately 5-10 mg / kg of the RNase-Fc fusion protein by intravenous injection. (Item 77) Use of an RNase-Fc fusion protein for use in the manufacture of a pharmaceutical product for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the use comprises the step of administering to the patient a dose of approximately 5-10 mg / kg of the RNase-Fc fusion protein by intravenous injection. (Item 78) An RNase-Fc fusion protein for use in a method for treating Sjögren's disease by improving cognitive effects in a human patient requiring treatment for Sjögren's disease, wherein the treatment comprises the step of administering an effective amount of the RNase-Fc fusion protein to the patient. (Item 79) Use of an RNase-Fc fusion protein for use in the manufacture of a pharmaceutical for treating Sjögren's disease by improving cognitive effects in a human patient requiring treatment for Sjögren's disease, the use comprising the step of administering an effective amount of the RNase-Fc fusion protein to the patient. (Item 80) An RNase-Fc fusion protein for use in a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the treatment comprises the step of administering to the patient an effective amount of the RNase-Fc fusion protein having the amino acid sequence shown in SEQ ID NO: 50. (Item 81) Use of an RNase-Fc fusion protein for use in the manufacture of a pharmaceutical for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, the use comprising the step of administering to the patient an effective amount of the RNase-Fc fusion protein having the amino acid sequence shown in SEQ ID NO: 50. (Item 82) An RNase-Fc fusion protein for use in a method for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the treatment comprises the step of administering an effective amount of a pharmaceutical composition to the patient, the composition comprising an RNase-Fc fusion protein having the amino acid sequence shown in SEQ ID NO: 50; and one or more pharmaceutically acceptable carriers and / or diluents. (Item 83) An RNase-Fc fusion protein for use in the manufacture of a pharmaceutical for treating Sjögren's disease by reducing fatigue in a human patient requiring treatment for Sjögren's disease, wherein the use comprises the step of administering an effective amount of a pharmaceutical composition to the patient, the composition comprising an RNase-Fc fusion protein having the amino acid sequence shown in SEQ ID NO: 50; and one or more pharmaceutically acceptable carriers and / or diluents. (Item 84) A method for treating Sjögren's disease in a patient requiring treatment for Sjögren's disease, comprising the step of administering an effective amount of an RNA nuclease agent to the patient, wherein the treatment results in a reduction of one or more inflammation-related genes. (Item 85) The method according to item 84, wherein the one or more inflammation-related genes are selected from the group consisting of IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4, and STAT5B. (Item 86) The method according to item 84, wherein the one or more inflammation-related genes are selected from the group consisting of IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R, and STAT5B. (Item 87) A method for treating Sjögren's disease in a patient requiring treatment for Sjögren's disease, comprising the step of administering an effective amount of an RNA nuclease agent to the patient, wherein the treatment results in an increase of one or more inflammation-related genes. (Item 88) The method according to item 86, wherein the one or more inflammation-related genes are selected from the group consisting of CXCL10 (IP-10), CD163, RIPK2, and CCR2. (Item 89) A method for treating Sjögren's disease in a patient requiring treatment for Sjögren's disease, comprising the step of administering an effective amount of an RNA nuclease agent to the patient, wherein the treatment results in an increase of one or more cytokines and an improvement in fatigue. (Item 90) The method according to item 89, wherein the cytokine is CXCL10. (Item 91) A method for identifying patients with Sjögren's disease as candidates for treatment with RNA nuclease agents, (a) A step of determining the inflammation-related gene expression profile in the sample obtained from the patient, (b) A step of comparing the inflammation-related gene expression profile determined in step (a) with the inflammation-related gene expression profile in a sample obtained from an appropriate control subject. A method comprising the following, wherein the inflammation-related gene expression profile indicates that the patient is a candidate for treatment with an RNA nuclease agent. (Item 92) The method according to item 91, wherein the inflammation-related gene is selected from the group consisting of MAP3K8, ACKR3, STAT1, STAT2, TRIM37, and ZNF606. (Item 93) Use of an RNA nuclease agent in the manufacture of a pharmaceutical product for the treatment of Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in a reduction of one or more inflammation-related genes. (Item 94) The use described in item 93, wherein the one or more inflammation-related genes are selected from the group consisting of IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4, and STAT5B. (Item 95) The use described in item 93, wherein one or more inflammation-related genes are selected from the group consisting of IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R, and STAT5B. (Item 96) RNA nuclease agents for use in a method for treating Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in a reduction of one or more inflammation-related genes. (Item 97) The RNA nuclease agent described in item 96, wherein one or more inflammation-related genes are selected from the group consisting of IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4, and STAT5B. (Item 98) The RNA nuclease agent described in item 96, wherein one or more inflammation-related genes are selected from the group consisting of IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R, and STAT5B. (Item 99) Use of an RNA nuclease agent in the manufacture of a pharmaceutical product for the treatment of Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in an increase of one or more inflammation-related genes. (Item 100) The use described in item 99, wherein the one or more inflammation-related genes are selected from the group consisting of CXCL10(IP-10), CD163, RIPK2, and CCR2. (Item 101) RNA nuclease agents for use in a method of treating Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in an increase of one or more inflammation-related genes. (Item 102) The RNA nuclease agent described in item 101, wherein one or more inflammation-related genes are selected from the group consisting of CXCL10 (IP-10), CD163, RIPK2, and CCR2. (Item 103) Use of an RNA nuclease agent in the manufacture of a pharmaceutical product for the treatment of Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in an increase of one or more cytokines and an improvement in fatigue. (Item 104) The use described in item 103, wherein the cytokine is CXCL10. (Item 105) An RNA nuclease agent for use in a method for treating Sjögren's disease, wherein the use comprises the step of administering an effective amount of the RNA nuclease agent to the patient, and the treatment results in an increase of one or more cytokines and an improvement in fatigue. (Item 106) The RNA nuclease agent described in item 105, wherein the cytokine is CXCL10. [Brief explanation of the drawing]

[0052] [Figure 1] Figure 1 shows the composition of RSLV-132, a homodimeric RNase-Fc fusion protein containing two polypeptides. Each polypeptide in the homodimer has a constituent RNase-Fc, and the wild-type human RNase 1 domain is operably linked without a linker to the N-terminus of the human IgG1 Fc domain, which includes an SCC hinge and CH2 mutations P238S and P331S.

[0053] [Figure 2] Figure 2 is a graph showing the improvement in ESSPRI scores in pSS patients treated with RSLV-132 compared to patients treated with placebo. ESSPRI scores were assessed in pSS patients treated with RSLV-132 and placebo at Trial 1 (baseline), days 29, 57, 85, and 99 / end of treatment. A reduction of at least 1 point in the ESSPRI score is clinically significant. [Figure 3] Figure 3 is a graph showing the improvement in ESSPRI scores in patients treated with RSLV-132 compared to patients treated with placebo. The change in ESSPRI score over time from baseline is shown on the y-axis. ESSPRI scores were assessed in patients treated with RSLV-132 and placebo at Trial 1 (baseline), days 29, 57, 85, and 99 / end of treatment. A decrease of at least 1 point in the ESSPRI score is clinically significant.

[0054] [Figure 4] Figure 4 is a graph showing the mean change from baseline in the fatigue components of the ESSPRI for the RSLV-132 and placebo groups (p=0.136). It shows the improvement in the fatigue components of the ESSPRI score in patients treated with RSLV-132 compared to patients treated with placebo. The change in the fatigue components of the ESSPRI score over time from baseline is shown on the y-axis. The ESSPRI score was assessed in patients treated with RSLV-132 and placebo at day 1 (baseline), 29, 57, 85, and 99 / end of treatment. Results were analyzed using separate one-way analysis of variance (ANOVA) models for each visit, each testing the null hypothesis (H0) that there was zero true mean difference between the treatment groups. Unadjusted alpha = 0.05. All standard error bars use the standard error at day 99.

[0055] [Figure 5A]Figure 5A is a graph showing the improvement in the fatigue component of the ESSPRI score in patients treated with RSLV-132 compared to patients treated with placebo. The fatigue component of the ESSPRI score is shown on the y-axis. The ESSPRI score was assessed in patients treated with RSLV-132 and placebo at Study 1 (baseline) and day 99.

[0056] [Figure 5B] Figure 5B is a graph showing the pain components of the ESSPRI score in patients treated with RSLV-132 compared to patients treated with placebo. The pain components of the ESSPRI score are shown on the y-axis. The ESSPRI score was assessed in patients treated with RSLV-132 and placebo at Study 1 (baseline) and day 99.

[0057] [Figure 5C] Figure 5C is a graph showing the dry components of the ESSPRI score in patients treated with RSLV-132 compared to patients treated with placebo. The dry components of the ESSPRI score are shown on the y-axis. The ESSPRI score was assessed in patients treated with RSLV-132 and placebo at Study 1 (baseline) and day 99.

[0058] [Figure 6] Figure 6 is a graph showing the mean change from baseline in FACIT for the RSLV-132 and placebo groups (p=0.92). It shows the improvement in FACIT fatigue score in patients treated with RSLV-132 compared to patients treated with placebo. FACIT fatigue score was assessed in patients treated with RSLV-132 and placebo at day 1 (baseline), 29, 57, 85, and 99 / end of treatment. An increase in FACIT fatigue score indicates improvement in fatigue. Results were analyzed using separate one-way analysis of variance (ANOVA) models at each visit, each testing the null hypothesis (H0) that there was zero true mean difference between the treatment groups. Unadjusted alpha = 0.05. Total standard error bars use the standard error at day 99.

[0059] [Figure 7] Figure 7 is a graph showing the improvement in ProF in patients treated with RSLV-132 compared to patients treated with placebo. The change in ProF score over time from baseline is shown on the y-axis. ProF scores were assessed in patients treated with RSLV-132 and placebo at day 1 (baseline), 29, 57, 85, and 99 / end of treatment. A decrease in ProF score indicates improvement in fatigue.

[0060] [Figure 8] Figure 8 is a graph showing the mean change in the mental fatigue component of ProF for the RSLV-132 and placebo groups (p=0.046). It shows the improvement in the mental component of ProF in patients treated with RSLV-132 compared to patients treated with placebo. The change in the mental component of ProF score over time from baseline is shown on the y-axis. The mental component of ProF score was assessed in patients treated with RSLV-132 and placebo at day 1 (baseline), 29, 57, 85, and 99 / end of treatment. A decrease in ProF score indicates improvement in fatigue. Results were analyzed using separate one-way analysis of variance (ANOVA) models at each visit, each testing the null hypothesis (H0) that there was zero true mean difference between treatment groups. Unadjusted alpha = 0.05. Total standard error bars use the standard error at day 99.

[0061] [Figure 9] Figure 9 is a graph showing the improvement in the physical components of ProF in patients treated with RSLV-132 compared to patients treated with placebo. The y-axis shows the change in the physical components of the ProF score over time from baseline. The physical components of the ProF score were assessed in patients treated with RSLV-132 and placebo at day 1 (baseline), 29, 57, 85, and 99 / end of treatment. A decrease in the ProF score indicates improvement in fatigue.

[0062] [Figure 10] Figures 10A and 10B show the results of the Digit Symbol Substitution Test (DSST) in patients treated with RSLV-132 and placebo. Patients underwent the DSST test at baseline (day 1) and day 99 of the trial. As shown in Figure 10A, "Total in 90 seconds" refers to the total number of symbols matched within 90 seconds. "Completion" refers to the time taken to complete the test, expressed in seconds. A statistically significant improvement in the time to complete the DSST test was observed in patients treated with RSLV-132 from the initial baseline test (day 1) to follow-up (day 99). Figure 10B is a graph showing the increase in time to completion for placebo-treated patients and the decrease in time to completion for RSLV-132-treated patients.

[0063] [Figure 11] Figure 11 shows the changes in gene expression at day 99 compared to day 1 (baseline) for RSLV-132 subjects who achieved or did not achieve a clinical response. The genes shown in the heatmap are those that showed a high degree of correlation with FACIT instrument performance (R2 > 0.6).

[0064] [Figure 12A] Figures 12A to 12C show the baseline (pre-administration of the study drug) gene expression patterns of RSLV-132 treated subjects who subsequently experienced MCII on day 99, compared to subjects who did not experience it. Genes with the best correlation to a given measurement method are shown. Figure 12A shows genes correlated with FACIT (R2>0.6). Figure 12B shows genes correlated with ProF (R2>0.6). Figure 12C shows genes correlated with ESSPRI (R2>0.6). [Figure 12B]Figures 12A to 12C show the baseline (pre-administration of the study drug) gene expression patterns of RSLV-132 treated subjects who subsequently experienced MCII on day 99, compared to subjects who did not experience it. Genes with the best correlation to a given measurement method are shown. Figure 12A shows genes correlated with FACIT (R2>0.6). Figure 12B shows genes correlated with ProF (R2>0.6). Figure 12C shows genes correlated with ESSPRI (R2>0.6). [Figure 12C] Figures 12A to 12C show the baseline (pre-administration of the study drug) gene expression patterns of RSLV-132 treated subjects who subsequently experienced MCII on day 99, compared to subjects who did not experience it. Genes with the best correlation to a given measurement method are shown. Figure 12A shows genes correlated with FACIT (R2>0.6). Figure 12B shows genes correlated with ProF (R2>0.6). Figure 12C shows genes correlated with ESSPRI (R2>0.6). [Modes for carrying out the invention]

[0065] This disclosure provides an RNase-containing nuclease fusion protein, including an RNase-Fc fusion protein, for digesting circulating RNA and RNA complexed with autoantibodies and immune complexes, thereby treating patients with primary Sjögren's syndrome (pSS). This disclosure also provides a method for treating diseases characterized by elevated levels of circulating RNA and / or RNA-containing autoantibodies, such as Sjögren's syndrome, and a method for treating symptoms of diseases characterized by elevated levels of circulating RNA and RNA-containing autoantibodies, such as Sjögren's syndrome-related fatigue, in human patients requiring such treatment. This disclosure also provides effective treatment and dosing regimens for administering an RNase-containing nuclease fusion protein, including an RNase-Fc fusion protein, to human patients with Sjögren's syndrome, including patients with pSS, who require such treatment.

[0066] This disclosure is based, at least in part, on the remarkable finding that treatment of patients with pSS with RNase-containing nuclease fusion proteins such as RSLV-132 reduces pSS-related fatigue in these patients. Without being constrained by theory, it is thought that the RNase-containing nuclease fusion proteins of this disclosure may digest circulating RNA, as well as RNA complexed with autoantibodies and immune complexes, in patients with autoimmune diseases, thereby reducing symptoms of autoimmune diseases, such as Sjögren's syndrome-related fatigue.

[0067] Without being constrained by theory, it is also conceivable that treatment of patients with Sjögren's syndrome with RNase-containing nuclease fusion proteins such as RSLV-132 reduces circulating RNA, whether it is associated with autoantibodies in circulation or is free, thereby reducing TLR activation and activation of several downstream inflammatory pathways. Therefore, treatment of patients with Sjögren's syndrome, including those with pSS, with RNase-containing nuclease fusion proteins such as RSLV-132 may reduce, decrease, or inhibit the activation of pro-inflammatory cascades, thereby reducing or decreasing the overall inflammatory characteristics of Sjögren's syndrome in patients, thereby reducing or decreasing symptoms associated with Sjögren's syndrome, including fatigue, pain, dryness, depression, and / or cognitive impairment.

[0068] Unexpectedly, treatment of pSS patients with RNase-containing nuclease fusion proteins such as RSLV-132 was found to result in improved fatigue in patients, as consistently demonstrated by three separate tests. In particular, when pSS patients were administered the EULAR Sjögren's Syndrome Patient Reported Index (ESSPRI) after treatment with RSLV-132, patients experienced a clinically significant decrease of 1 point or more in the fatigue component of the ESSPRI score. The decrease in the ESSPRI score is associated with improved fatigue. Administration of the Functional Assessment of Chronic Illness Test (FACIT) in these patients after treatment with RSLV-132 resulted in an increase in the FACIT fatigue score, associated with reduced fatigue. Similarly, administration of the Profile of Fatigue (ProF) test after treatment with RSLV-132 resulted in a decrease in the ProF score, associated with reduced fatigue.

[0069] Furthermore, surprisingly, it was discovered that after treatment of pSS patients with RLSV-132, patients demonstrated improved cognitive abilities, as measured by the Digital Sign Substitution Test (DSST). Therefore, this disclosure provides an RNase-containing nuclease fusion protein, including an RNase-Fc fusion protein, which is useful in methods for treating Sjögren's syndrome, methods for reducing Sjögren's syndrome-related fatigue, and methods for improving cognitive abilities in patients with Sjögren's syndrome. This disclosure also provides a composition comprising an RNase-Fc fusion protein and one or more pharmaceutically acceptable carriers and / or diluents, which is useful in methods for treating Sjögren's syndrome, methods for reducing Sjögren's syndrome-related fatigue, and methods for improving cognitive abilities in patients with Sjögren's syndrome. In some embodiments, the RNase-Fc fusion protein is RSLV-132. In some embodiments, the RNase-Fc fusion protein is administered to human patients at doses of approximately 5–10 mg / kg, approximately 2–8 mg / kg, approximately 3–6 mg / kg, approximately 3 mg / kg, approximately 5 mg / kg, or approximately 10 mg / kg.

[0070] Furthermore, it was discovered that patients with primary Sjögren's syndrome (pSS) who achieved a clinical response after treatment with RNA nucleases (e.g., RSLV-132) exhibited decreased expression of inflammation-related genes. For example, the expression of IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4, and STAT5B was decreased in patients treated with RNA nucleases (e.g., RSLV-132) who experienced a clinical response. It was also discovered that patients who achieved a clinical response after treatment with RSLV-132 exhibited increased expression of inflammation-related genes. For example, the expression of CXCL10 (IP-10), CD163, RIPK2, and CCR2 was increased in patients treated with RNA nucleases (e.g., RSLV-132) who experienced a clinical response.

[0071] Further findings revealed that distinct gene expression profiles existed prior to RNA nuclease (e.g., RSLV-132) administration in patients with autoimmune diseases (e.g., primary Sjögren's syndrome (pSS)) who subsequently developed a positive clinical response to RNA nucleases (e.g., RSLV-132). For example, specific profiles emerged among RSLV-132 responders when baseline gene expression correlated with FACIT, ProF, or ESSPRI. Decreased expression of STAT1 and STAT2 correlated with FACIT testing, increased expression of ZNF606 and decreased expression of TRIM37 correlated with ProF testing, and increased expression of ACKR3 and decreased expression of MAPK3K8 correlated with ESSPRI testing.

[0072] Without being constrained by theory, specific RNA molecules that promote chronic activation of inflammatory pathways may be present in the circulation of some patients. Therefore, the removal of specific circulating non-coding RNAs and pro-inflammatory RNAs by RNA nucleases (e.g., RSLV-132) is generally considered to contribute to the treatment of patients with autoimmune diseases (e.g., pSS). Thus, it may be possible to use "gene expression fingerprinting" to identify patients who would benefit most from treatment with RNA nucleases such as RSLV-132.

[0073] definition Unless otherwise specified, terms used in the claims and specification are defined as set forth below.

[0074] "Amino acids" refer to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code, as well as later modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., hydrogen-bonded carbon, carboxyl group, amino group, and R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

[0075] Amino acids may be referred herein by either their commonly known three-letter or one-letter symbols, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may similarly be referred by their commonly accepted single-letter codes.

[0076] "Amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different "substitute" amino acid residue. "Amino acid insertion" refers to the incorporation of at least one additional amino acid into a given amino acid sequence. Insertions will typically consist of the insertion of one or two amino acid residues, but larger "peptide insertions," such as the insertion of about 3 to about 5 or even up to about 10, 15, or 20 amino acid residues, are possible. The inserted residue(s) may be naturally occurring or non-naturally occurring, as disclosed above. "Amino acid deletion" refers to the removal of at least one amino acid residue from a given amino acid sequence.

[0077] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimeticities of corresponding naturally occurring amino acids.

[0078] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides, and polymers of these in either single-stranded or double-stranded forms. Unless otherwise specified, this term encompasses nucleic acids containing known analogs of native nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a similar manner to naturally occurring nucleotides. Unless otherwise noted, a particular nucleic acid sequence implicitly includes not only the explicitly shown sequence but also its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res 1991;19:5081; Ohtsuka et al., JBC 1985;260:2605-8; Rossolini et al., Mol Cell Probes 1994;8:91-8). With respect to arginine and leucine, the modification at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by gene.

[0079] The polynucleotides of the present invention may consist of any polyribonucleotide or polydeoxyribonucleotide that can be unmodified RNA or DNA, or modified RNA or DNA. For example, polynucleotides may consist of hybrid molecules including single- and double-stranded DNA, DNA which is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA which is a mixture of single- and double-stranded regions, single-stranded, or more typically double-stranded, or a mixture of single- and double-stranded regions. In addition, polynucleotides may consist of triple-stranded regions that include RNA or DNA, or both RNA and DNA. Polynucleotides may also contain one or more modified bases or DNA or RNA backbone that have been modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; therefore, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms.

[0080] As used herein, the terms “operably coupled” or “operably coupled” refer to a juxtaposition of components described in a relationship that enables them to function in the manner they are intended.

[0081] As used herein, the terms “glycosylation” or “glycosylated” refer to the process or result of adding a sugar moiety to a molecule.

[0082] As used herein, the term “modified glycosylation” refers to a molecule that is aglycosylated, deglycosylated, or underglycosylated.

[0083] As used herein, “glycosylation site(s)” refers to both a site that is potentially able to accept a carbohydrate moiety and a site within a protein to which the carbohydrate moiety is actually attached, and includes any amino acid sequence that can act as an acceptor for oligosaccharides and / or carbohydrates.

[0084] As used herein, the terms “undglycosylated” or “undglycosylated” refer to the production of a molecule in an unglycosylated form (for example, by manipulating a protein or polypeptide to lack an amino acid residue that functions as a glycosylation acceptor). Alternatively, a protein or polypeptide can be expressed, for example, in E. coli to produce an undglycosylated protein or polypeptide.

[0085] As used herein, the terms “desglycosylation” or “desglycosylated” refer to the process or result of the enzymatic removal of a sugar portion from a molecule.

[0086] As used herein, the terms “insufficiently glycosylated” or “insufficiently glycosylated” refer to a molecule in which one or more carbohydrate structures that would normally be present when produced in a mammalian cell are omitted, removed, modified, or shielded.

[0087] As used herein, the terms “Fc region” and “Fc domain” refer to a portion of a native immunoglobulin formed by the Fc domains (or Fc parities) of its two heavy chains, without the antigen-binding variable region. In some embodiments, the Fc domain begins in the hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc domain includes at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc domain includes at least one of the following: a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or variants, parities, or fragments thereof. In other embodiments, the Fc domain includes a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In one embodiment, the Fc domain includes a hinge domain (or parity thereof) fused to a CH3 domain (or parity thereof). In another embodiment, the Fc domain includes a CH2 domain (or parity thereof) fused to a CH3 domain (or parity thereof). In another embodiment, the Fc domain consists of a CH3 domain or a portion thereof. In another embodiment, the Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In another embodiment, the Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In another embodiment, the Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In one embodiment, the Fc domain lacks at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). In one embodiment, the Fc domain of the present invention includes at least a portion of an Fc molecule known in the art as required for FcRn binding. In one embodiment, the Fc domain of the present invention includes at least a portion of an Fc molecule known in the art as required for protein A binding. In one embodiment, the Fc domain of the present invention includes at least a portion of an Fc molecule known in the art as required for protein G binding. As used herein, an Fc domain generally refers to a polypeptide comprising all or part of an Fc domain of an immunoglobulin heavy chain.Examples of such molecules include, but are not limited to, polypeptides containing the entire CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides containing only the hinge, CH2, and CH3 domains. Fc domains can originate from immunoglobulins of any species and / or any subtype, including, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. Fc domains encompass native Fc and Fc variant molecules. As with Fc variants and native Fc, the term Fc domain includes molecules in monomeric or polymeric form, whether digested from whole antibodies or produced by other means.

[0088] As described herein, those skilled in the art will understand that any of the Fc domains of naturally occurring immunoglobulin molecules can be modified so that their amino acid sequences are altered.

[0089] The Fc domain of the RNase-Fc fusion protein of this disclosure may be derived from different immunoglobulin molecules. For example, the Fc domain of the RNase-Fc fusion protein may include CH2 and / or CH3 domains derived from the IgG1 molecule, as well as a hinge region derived from the IgG3 molecule. In another example, the Fc domain may include a chimeric hinge region partially derived from the IgG1 molecule and partially derived from the IgG3 molecule. In yet another example, the Fc domain may include a chimeric hinge partially derived from the IgG1 molecule and partially derived from the IgG4 molecule. The wild-type human IgG1 Fc domain has the amino acid sequence shown in SEQ ID NO: 20.

[0090] As used herein, the term “serum half-life” refers to the time required for the concentration of an in vivo serum RNase-Fc fusion protein to decrease by 50%. A shorter serum half-life for an RNase-Fc fusion protein means that it takes less time to exert its therapeutic effect.

[0091] As used herein, the term “RNA nuclease” refers to a drug containing an RNase domain. In some embodiments, the RNA nuclease is an RNase-containing nuclease fusion protein. In some embodiments, the RNA nuclease is an RNase-Fc fusion protein. In some embodiments, the RNase domain of the RNA nuclease is human pancreatic RNase 1. In some embodiments, the RNA nuclease is a polypeptide. In some embodiments, the RNA nuclease is RSLV-132.

[0092] As used herein, the term “RNase-containing nuclease fusion protein” refers to a polypeptide comprising at least one nuclease domain operably linked to a PK (pharmacokinetic) portion, such as an Fc domain or a variant or fragment thereof, with or without a linker, and the nucleic acid encoding such a polypeptide. In some embodiments, an RNase-containing nuclease fusion protein is an “RNase-Fc fusion protein,” which refers to a polypeptide comprising at least one nuclease domain operably linked to an Fc domain or a variant or fragment thereof, with or without a linker, and the nucleic acid encoding such a polypeptide. In some embodiments, an RNase-Fc fusion protein is a polypeptide comprising at least two nuclease domains operably linked to an Fc domain or a variant or fragment thereof, with or without a linker, and the nucleic acid encoding such a polypeptide. In some embodiments, the nuclease domain is human RNase 1. In some embodiments, an RNase-Fc fusion protein comprises one or more RNase domains and one or more Fc domains. In some embodiments, the RNase-Fc fusion protein comprises one or more RNase-domains, one or more Fc domains, and one or more DNase domains. In some embodiments, the RNase-Fc fusion protein comprises an RNase 1 domain operably ligated to the N or C terminus of an Fc domain, and a DNase domain operably ligated to the N or C terminus of an Fc domain. In some embodiments, the RNase-Fc fusion protein is a tandem RNase-Fc fusion protein, for example, in which one or more RNase 1 domains and / or one or more DNase domains are tandemly ligated to either the N or C terminus of one or more Fc domains. In some embodiments, the RNase-Fc fusion protein is a homodimeric RNase-Fc fusion protein (two identical polypeptides).In some embodiments, the RNase-Fc fusion protein is a heterodimeric RNase-Fc fusion protein (two different polypeptides). In some embodiments, the domains of the RNase-Fc fusion protein are operably linked with a linker domain. In some embodiments, the domains of the RNase-Fc fusion protein are operably linked without a linker domain.

[0093] As used herein, the term “tandem RNase-Fc fusion protein” refers to a polypeptide comprising at least two nuclease domains linked in tandem (from N-terminus to C-terminus), and an Fc domain or a variant or fragment thereof, as well as the nucleic acid encoding such a polypeptide. In some embodiments, the tandem RNase-Fc fusion protein is a polypeptide comprising at least two RNase 1 domains operably linked in tandem to at least one Fc domain. In some embodiments, the tandem RNase-Fc fusion protein is a polypeptide comprising at least one DNase 1 domain and at least one RNase 1 domain operably linked in tandem to at least one Fc domain. In some embodiments, the tandem RNase-Fc fusion protein comprises, from N-terminus to C-terminus, a DNase 1 domain, a first linker, an RNase 1 domain, a second linker, and an Fc domain or a variant or fragment thereof.

[0094] As used herein, the term “heterodimerated RNase-Fc fusion protein” refers to a heterodimer comprising a first and second polypeptide comprising at least two nuclease domains and two Fc domains, variants or fragments thereof, together, as well as the nucleic acid encoding such polypeptides. In some embodiments, the heterodimer comprises a first RNase 1 domain operably linked to the N or C terminus of the first Fc domain with or without a linker, such that the first RNase 1 and second RNase 1 domains are located at the same end (N or C terminus) of the heterodimer, and a second RNase 1 domain operably linked to the N or C terminus of the second Fc domain with or without a linker. In some embodiments, the heterodimer comprises a first RNase 1 domain operably linked to the N terminus of the first Fc domain with or without a linker, and a second RNase 1 domain operably linked to the C terminus of the second Fc domain with or without a linker. In some embodiments, the first RNase 1 domain is operably linked to the C-terminus of the first Fc domain with or without a linker, and the second RNase 1 domain is operably linked to the N-terminus of the second Fc domain with or without a linker. In some embodiments, the first and second RNase 1 domains of the heterodimer are different. In some embodiments, the heterodimer RNase-Fc fusion protein is a heterodimer comprising at least one DNase1 domain and at least one RNase1 domain, operably linked to at least one Fc domain, where the DNase1 domain is operably linked to the N or C terminus of the first Fc domain with or without a linker, and the RNase1 domain is operably linked to the N or C terminus of the same (first Fc domain) or a different Fc domain (second Fc domain) with or without a linker, so that the DNase1 domain and the RNase1 domain are located at opposite ends (N or C terminus) of either the same (first Fc domain) or a different Fc domain (second Fc domain).In some embodiments, the heterodimer contains DNase.

[0095] As used herein, the term “homodimerated RNase-Fc fusion protein” refers to a homodimer comprising a first and second polypeptide comprising at least two identical nuclease domains and two Fc domains, variants or fragments thereof, together, as well as the nucleic acid encoding such polypeptides. In some embodiments, the homodimer comprises a first RNase 1 domain operably linked to the N or C terminus of the first Fc domain with or without a linker, and a second RNase 1 domain operably linked to the N or C terminus of the second Fc domain with or without a linker, so that the first and second RNase 1 domains are located at the same terminus (N or C terminus) of the homodimer. In some embodiments, the first and second RNase 1 domains of the homodimer are identical. In some embodiments, the homodimer includes an RNase 1 domain operably linked to the N or C terminus of the Fc domain with or without a linker, and a DNase domain operably linked to the N or C terminus of the Fc domain with or without a linker. In some embodiments, the RNase 1 and DNase domains are located at the same terminus (N or C terminus) of the Fc domain. In some embodiments, the RNase 1 and DNase domains are located at opposite terms of the Fc domain.

[0096] As used herein, the term “dimer” refers to a macromolecular complex formed by two macromolecules (e.g., polypeptides). “Homodimer” refers to a dimer formed by two identical macromolecules (e.g., polypeptides). “Heterodimer” refers to a dimer formed by two different macromolecules (e.g., polypeptides).

[0097] As used herein, the term “variant” refers to a polypeptide derived from a wild-type nuclease (e.g., RNase) or Fc domain that differs from the wild-type due to one or more alterations (singular or plural) at one or more positions, i.e., substitutions, insertions, and / or deletions. Substitution means the replacement of an amino acid occupying a position with a different amino acid. Deletion means the removal of an amino acid occupying a position. Insertion means the addition of one or more amino acids, such as one to three, directly adjacent to an amino acid occupying a position. Variant polypeptides necessarily have less than 100% sequence identity or similarity to the wild-type polypeptide. In some embodiments, the variant polypeptide may have an amino acid sequence having, for example, about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the wild-type polypeptide over the length of the variant polypeptide, or about 80% to less than 100%, about 85% to less than 100%, or about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or about 95% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the wild-type polypeptide.

[0098] In certain embodiments, RNase-Fc fusion proteins utilize one or more “linker domains,” such as polypeptide linkers. As used herein, the term “linker domain” refers to one or more amino acids that connect two or more peptide domains in a linear polypeptide sequence. As used herein, the term “polypeptide linker” refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) that connects two or more polypeptide domains in a linear amino acid sequence of a protein. For example, a polypeptide linker can be used to operably link a nuclease domain (e.g., RNase) to an Fc domain. Such polypeptide linkers provide flexibility to the polypeptide molecule in some embodiments. In some embodiments, a polypeptide linker is used to connect (e.g., genetically fuse) the RNase domain to the Fc domain. RNase-Fc fusion proteins may contain one or more linker domains or peptide linkers. Various peptide linkers are known in the art.

[0099] As used herein, the term “gly-ser polypeptide linker” refers to a peptide consisting of glycine and serine residues. An exemplary gly / ser polypeptide linker comprises the amino acid sequence (Gly4Ser)n. In some embodiments, n is one or more, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (e.g., (Gly4Ser)10). Another exemplary gly / ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser)n. In some embodiments, n is one or more, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (e.g., Ser(Gly4Ser)10).

[0100] As used herein, the terms “coupled,” “conjugated,” “linked,” “fused,” and “fused” are interchangeable. These terms refer to the jointing of two or more elements, components, or domains by any means, including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art.

[0101] A polypeptide or amino acid sequence "derived from" a designated polypeptide or protein refers to the origin of the polypeptide. Preferably, a polypeptide or amino acid sequence derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, the portion consisting of at least 10 to 20 amino acids, preferably at least 20 to 30 amino acids, more preferably at least 30 to 50 amino acids, or it can otherwise be identified as originating from that sequence by those skilled in the art. A polypeptide derived from another polypeptide may have one or more amino acid residues that have one or more mutations compared to the starting polypeptide, for example, substitution with another amino acid residue, or insertion or deletion of one or more amino acid residues.

[0102] In one embodiment, there is a difference of one amino acid between the starting polypeptide sequence and the sequence derived therefrom. Sequence identity or similarity is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., the same residue) to the starting amino acid residue after the sequences have been aligned and gaps introduced if necessary to achieve maximum percentage sequence identity.

[0103] In one embodiment, the polypeptides of the Disclosure consist of, or consist of, the amino acid sequences listed in the sequence list or sequence listing disclosed herein, and functionally active variants thereof, or consist of, essentially, or include thereof. In one embodiment, the polypeptide includes an amino acid sequence that is at least 80%, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequences listed in the sequence list or sequence listing disclosed herein. In some embodiments, the polypeptide includes a neighboring amino acid sequence that is at least 80% identical, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a neighboring amino acid sequence listed in the sequence list or sequence listing disclosed herein. In some embodiments, the polypeptide comprises an amino acid sequence having at least 10, for example, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 200, at least 300, at least 400 or at least 500 (or any integer within these numbers) adjacent amino acids of an amino acid sequence as described in the sequence list or sequence listing disclosed herein.

[0104] In some embodiments, the RNase-Fc fusion proteins of the Disclosure are encoded by nucleotide sequences. The nucleotide sequences of the Disclosure may be useful for a number of applications, including cloning, gene therapy, protein expression and purification, mutagenesis, DNA vaccination of hosts requiring it, antibody generation for passive immunization, PCR, primer and probe generation, siRNA design and generation (see, for example, the Dharmacon siDesign website), and others. In some embodiments, the nucleotide sequences of the Disclosure comprise, or are essentially comprised of, nucleotide sequences encoding the amino acid sequence of an RNase-Fc fusion protein selected from a sequence listing or sequence list. In some embodiments, the nucleotide sequence includes a nucleotide sequence that is at least 80%, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence encoding an amino acid sequence in a sequence list or sequence listing disclosed herein. In some embodiments, the nucleotide sequence includes a neighboring nucleotide sequence that is at least 80%, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a neighboring nucleotide sequence encoding an amino acid sequence as described in the sequence list or sequence listing disclosed herein.In some embodiments, the nucleotide sequence includes a nucleotide sequence having at least 10, for example, at least 15, for example, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 200, at least 300, at least 400 or at least 500 (or any integer within these numbers) adjacent nucleotides of a nucleotide sequence encoding an amino acid sequence as described in the sequence list or sequence listing disclosed herein.

[0105] Those skilled in the art will also understand that it is possible to modify RNase-Fc fusion proteins so that their components (e.g., nuclease domain, linker domain, and Fc domain) are derived from or deviate from the naturally occurring sequences, while retaining the desired activity of the natural sequence. For example, nucleotide or amino acid substitutions can be made that result in conservative substitutions or changes to "non-essential" amino acid residues. Isolated nucleic acid molecules encoding non-natural variants can be prepared by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of an RNase-Fc fusion protein so that one or more amino acid substitutions, additions, or deletions are introduced into the encoding protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0106] RNase-Fc fusion proteins may contain one or more amino acid residues, e.g., essential or non-essential amino acid residues, with conserved amino acid substitutions. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the Art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in the RNase-Fc fusion protein are preferably replaced with other amino acid residues derived from the same side-chain family. In another embodiment, the amino acid sequence can be replaced with a structurally similar sequence that differs in the order and / or composition of the side-chain family members. Alternatively, in another embodiment, mutations can be introduced randomly along the entire or partial coding sequence by saturation mutagenesis or the like, and the resulting mutants can be incorporated into the RNase-Fc fusion protein and screened for their ability to bind to a desired target.

[0107] As used herein, the term “innate immune system regulators” refers to any gene, protein, nucleic acid or microRNA associated with the expression or regulation of the innate immune response, including cytokine and chemokine expression and secretion; dendritic cell activation; and complement cascades. In some embodiments, innate immune system regulators include inflammation-related molecules. In some embodiments, innate immune system regulators include inflammation-related genes. In some embodiments, innate immune system regulators include inflammation-related proteins.

[0108] In some embodiments, the regulators of the innate immune system include genes or proteins involved in the regulation of signaling, interferon family members, complement, antigen processing, signaling, ubiquitination, chemotaxis, cell adhesion, and polymerase activity.

[0109] The term "innate immune system" refers to a non-specific defense mechanism against antigens. The innate immune response is not driven by specificity to a particular antigen, but rather by the presence of the antigen. Functions of the innate immune system include acting as a physical and chemical barrier against infectious pathogens, recognition and removal of foreign substances by leukocytes, dendritic cell activation, cytokine and chemokine secretion, activation of the complement cascade, and activation of the adaptive immune system.

[0110] As used herein, the term “inflammation-related molecule” refers to a molecule that functions in inflammation or an inflammatory response. In some embodiments, the inflammation-related molecule is a pro-inflammatory molecule. In some embodiments, the inflammation-related molecule is an anti-inflammatory molecule. In some embodiments, the inflammation-related molecule is an inflammation-related gene. In some embodiments, the inflammation-related molecule is an inflammation-related protein. In some embodiments, the inflammation-related molecule is an inflammation-related cytokine. In some embodiments, the inflammation-related molecule is an inflammation mediator.

[0111] As used herein, the term “inflammation-related gene” refers to a gene that functions in inflammation or an inflammatory response. In some embodiments, the inflammation-related gene is a pro-inflammatory gene. In some embodiments, the inflammation-related gene is an anti-inflammatory gene. In some embodiments, the inflammation-related gene encodes an inflammation-related protein. In some embodiments, the inflammation-related gene encodes a cytokine.

[0112] As used herein, the term “inflammation-related protein” refers to a protein that functions in inflammation or an inflammatory response. In some embodiments, the inflammation-related protein is a pro-inflammatory protein. In some embodiments, the inflammation-related protein is an anti-inflammatory protein. In some embodiments, the inflammation-related protein is a cytokine.

[0113] As used herein, the term “pro-inflammatory molecule” refers to a molecule that enhances or stimulates an inflammatory response. In some embodiments, the pro-inflammatory molecule is a “pro-inflammatory gene.” In some embodiments, the pro-inflammatory molecule is a “pro-inflammatory protein.” In some embodiments, the pro-inflammatory gene encodes a pro-inflammatory protein. In some embodiments, the pro-inflammatory molecule is an “inflammatory cytokine.”

[0114] In some embodiments, the term "stimulating an inflammatory response" refers to stimulating the production of inflammatory cytokines.

[0115] As used herein, the term “inflammatory cytokine” refers to signaling molecules (cytokines) secreted by immune cells (e.g., helper T cells and macrophages) that play a role in the inflammatory response.

[0116] As used herein, the term “gene expression profile” refers to a technique for identifying genes expressed in a sample and / or determining the degree of their expression at a specified time. The term “inflammation-related gene expression profile” refers to a gene expression profile for identifying inflammation-related genes that are expressed and / or determining the degree of their expression at a specified time.

[0117] The term "remission" refers to any therapeutically beneficial outcome in the treatment of a disease condition, such as an autoimmune disease condition (e.g., SLE, Sjögren's syndrome), including prevention, reduction of severity or progression, improvement or cure.

[0118] As used herein, the terms “primary Sjögren’s syndrome (pSS),” “Sjögren’s syndrome,” “Sjögren’s disease,” and “Sjögren” are interchangeable.

[0119] The term "in situ" refers to the process that occurs in living cells that are separated from a living organism and grow, for example, in tissue culture.

[0120] The term "in vivo" refers to processes that occur in living organisms.

[0121] The terms “mammal,” “subject,” or “patient,” as used herein, include both human and non-human animals, including but not limited to humans, non-human primates, dogs, cats, mice, cattle, horses, and pigs.

[0122] In the context of two or more nucleic acid or polypeptide sequences, the term "percent identity" refers to two or more sequences or subsequences that, when compared and aligned, have a specified percentage of nucleotide or amino acid residues that are identical in terms of maximum match, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the percentage "identity" may exist across regions of the sequences being compared, for example, across functional domains, or across the entire length of the two sequences being compared.

[0123] For sequence comparison, typically one sequence acts as the reference sequence compared to the sequence under comparison. When using a sequence comparison algorithm, the sequence under comparison and the reference sequence are input into the computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the sequence(s) under comparison compared to the reference sequence(s) based on the specified program parameters.

[0124] For example, optimal sequence alignment for comparison can be achieved using the local homology algorithm of Smith & Waterman, Adv Appl Math 1981;2:482, the homology alignment algorithm of Needleman & Wunsch, J Mol Biol 1970;48:443, the similarity search method of Pearson & Lipman, PNAS 1988;85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, within the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (generally, see Ausubel et al., below).

[0125] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J Mol Biol 1990;215:403-10. Software for performing BLAST analysis is available through the National Center for Biotechnology Information website.

[0126] The term "sufficient amount" means an amount sufficient to produce the desired effect.

[0127] The term "therapeutic dose" refers to the amount of a drug effective in achieving remission of the symptoms of a disease. Since preventive measures can be considered as treatments, the therapeutic dose may also be called the "preventive dose."

[0128] Those skilled in the art will understand that the term "approximately" can vary to some extent depending on the context in which it is used. Where the use of this term is not apparent to those skilled in the art, even considering the context in which it is used, "approximately" will mean within plus or minus 10% of a given value.

[0129] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise.

[0130] RNase-containing nuclease fusion protein The RNase-containing nuclease fusion proteins of this disclosure, including RNase-Fc fusion proteins, include at least one enzymatically active RNase domain or a fragment or variant, such as human RNase 1 or a fragment or variant, which is operably ligated to a PK moiety that provides a scaffold and / or extends the in vivo half-life of the RNase domain compared to a nuclease domain without such a PK moiety. In some embodiments, the RNase-containing nuclease fusion protein is an RNase-Fc fusion protein that includes at least one enzymatically active RNase domain or a fragment or variant, such as human RNase 1 or a fragment or variant, which is operably ligated to an Fc domain, such as human IgG1 Fc domain or a variant or fragment, which alters the serum half-life of the nuclease molecule to which it is fused compared to a nuclease molecule not fused to the Fc domain or a variant or fragment.

[0131] In some embodiments, the RNase-containing nuclease fusion proteins of this disclosure, including RNase-Fc fusion proteins, are operably linked to an Fc domain or a variant or fragment thereof via a linker domain. In some embodiments, the linker domain is a linker peptide. In some embodiments, the linker domain is a linker nucleotide.

[0132] In some embodiments, the RNase-containing nuclease fusion proteins of this disclosure, including RNase-Fc fusion proteins, include a leader sequence, e.g., a leader peptide. In some embodiments, the leader molecule is a leader peptide located at the N-terminus of the nuclease domain. In some embodiments, the RNase-Fc fusion protein includes a leader peptide at the N-terminus of the molecule, which is subsequently cleaved from the interferon receptor Fc construct. Methods for generating nucleic acid sequences encoding a leader peptide to be fused to a recombinant protein are well known in the art. In some embodiments, the RNase-Fc fusion protein can be expressed with or without the leader fused to its N-terminus. The protein sequence of the RNase-Fc fusion protein of this disclosure after cleavage of the fused leader peptide can be predicted and / or inferred by those skilled in the art.

[0133] In some embodiments, the leader is a VK3 leader peptide (VK3LP), which is fused to the N-terminus of an RNase-Fc fusion protein. Such a leader sequence can improve the level of synthesis and secretion of RNase-Fc fusion proteins in mammalian cells. In some embodiments, the leader is cleaved to produce an RNase-Fc fusion protein. In some embodiments, the RNase-Fc fusion protein of this disclosure is expressed without the leader peptide fused to its N-terminus, and the resulting RNase-Fc fusion protein has an N-terminal methionine.

[0134] In some embodiments, the RNase-Fc fusion protein of this disclosure includes a VK3 leader peptide fused to the N-terminus of the RNase-Fc fusion protein, for example, SEQ ID NO: 49 (RSLV-132). In some embodiments, the RNase-Fc fusion protein of this disclosure does not include a leader sequence, for example, SEQ ID NO: 50 (RSLV-132).

[0135] In some embodiments, the RNase-Fc fusion protein includes an RNase domain operably ligated to the N or C terminus of the Fc domain or a variant or fragment thereof. In some embodiments, the RNase-Fc fusion protein includes both an RNase domain and a DNase domain. In some embodiments, the RNase-Fc fusion protein comprises two nuclease domains (e.g., two RNase domains) that are tadem-operably linked to one another and further operably linked to the N or C terminus of the same or different Fc domain or variant or fragment thereof.

[0136] The sequence listing shows sequences of RNase-Fc fusion proteins that exemplify various configurations.

[0137] In some embodiments, the RNase-Fc fusion protein is a multinuclease protein (e.g., two RNA nucleases, or RNase and DNase) fused to the same or different Fc domain or variant or fragment thereof that specifically binds to an extracellular immune complex.

[0138] In one embodiment, the nuclease domain is operably linked to the N-terminus of the Fc domain or its variant or fragment (e.g., chemically conjugated or genetically fused (e.g., directly or via a polypeptide linker)). In another embodiment, the nuclease domain is operably linked to the C-terminus of the Fc domain or its variant or fragment (e.g., chemically conjugated or genetically fused (e.g., directly or via a polypeptide linker)). In yet another embodiment, the nuclease domain is operably linked via the amino acid side chain of the Fc domain or its variant or fragment (e.g., chemically conjugated or genetically fused (e.g., directly or via a polypeptide linker)).

[0139] In certain embodiments, the RNase-Fc fusion protein of this disclosure comprises two or more nuclease domains and at least one Fc domain or a variant or fragment thereof. For example, a nuclease domain may be operably ligated to both the N-terminus and C-terminus of the same or different Fc domain or variant or fragment, along with a linker as needed between the nuclease domain and the Fc domain, its variant or fragment. In some embodiments, the nuclease domains are identical (e.g., RNase and RNase). In other embodiments, the nuclease domains are different (e.g., two different RNA nucleases or RNase and DNase).

[0140] In some embodiments, two or more nuclease domains are operably linked to each other in series (e.g., via a polypeptide linker), and tandem-aligned nuclease domains are operably linked to either the N-terminus or C-terminus of the same or different Fc domain or its variant or fragment (e.g., chemically conjugated (e.g., directly or via a polypeptide linker) or genetically fused). In other embodiments, tandem-aligned nuclease domains are operably linked to both the N-terminus and C-terminus of the same Fc domain or its variant or fragment. In some embodiments, nuclease domains are operably linked in tandem (e.g., N-RNase-RNase-C, N-RNase-DNase-C, or N-DNase-RNase-C) to the N-terminus or C-terminus of the same or different Fc domain with or without a linker. In some embodiments, the RNase-Fc fusion protein forms homodimers or heterodimers.

[0141] In other embodiments, one or more nuclease domains are inserted between two Fc domains or variants or fragments thereof. For example, one or more nuclease domains may form all or part of the polypeptide linker of the RNase-Fc fusion protein of the Disclosure.

[0142] In some embodiments, the RNase-Fc fusion protein comprises at least two nuclease domains (e.g., RNase and RNase or RNase and DNase), at least one linker domain, and at least one Fc domain or a variant or fragment thereof.

[0143] In some embodiments, the RNase-Fc fusion protein of this disclosure comprises an Fc domain or a variant or fragment thereof as described herein, thereby increasing the serum half-life and bioavailability of the RNase-Fc fusion protein. In some embodiments, the RNase-Fc fusion protein comprises one or more polypeptides, for example, a polypeptide comprising an amino acid sequence shown in any of SEQ ID NOs: 44-58.

[0144] Those skilled in the art will understand that other configurations of nuclease domains and Fc domains are possible by the inclusion of linkers as needed between nuclease domains and / or between nuclease domains and Fc domains. They will also understand that the domain orientation of the nuclease domains can be altered, as long as they are active in the particular configuration examined.

[0145] In certain embodiments, the RNase-Fc fusion protein of the Disclosure has at least one nuclease domain specific to a target molecule that mediates a biological effect. In other embodiments, binding of the RNase-Fc fusion protein of the Disclosure to a target molecule (e.g., RNA or DNA) results in the reduction or efflux of the target molecule, for example, from a cell, tissue or circulation.

[0146] In other embodiments, the RNase-Fc fusion protein of the Disclosure may be assembled together, or with other polypeptides, to form a binding protein having two or more polypeptides ("multimer"), where at least one polypeptide of the multimer is the RNase-Fc fusion protein of the Disclosure. Exemplary multimer forms include modified binding proteins such as dimers, trimers, tetramers, and hexamers. In one embodiment, the polypeptides of the multimer are the same (i.e., modified homomeric binding proteins, e.g., homodimer, homotetramer). In another embodiment, the polypeptides of the multimer are different (e.g., heteromeric). In one embodiment, the RNase-Fc fusion protein of the Disclosure is assembled together to form a dimer. In one embodiment, the dimer is a homodimer. In one embodiment, the dimer is a heterodimer.

[0147] In some embodiments, the RNase-Fc fusion protein has a serum half-life that is at least about 1.5 times, for example, at least 3 times, at least 5 times, at least 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1000 times, or 1000 times or more, compared to the corresponding nuclease molecule that is not fused to the Fc domain or its variant or fragment. In other embodiments, the RNase-Fc fusion protein has a serum half-life that is reduced to at most about two-thirds, for example, at most one-third, at most one-fifth, at most one-tenth, at most about one-twentieth, at most about one-fiftieth, at most about one-hundredth, at most about one-two-hundredth, at most about one-two-hundredth, at most about one-two-hundredth, at most about one-three-hundredth, at most about one-four-hundredth, at most about one-fiftyth, or one-fiftyth or lower, compared to the corresponding nuclease molecule that is not fused to the Fc domain or its variant or fragment. The serum half-life of the RNase-Fc fusion protein of this disclosure can be determined using methods commonly recognized in the art.

[0148] In some embodiments, the RNase activity in the RNase-Fc fusion protein is about one-tenth or more lower than the activity of the control RNase molecule, for example, one-ninth, one-eighth, one-seventh, one-sixth, one-fifth, one-quarter, one-third, or one-half lower. In some embodiments, the RNase activity in the RNase-Fc fusion protein is approximately equal to the activity of the control RNase molecule.

[0149] In some embodiments, the RNase-Fc fusion protein may be active against extracellular immune complexes containing, for example, DNA and / or RNA deposited in soluble or insoluble forms.

[0150] In some embodiments, the activity of the RNase-Fc fusion protein is detectable in vitro and / or in vivo.

[0151] In another embodiment, a multifunctional RNase molecule is provided which is bound to an enzyme, or another antibody having binding specificity such as scFv that targets RNA or DNA, or to a second nuclease domain having the same or different specificity as the first domain.

[0152] In some embodiments, the linker domain includes (gly4ser)3,4, or5 variants that alter the linker length by a 5-amino acid progression. In other embodiments, the linker domain is approximately 18 amino acids long and includes an N-linked glycosylation site that may be sensitive to protease cleavage in vivo. In some embodiments, the N-linked glycosylation site can protect the RNase-Fc fusion protein from cleavage in the linker domain. In some embodiments, the N-linked glycosylation site can assist in the separation of folding of independent functional domains that are separated by the linker domain.

[0153] In some embodiments, the linker domain is the NLG linker (VDGASSPVNVSSPSVQDI) (SEQ ID NO: 37).

[0154] In some embodiments, the RNase-Fc fusion protein comprises substantially all or at least an enzymatically active fragment of DNase. In some embodiments, the DNase is type I secretory DNase, preferably human DNase, e.g., mature human pancreatic DNase 1 (UniProtKB entry P24855, SEQ ID NO: 6). In some embodiments, the naturally occurring variant allele A114F (SEQ ID NO: 8), which exhibits reduced actin sensitivity, is included in the DNase 1 of the RNase-Fc fusion protein (see Pan et al., JBC 1998;273:18374-81; Zhen et al., BBRC 1997;231:499-504; Rodriguez et al., Genomics 1997;42:507-13). In other embodiments, the naturally occurring variant allele G105R (SEQ ID NO: 9), which exhibits higher DNase activity compared to wild-type DNase 1, is included in the DNase 1 of the RNase-Fc fusion protein (see Yasuda et al., Int J See Biochem Cell Biol 2010;42:1216-25). In some embodiments, this mutation is introduced into an RNase-Fc fusion protein to produce a more stable derivative of human DNase1. In some embodiments, the DNase is either human wild-type DNase1 or human DNase1 A114F (i.e., human DNase1 N18S / N106S / A114F, SEQ ID NO: 11) mutated to remove all potential N-linked glycosylation sites, i.e., the asparagine residues at positions 18 and 106 of the DNase1 domain shown in SEQ ID NO: 6 (which correspond to the asparagine residues at positions 40 and 128, respectively, of full-length pancreatic DNase1 with a natural leader (SEQ ID NO: 5)).

[0155] In some embodiments, the DNase is human DNase 1 containing one or more basic (i.e., positively charged) amino acid substitutions to enhance DNase functionality and chromatin cleavage. In some embodiments, basic amino acids are introduced to the DNA-binding surface of human DNase 1 to enhance binding to charged phosphate on the DNA substrate (see U.S. Patent No. 7,407,785; U.S. Patent No. 6,391,607). This overactive DNase 1 may be referred to as a "chromatin cutter."

[0156] In some embodiments, one, two, three, four, five, or six basic amino acid substitutions are introduced into DNase1. For example, one or more of the following residues are mutated to enhance DNA binding: Gln9, Glu13, Thr14, His44, Asn74, Asn110, Thr205. In some embodiments, one or more of the above amino acids are substituted with basic amino acids, such as arginine, lysine, and / or histidine. For example, human DNase may contain one or more of the following substitutions: Q9R, E13R, T14K, H44K, N74K, N110R, T205K. In some embodiments, human DNase1 also contains the A114F substitution that reduces actin sensitivity (see U.S. Patent No. 6,348,343). In one embodiment, human DNase1 includes the following substitutions: E13R, N74K, A114F, and T205K.

[0157] In some embodiments, human DNase1 further includes mutations to remove potential glycosylation sites, e.g., asparagine residues at positions 18 and 106 of the DNase1 domain shown in SEQ ID NO: 6 (which correspond to asparagine residues at positions 40 and 128, respectively, of full-length pancreatic DNase1 with a native leader). In one embodiment, human DNase1 includes the following substitution: E13R / N74K / A114F / T205K / N18S / N106S.

[0158] In some embodiments, DNase is DNase1-like (DNaseL) enzymes 1-3 (UniProtKB entry Q13609; SEQ ID NO: 15). In some embodiments, DNase is 3-prime repair exonuclease 1 (TREX1; UniProtKB entry Q9NSU2; SEQ ID NO: 16). In some embodiments, DNase is DNase2. In some embodiments, DNase2 is DNAse2α (i.e., DNase2; UnitProtKB entry O00115; SEQ ID NO: 18) or DNase2β (i.e., DNase2-like acid DNase; UnitProtKB entry Q8WZ79; SEQ ID NO: 19). In some embodiments, the N-linked glycosylation site of DNase1L3, TREX1, DNase2α, or DNase2β is mutated to remove a potential N-linked glycosylation site. In some embodiments, a DNase-linker-Fc domain containing 20 or 25aa linker domains is constructed.

[0159] In some embodiments, the DNase activity in the RNase-Fc fusion protein is approximately one-tenth or more of the activity of the control DNase molecule, such as one-ninth, one-eighth, one-seventh, one-sixth, one-fifth, one-quarter, one-third, or one-half. In some embodiments, the DNase activity in the RNase-Fc fusion protein is approximately equal to the activity of the control DNase molecule.

[0160] In some embodiments, the RNase-Fc fusion protein of this disclosure comprises human RNase 1. In some embodiments, the RNase-Fc fusion protein comprises a wild-type human RNase 1 domain. In some embodiments, the RNase-Fc fusion protein comprises human pancreatic RNase 1 of the RNase A family (UniProtKB entry P07998; SEQ ID NO: 1). In some embodiments, the RNase-Fc fusion protein comprises the mature form of human pancreatic RNase 1 shown in SEQ ID NO: 2. In some embodiments, the RNase-Fc domain comprises a human RNase 1 domain with one or more mutations. In some embodiments, human RNase 1 is mutated to remove asparagine residues at positions 34, 76, and 88 of the RNase 1 domain shown in SEQ ID NO: 2, corresponding to any potential N-linked glycosylation site, i.e., asparagine residues at positions 62, 104, and 116, respectively, of full-length pancreatic RNase 1 with a native leader (SEQ ID NO: 1) (human RNase 1 N34S / N76S / N88S, SEQ ID NO: 4). In some embodiments, an RNase1-linker-Fc containing a 20 or 25aa linker domain is prepared.

[0161] In some embodiments, the RNase-Fc fusion protein contains mammalian RNase 1. In some embodiments, the RNase-Fc fusion protein contains primate RNase 1. In some embodiments, the RNase-Fc fusion protein contains rodent RNase 1. In some embodiments, the RNase-Fc fusion protein contains mouse RNase It includes 1. In some embodiments, the RNase-Fc fusion protein includes rat RNase 1. In some embodiments, the RNase-Fc fusion protein includes monkey RNase 1. In some embodiments, the RNase-Fc fusion protein includes goat RNase 1. In some embodiments, the RNase-Fc fusion protein includes rabbit RNase 1. In some embodiments, the RNase-Fc fusion protein includes horse RNase 1. In some embodiments, the RNase-Fc fusion protein includes canine RNase 1. In some embodiments, the RNase 1 domain is a mutant RNase 1 domain.

[0162] In some embodiments, the RNase-Fc fusion protein includes an RNase molecule bound to an Fc domain that specifically binds to an extracellular immune complex. In some embodiments, the Fc domain does not effectively bind to the Fcγ receptor. In one embodiment, the RNase-Fc fusion protein does not effectively bind to C1q. In other embodiments, the RNase-Fc fusion protein includes an in-frame Fc domain derived from IgG1. In other embodiments, the RNase-Fc fusion protein further includes mutations in the hinge, CH2 and / or CH3 domains. In other embodiments, the mutations are P238S, P331S or N297S and may include mutations in one or more of the three hinge cysteines. In some such embodiments, the mutations are located in one or more of the three hinge cysteines at residues 220, 226 and 229, numbered according to the EU index, e.g., serine substitution of one or more cysteine ​​residues, e.g., C220S, C226S and / or C229S. In some embodiments, one of the three hinge region cysteine ​​is replaced by serine, e.g., C220S, also referred to herein as the “SCC hinge”. In some embodiments, all three hinge region cysteine ​​are replaced by serine, e.g., C220S, C226S, and C229S, also referred to herein as the “SSS hinge”. In other embodiments, the RNase-Fc fusion protein contains the SCC hinge but is otherwise wild-type human IgG1 Fc CH2 and CH3 domains, efficiently binding to the Fc receptor and facilitating the uptake of the RNase-Fc fusion protein into the endocytotic compartment of the bound cell. In other embodiments, the RNase-Fc fusion protein has activity toward single-stranded and / or double-stranded RNA substrates.

[0163] In some embodiments, the RNase-Fc fusion protein includes a mutant Fc domain. In some embodiments, the RNase-Fc fusion protein includes a mutant IgG1 Fc domain. In some embodiments, the mutant Fc domain includes one or more mutations in the hinge, CH2, and / or CH3 domains. In some embodiments, the mutant Fc domain includes the P238S mutation. In some embodiments, the mutant Fc domain includes the P331S mutation. In some embodiments, the mutant Fc domain includes both the P238S and P331S mutations. In some embodiments, the mutant Fc domain includes P238S and / or P331S and may include mutations in one or more of the three hinge cysteines. In some embodiments, the mutant Fc domain includes P238S and / or P331S, and / or one or more mutations in the three hinge cysteines. In some embodiments, the mutant Fc domain includes mutations in P238S and / or P331S, and / or three hinge cysteines to become SSS or one hinge cysteine ​​to become SCC. In some embodiments, the mutant Fc domain includes mutations in P238S and P331S, and three hinge cysteines. In some embodiments, the mutant Fc domain includes P238S and P331S, and either SCC or SSS. In some embodiments, the mutant Fc domain includes P238S and P331S, and SCC. In some embodiments, the mutant Fc domain includes P238S SSS. In some embodiments, the mutant Fc domain includes P331S, and either SCC or SSS. In some embodiments, the mutant Fc domain includes mutations in one or more of the three hinge cysteines. In some embodiments, the mutant Fc domain includes mutations in three hinge cysteines. In some embodiments, the mutant Fc domain contains mutations in three hinge cysteine ​​molecules to result in SSS. In some embodiments, the mutant Fc domain contains a mutation in one of the three hinge cysteine ​​molecules to result in SCC. In some embodiments, the mutant Fc domain contains either SCC or SSS. In some embodiments, the mutant Fc domain is as shown in any of Sequence IDs 21-28.In some embodiments, the RNase-Fc fusion protein is as shown in any of SEQ ID NOs: 44-58. In some embodiments, the RNase-Fc fusion protein comprises a mutant human IgG1 Fc domain containing SCC, P238S, and P331S, or a wild-type human RNase1 domain ligated to a mutant human IgG1 Fc domain containing SSS, P238S, and P331S. In some embodiments, the RNase-Fc fusion protein is shown in SEQ ID NOs: 45-46. In some embodiments, the RNase-Fc fusion protein is shown in SEQ ID NO: 50.

[0164] In some embodiments, the RNase-Fc fusion protein comprises a wild-type human RNase1 domain linked via a (Gly4Ser)4 linker domain to a mutant human IgG1 Fc domain containing SCC, P238S, and P331S, or to a mutant human IgG1 Fc domain containing SSS, P238S, and P331S. In some embodiments, the RNase-Fc fusion protein is shown in SEQ ID NOs. 47-48.

[0165] In some embodiments, the RNase-Fc fusion protein comprises a human DNase1 G105R A114F domain ligated via a (Gly4Ser)4 linker domain to a mutant human IgG1 Fc domain containing SCC, P238S, and P331S, which is ligated via an NLG linker domain to a wild-type human RNase1 domain. In some embodiments, the RNase-Fc fusion protein comprises a human DNase1 G105R A114F domain ligated via a (Gly4Ser)4 linker domain to a mutant human IgG1 Fc domain containing SSS, P238S, and P331S, which is ligated via an NLG linker domain to a wild-type human RNase1 domain. In some embodiments, the RNase-Fc fusion protein is shown in SEQ ID NOs. 51-52.

[0166] In some embodiments, the RNase-Fc fusion protein is human DNase1 G105R The RNase-Fc fusion protein comprises a wild-type human RNase1 domain linked via a (Gly4Ser)4 linker domain to a mutant human IgG1 Fc domain containing SCC, P238S, and P331S, which is linked to the A114F domain via an NLG linker domain. In some embodiments, the RNase-Fc fusion protein comprises a wild-type human RNase1 domain linked via a (Gly4Ser)4 linker domain to a mutant human IgG1 Fc domain containing SSS, P238S, and P331S, which is linked to the human DNase1 G105R A114F domain via an NLG linker domain. In some embodiments, the RNase-Fc fusion protein is shown in SEQ ID NOs. 53-54.

[0167] In some embodiments, the RNase-Fc fusion protein is human DNase1 G105R The RNase-Fc fusion protein comprises a wild-type human RNase1 domain linked to a mutant human IgG1 Fc domain containing SCC, P238S, and P331S, which is linked to the A114F domain via an NLG linker domain. In some embodiments, the RNase-Fc fusion protein comprises a wild-type human RNase1 domain linked to a mutant human IgG1 Fc domain containing SSS, P238S, and P331S, which is linked to the human DNase1 G105R A114F domain via an NLG linker domain. In some embodiments, the RNase-Fc fusion protein is shown in SEQ ID NOs. 55-58.

[0168] In some embodiments, the activity of RNase-Fc fusion proteins can be detected in vitro and / or in vivo.

[0169] In some configurations, the RNase-Fc fusion protein contains an RNase domain and an Fc domain, with the RNase1 domain located on the COOH side of Fc. In other configurations, the RNase-Fc fusion protein contains an RNase domain and an Fc domain, with the RNase1 domain located on the NH2 side of Fc. In some configurations, the RNase-Fc fusion protein contains RNase-Fc;Fc-RNase;Fc-linker-RNase;RNase-linker-Fc-DNase;DNase-Fc-RNase;RNase-linker-Fc-linker-DNase;DNase-linker-Fc-linker-RNase;RNase-Fc-linker-DNase;DNase-Fc-linker-RNase;RNase-linker-Fc-DNase;DNase-linker-Fc-RNase.

[0170] In some embodiments, the fusion junction between the enzyme domain and other domains of the RNase-Fc fusion protein is optimized.

[0171] In some embodiments, the targets of the RNase enzyme activity of the RNase-Fc fusion protein are primarily extracellular, consisting of, for example, RNA contained in immune complexes with anti-RNP autoantibodies and RNA expressed on the surface of apoptotic cells. In some embodiments, the RNase-Fc fusion protein is active in the acidic environment of intracellular vesicles. In some embodiments, the RNase-Fc fusion protein includes a wild-type (wt) Fc domain to allow the molecule to bind to FcR and enter the endocytic compartment through an entry pathway used by immune complexes. In some embodiments, the RNase-Fc fusion protein, including the Fc domain or a variant or fragment thereof, is adapted to be active both extracellularly and in the endocytic environment (where TLR7 may be expressed). In some embodiments, this allows the RNase-Fc fusion protein, including the wild-type Fc domain or a variant or fragment thereof, to halt TLR7 signaling by RNA that activates TLR7 after a pre-phagocytosed immune complex or viral infection. In some embodiments, the wild-type RNase of the RNase-Fc fusion protein is not resistant to inhibition by cytoplasmic inhibitors of RNase. In some embodiments, the wild-type RNase of the RNase-Fc fusion protein is inactive in the cytoplasm of cells.

[0172] In some embodiments, the RNase-Fc fusion protein contains an RNase. In some embodiments, the RNase-Fc fusion protein contains both a DNase and an RNase. In some embodiments, these RNase-Fc fusion proteins digest or degrade immune complexes containing RNA, DNA, or a combination of both RNA and DNA, and are extracellularly active, thus improving the treatment of Sjögren's disease. In some embodiments, the RNase-Fc fusion proteins of this disclosure reduce fatigue in patients with Sjögren's disease.

[0173] In some embodiments, the Disclosure provides nucleic acids encoding one or more RNase-Fc fusion proteins for use in gene therapy methods for treating or preventing disorders, diseases, and conditions. The gene therapy methods relate to the introduction of RNase-Fc fusion protein nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into animals requiring expression of the polypeptide(s) of the Disclosure. The methods may include the introduction of one or more polynucleotides encoding the RNase-Fc fusion proteins of the Disclosure, operably linked to promoters and any other genetic elements necessary for the expression of the RNase-Fc fusion proteins by the target tissue.

[0174] In gene therapy applications, RNase-Fc fusion protein genes are introduced into cells to achieve in vivo synthesis of therapeutically effective gene products. "Gene therapy" includes both conventional gene therapy, where a single treatment achieves a permanent effect, and the administration of gene therapy agents involving one or repeated doses of therapeutically effective DNA or mRNA. Oligonucleotides can be modified, for example, by substituting their negatively charged phosphate diester groups with uncharged groups, to enhance their uptake.

[0175] FC Domain In some embodiments, a polypeptide comprising one or more nuclease domains or variants or fragments thereof is operably linked to an Fc domain, with or without a linker domain, which functions as a scaffold and as a means of increasing the serum half-life of the polypeptide. In some embodiments, one or more nuclease domains and / or Fc domains are unglycosylated, deglycosylated, or insufficiently glycosylated. In some embodiments, the Fc domain is a mutant or variant Fc domain, or a fragment of an Fc domain.

[0176] Suitable Fc domains are well known in the art and include, but are not limited to, those disclosed in WO2011 / 053982, WO02 / 060955, WO02 / 096948, WO05 / 047327, WO05 / 018572 and US2007 / 0111281 (the foregoing is incorporated herein by reference). It is within the capabilities of those skilled in the art to use conventional methods (e.g., cloning, conjugation) to introduce Fc domains into RNase-Fc fusion proteins (with or without altered glycosylation) disclosed herein.

[0177] In some embodiments, the Fc domain is a wild-type human IgG1 Fc, such as the one shown in Sequence ID No. 20. In some embodiments, the Fc domain is a human IgG1 Fc domain having one or more mutations.

[0178] In some embodiments, the Fc domain is wild-type human IgG4 Fc, such as those shown in SEQ ID NOs. 30-31. In some embodiments, the Fc domain is a human IgG4 Fc domain having one or more mutations.

[0179] In some embodiments, the Fc domain is altered or modified by mutations resulting in, for example, amino acid addition, deletion, or substitution. As used herein, the term “Fc domain variant” refers to an Fc domain having at least one amino acid modification, such as an amino acid substitution, compared to the wild-type Fc from which the Fc domain is derived. For example, if the Fc domain is derived from a human IgG1 antibody, the variant includes at least one amino acid mutation (e.g., substitution) compared to the wild-type amino acid at the corresponding position in the human IgG1 Fc region. The amino acid substitution(s) of the Fc variant may be located at a position within the Fc domain that corresponds to a position number (numbering according to the EU index) assigned to the residue in the Fc region of the antibody.

[0180] In one embodiment, the Fc variant includes one or more amino acid substitutions at amino acid positions located in or part of the hinge region. In another embodiment, the Fc variant includes one or more amino acid substitutions at amino acid positions located in or part of the CH2 domain. In yet another embodiment, the Fc variant includes one or more amino acid substitutions at amino acid positions located in or part of the CH3 domain. In yet another embodiment, the Fc variant includes one or more amino acid substitutions at amino acid positions located in or part of the CH4 domain.

[0181] In some embodiments, the Fc domain includes one or more of the following amino acid substitutions: T350V, L351Y, F405A, and Y407V. In some embodiments, the Fc domain includes one or more of the following amino acid substitutions: T350V, T366L, K392L, and T394W.

[0182] In some embodiments, the human IgG1 Fc region has a mutation at N83 (i.e., N297 according to Kabat numbering), resulting in a non-glycosylated Fc region (e.g., Fc N83S; SEQ ID NO: 21). In some embodiments, the human IgG1 Fc domain contains mutations in one or more of the three hinge region cysteines (residues 220, 226, and 229, numbered according to the EU index). In some embodiments, one or more of the three hinge cysteines in the Fc domain can be mutated to SCC (SEQ ID NO: 24) or SSS (SEQ ID NO: 25), where "S" represents a serine amino acid substitution of cysteine ​​(CCC refers to the three cysteines present in the wild-type hinge domain). Therefore, "SCC" indicates that only the first cysteine ​​out of the three hinge region cysteines (residues 220, 226, and 229, numbered according to the EU index) has been substituted with serine, while "SSS" indicates that all three cysteines in the hinge region have been substituted with serine (residues 220, 226, and 229, numbered according to the EU index).

[0183] In some embodiments, the Fc domain is a human IgG1 Fc domain having one or more mutations.

[0184] In some embodiments, the mutant Fc domain contains one or more mutations in the hinge, CH2, and / or CH3 domains.

[0185] In some embodiments, the Fc domain is a human IgG4 Fc domain having one or more mutations. In some embodiments, the mutations in the IgG4 Fc domain include one or more mutations selected from the following group of mutations: F296Y, E356K, R409K, and H345R. In some embodiments, the mutations in the IgG4 Fc domain include one or more mutations selected from the following group of mutations: F296Y, R409K, and K439E. In some embodiments, the RNase-Fc fusion protein disclosed herein comprises a first polypeptide comprising a mutant IgG4 Fc domain in which the Fc domain comprises mutations F296Y, E356K, R409K, and H345R, and a second polypeptide comprising a mutant IgG4 Fc domain in which the CH3 domain comprises mutations F296Y, R409K, and K439E. In some embodiments, the mutant IgG4 Fc domain includes one or more mutations in the hinge, CH2, and / or CH3 domains.

[0186] CH2 substitution In some embodiments, the mutant Fc domain contains the P238S mutation. In some embodiments, the mutant Fc domain contains the P331S mutation. In some embodiments, the mutant Fc domain contains both the P238S and P331S mutations. In some embodiments, the mutant Fc domain, numbered according to the EU index, contains P238S and / or P331S, and may also contain mutations in one or more of the three hinge cysteine ​​residues (residues 220, 226, and 229). In some embodiments, the mutant Fc domain, numbered according to the EU index, contains P238S and / or P331S, and / or one or more mutations in the three hinge cysteine ​​residues (residues 220, 226, and 229). In some embodiments, the mutant Fc domain includes mutations in the hinge cysteine ​​to result in P238S and / or P331S and / or SCC, or in three hinge cysteines to result in SSS. In some embodiments, the mutant Fc domain includes mutations in P238S and P331S, as well as in at least one of the three hinge cysteines. In some embodiments, the mutant Fc domain includes P238S and P331S and SCC. In some embodiments, the mutant Fc domain includes P238S and P331S and SSS. In some embodiments, the mutant Fc domain includes P238S and SCC or SSS. In some embodiments, the mutant Fc domain includes P331S and SCC or SSS (full numbering according to the EU index).

[0187] In some embodiments, the mutant Fc domain includes a mutation at an N-linked glycosylation site such as N297, for example, a substitution of asparagine with another amino acid such as serine, e.g., N297S. In some embodiments, the mutant Fc domain includes a mutation at an N-linked glycosylation site such as N297, for example, a substitution of asparagine with another amino acid such as serine, e.g., N297S, and a mutation in one or more of the three hinged cysteines. In some embodiments, the mutant Fc domain includes a mutation at an N-linked glycosylation site such as N297, for example, a substitution of asparagine with another amino acid such as serine, e.g., N297S, and a mutation in one of the three hinged cysteines to become SCC or in all three cysteines to become SSS. In some embodiments, the mutant Fc domain includes one or more mutations in the CH2 domain that reduce FcγR binding and / or complement activation, such as mutations at N-linked glycosylation sites, e.g., substitution of asparagine with another amino acid, e.g., serine, e.g., N297, and mutations at P238 or P331 or both, e.g., P238S or P331S, or both P238S and P331S. In some embodiments, such mutant Fc domains may further include mutations in the hinge region, e.g., SCC or SSS (full numbering according to the EU index). In some embodiments, the mutant Fc domain is as shown in the sequence listing or sequence list herein.

[0188] CH3 substitution In some embodiments, heterodimers are formed by mutations in the CH3 domain of the Fc domain in the RNase-Fc fusion proteins disclosed herein. The heavy chain was first manipulated for heterodimerization using a “knob-into-hole” strategy (Rigway B, et al., Protein Eng., 9 (1996) pp. 617-621, incorporated herein by reference). The term “knob-into-hole” refers to a technique that directs the in vitro or in vivo integral pairing of two polypeptides by introducing a pertuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the interface where they interact. See, for example, WO96 / 027011, WO98 / 050431, US5,731,168, US2007 / 0178552, WO2009089004, US20090182127. In particular, heterodimers can be formed using combinations of mutations in the CH3 domain, for example, S354C, T366W in the "knob" heavy chain and Y349C, T366S, L368A, Y407V in the "hole" heavy chain. Another example is T366Y in the "knob" heavy chain and Y407T in the "hole" heavy chain. In some embodiments, the heterodimeric RNase-Fc fusion protein disclosed herein comprises a first CH3 domain having the knob mutation T366W, and a second CH3 domain having the hole mutations T366S, L368A, and Y407V (numbered according to the EU index). In some embodiments, the RNase-Fc fusion protein disclosed herein comprises a first CH3 domain having the knob mutation T366Y, and a second CH3 domain having the hole mutation Y407T.

[0189] In some embodiments, the CH3 mutations are those described in US2012 / 0149876A1, US2017 / 0158779, US9574010 and US9562109, which are incorporated herein by reference; and those described in Von Kreudenstein, TS et al. mABs, 5 (2013), pp. 646-654, which are incorporated herein by reference, and include the following mutations: T350V, L351Y, F405A and Y407V (first CH3 domain); and T350V, T366L, K392L, T394W (second CH3 domain). In some embodiments, the heterodimeric RNase-Fc fusion proteins disclosed herein include a first CH3 domain having T350V, L351Y, F405A and Y407V mutations, and a second CH3 domain having T350V, T366L, K392L and T394W mutations (numbered according to the EU index).

[0190] In some embodiments, heterodimers are formed by mutations in the CH3 domain of the Fc domain in the RNase-Fc fusion proteins disclosed herein. In particular, combinations of mutations in the CH3 domain can be used to form heterodimers with high heterodimer stability and purity; see, for example, Von Kreudenstein et al., mAbs 5:5, 646-654; September-October 2013, and US2012 / 0149876A1, US2017 / 0158779, US9574010 and US9562109, each of which is incorporated herein by reference. In some embodiments, the mutations in the Fc domain include one or more mutations selected from the following group of mutations: T350V, L351Y, F405A and Y407V. In some embodiments, the mutations in the Fc domain include one or more mutations selected from the following group of mutations: T350V, T366L, K392L, and T394W. In some embodiments, the RNase-Fc fusion protein disclosed herein includes a CH3 domain having mutations T350V, L351Y, F405A, and Y407V. In some embodiments, the RNase-Fc fusion protein disclosed herein includes a CH3 domain having mutations T350V, T366L, K392L, and T394W. In some embodiments, the RNase-Fc fusion protein disclosed herein includes a first polypeptide comprising a mutant Fc domain in which the CH3 domain comprises mutations T350V, L351Y, F405A, and Y407V, and a second polypeptide comprising a mutant Fc domain in which the CH3 domain comprises mutations T350V, T366L, K392L, and T394W.

[0191] Other mutations in the CH3 domain of the Fc domain are intended to preferentially form heterodimers. See, for example, Von Kreudenstein et al., mAbs 5:5, 646-654; September-October 2013, which is incorporated herein by reference. In some embodiments, the mutations in the Fc domain of the first polypeptide include one or more mutations selected from the following group of mutations: T350V, L351Y, F405A, and Y407V, and the mutations in the Fc domain of the second polypeptide include one or more mutations selected from the following group of mutations: T350V, T366L, K392M, and T394W. In some embodiments, the mutations in the Fc domain of the first polypeptide include one or more mutations selected from the following group of mutations: L351Y, F405A, and Y407V, and the mutations in the Fc domain of the second polypeptide include one or more mutations selected from the following group of mutations: T366L, K392M, and T394W.

[0192] In some embodiments, the CH3 mutations are those described by Moore, GL et al. (mABs, 3 (2011), pp. 546-557) and include the following mutations: S364H and F405A (first CH3 domain); and Y349T and T394F (second CH3 domain). In some embodiments, the heterodimeric RNase-Fc fusion proteins disclosed herein include a first CH3 domain with the S364H and F405A mutations, and a second CH3 domain with the Y349T and T394F mutations (numbered according to the EU index).

[0193] In some embodiments, the CH3 mutations are those described by Gunasekaran, K. et al. (J. Biol. Chem., 285 (2010), pp. 19637-19646) and include the following mutations: K409D and K392D (first CH3 domain); and D399K and E365K (second CH3 domain). In some embodiments, the RNase-Fc fusion proteins disclosed herein include a first CH3 domain with the K409D and K392D mutations, and a second CH3 domain with the D399K and E365K mutations (numbered according to the EU index).

[0194] The RNase-Fc fusion proteins of this disclosure may utilize Fc variants known in the art that confer effector function and / or alter FcR binding. For example, each of these is incorporated herein by reference: International PCT Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO03 / 074569A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351 A2, WO04 / 074455A2, WO04 / 099249A2, WO05 / 040217A2, WO04 / 044859, WO05 / 070963A1, WO05 / 077 981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350A2, and WO06 / 085967A2; National patent application publication numbers US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056; or US Patent Application Publication No. 5,648,260; A change (e.g., substitution) at one or more of the amino acid positions disclosed in Patent No. 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; and Patent No. 7,317,091. In one embodiment, a specific change (e.g., a specific substitution of one or more amino acids disclosed in the Art) can be at one or more of the disclosed amino acid positions.In another embodiment, different changes can be made at one or more of the disclosed amino acid positions (e.g., different substitutions of one or more amino acid positions disclosed in the Art).

[0195] Other amino acid mutations in the Fc domain are intended to reduce binding to the Fc gamma receptor and Fc gamma receptor subtypes. The assignment of amino acid residue numbers to the Fc domain follows Kabat's definition. For example, each of these is incorporated herein by reference for all purposes: Sequences of Proteins of Immunological Interest (Table of Contents, Introduction and Constant Region Sequences sections), 5th edition, Bethesda, MD:NIH vol. 1:647-723 (1991); Kabat et al. al., “Introduction” Sequences of Proteins of Immunological Interest, US Dept of See Health and Human Services, NIH, 5th edition, Bethesda, MD vol. 1:xiii-xcvi (1991); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989).

[0196] For example, the positions of the Fc region are 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 322, 324, 327, 329, 330, 331, 333, 334, Mutations in 335, 337, 338, 340, 356, 360, 373, 376, 378, 379, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439 can alter the binding as described in U.S. Patent No. 6,737,056 issued on 18 May 2004, which is incorporated herein by reference in its entirety. This patent reports that changing Pro331 to Ser in IgG3 resulted in one-sixth the affinity compared to unmutated IgG3, demonstrating the involvement of Pro331 in Fc gamma RI binding. In addition, amino acid modifications at positions 234, 235, 236 and 237, 297, 318, 320 and 322 are disclosed as potentially altering receptor binding affinity in US5,624,821, published on April 29, 1997, which is incorporated herein by reference in its entirety (numbered according to the EU index).

[0197] Further variations intended for use include, for example, the variations described in U.S. Patent Application Publication No. 2006 / 0235208, published on October 19, 2006, which is incorporated herein by reference in its entirety. This publication covers 232G, 234G, 234H, 235D, 235G, 235H, 236I, 236N, 236P, 236R, 237K, 237L, 237N, 237P, 238K, 239R, 265G, 267R, 269R, 270H, 297S, 299A, 299I, 299V, 325A, 325L, 327R, 328R, 329K, 330I, 330L, 330N, 330P, 330R, and 331L (numbering follows the EU index), as well as the double variants 236R / 237K, 236R / 325L This document describes Fc variants that exhibit reduced binding to the Fc gamma receptor, reduced antibody-dependent cell-mediated cytotoxicity, or reduced complement-dependent cytotoxicity, including 236R / 328R, 237K / 325L, 237K / 328R, 325L / 328R, 235G / 236R, 267R / 269R, 234G / 235G, 236R / 237K / 325L, 236R / 325L / 328R, 235G / 236R / 237K, and 237K / 325L / 328R, which include at least one amino acid modification in the Fc region. Other variants intended for use described in this publication include 227G, 234D, 234E, 234G, 234I, 234Y, 235D, 235I, 235S, 236S, 239D, 246H, 255Y, 258H, 260H, 2641, 267D, 267E, 268D, 268E, 272H, 272I, 272R, 281D, 282G, 283H, 284E, 293R, 295E, 304T, 324G, 324I, 327D, 327A, 328A, 328D, 328E , 328F, 328I, 328M, 328N, 328Q, 328T, 328V, 328Y, 330I, 330L, 330Y, 332D, 332E, 335D, insertion of G between positions 235 and 236, insertion of A between positions 235 and 236, insertion of S between positions 235 and 236, insertion of T between positions 235 and 236, insertion of N between positions 235 and 236, insertion of D between positions 235 and 236, insertion of V between positions 235 and 236,Insertion of L between positions 235 and 236, insertion of G between positions 235 and 236, insertion of A between positions 235 and 236, insertion of S between positions 235 and 236, insertion of T between positions 235 and 236, insertion of N between positions 235 and 236, insertion of D between positions 235 and 236, insertion of V between positions 235 and 236, insertion of L between positions 235 and 236, between positions 297 and 298 This includes the insertion of G, the insertion of A between positions 297 and 298, the insertion of S between positions 297 and 298, the insertion of D between positions 297 and 298, the insertion of G between positions 326 and 327, the insertion of A between positions 326 and 327, the insertion of T between positions 326 and 327, the insertion of D between positions 326 and 327, and the insertion of E between positions 326 and 327 (numbering follows the EU index). Furthermore, the variants described in U.S. Patent Application Publication No. 2006 / 0235208 are 227G / 332E, 234D / 332E, 234E / 332E, 234Y / 332E, 234I / 332E, 234G / 332E, 235I / 332E, 235S / 332E, 235D / 332E, 235E / 332E, 236S / 332E, and 236A / 332E, 236S / 332D, 236A / 332D, 239D / 268E, 246H / 332E, 255Y / 332E, 258H / 332E, 260H / 332E, 264I / 332E, 267E / 332E, 267D / 332E, 268D / 332D, 268E / 332D, 268E / 332E, 268D / 332E, 268E / 3 30Y, 268D / 330Y, 272R / 332E, 272H / 332E, 283H / 332E, 284E / 332E, 293R / 332E, 295E / 332E, 30 4T / 332E, 324I / 332E, 324G / 332E, 324I / 332D, 324G / 332D, 327D / 332E, 328A / 332E, 328T / 332 E, 328V / 332E, 328I / 332E, 328F / 332E, 328Y / 332E, 328M / 332E, 328D / 332E, 328E / 332E, 328N / 332E, 328Q / 332E, 328A / 332D, 328T / 332D, 328V / 332D, 328I / 332D, 328F / 332D, 328Y / 332D,328M / 332D, 328D / 332D, 328E / 332D, 328N / 332D, 328Q / 332D, 330L / 332E, 330Y / 332E, 330I / 332E , 332D / 330Y, 335D / 332E, 239D / 332E, 239D / 332E / 330Y, 239D / 332E / 330L, 239D / 332E / 330I, 239D / 332E / 268E, 239D / 332E / 268D, 239D / 332E / 327D, 239D / 332E / 284E, 239D / 268E / 330Y, 239D / 332 E / 268E / 330Y, 239D / 332E / 327A, 239D / 332E / 268E / 327A, 239D / 332E / 330Y / 327A, 332E / 330Y / 268 E / 327A, 239D / 332E / 268E / 330Y / 327A, Insertion G>297-298 / 332E, Insertion A>297-298 / 332E, Insertion S>297-298 / 332E, Insertion D>297-298 / 332E, Insertion G>326-327 / 332E, Insertion A>326-327 / 332E, Insertion T>326-327 / 332E, Insertion D>326-327 / 332E, Insertion E>326-327 / 332E, Insertion G>235-236 / 332E, Insertion A>235-236 / 332E, Insertion S>235-236 / 332E, Insertion T>235-2 Includes 36 / 332E, insertion N>235-236 / 332E, insertion D>235-236 / 332E, insertion V>235-236 / 332E, insertion L>235-236 / 332E, insertion G>235-236 / 332D, insertion A>235-236 / 332D, insertion S>235-236 / 332D, insertion T>235-236 / 332D, insertion N>235-236 / 332D, insertion D>235-236 / 332D, insertion V>235-236 / 332D, and insertion L>235-236 / 332D (numbering according to the EU index), intended for use. The variants L234A / L235A are described, for example, in U.S. Patent Application Publication No. 2003 / 0108548, published on June 12, 2003, which is incorporated herein by reference in its entirety. In embodiments, the described modifications are included either individually or in combination (numbered according to the EU index).

[0198] PK moiety In some embodiments, the RNase is operably linked to a PK moiety that functions as a scaffold and as a means for increasing the serum half-life of the RNase.

[0199] Suitable PK moieties are well known in the art, and include, but are not limited to, albumin, transferrin, Fc and variants thereof, and polyethylene glycol (PEG) and derivatives thereof. Suitable PK moieties include HSA or a variant or fragment thereof, such as those disclosed in US 5,876,969, WO2011 / 124718 and WO2011 / 0514789; Fc and Fc variants, such as those disclosed in WO2011 / 053982, WO02 / 060955, WO02 / 096948, WO05 / 047327, WO05 / 018572 and US 2007 / 0111281; transferrin or a variant or fragment thereof disclosed in US 7,176,278 and US 8,158,579; and PEG or a derivative, such as those disclosed in Zalipsky et al. ("Use of Functionalized Poly(Ethylene Glycols) for Modification of Polypeptides" in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, Plenus Press, New York (1992)) and Zalipsky et al. Advanced Drug Reviews 1995:16: 157-182), as well as U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, 4,179,337 and 5,932,462, the contents of which are incorporated herein by reference, but are not limited thereto. Operably linking a PK moiety to the RNase of the present invention (e.g., cloning, conjugation) using conventional methods is within the ability of those skilled in the art.

[0200] In some embodiments, the PK moiety is naturally non-glycosylated HSA.

[0201] In some embodiments, the PK moiety is wild-type Fc (SEQ ID NO: 20).

[0202] In certain embodiments, the Fc domain is altered or modified, for example, by amino acid mutation (e.g., addition, deletion or substitution). As used herein, the term "Fc domain variant" refers to an Fc domain that has at least one amino acid modification, such as an amino acid substitution, compared to the wild-type Fc from which the Fc domain is derived. For example, when the Fc domain is derived from a human IgG1 antibody, the variant comprises at least one amino acid mutation (e.g., substitution) compared to the wild-type amino acid at the corresponding position in the human IgG1 Fc region. For example, when the Fc domain is derived from a human IgG4 antibody, the variant comprises at least one amino acid mutation (e.g., substitution) at the corresponding position in the human IgG4 Fc region compared to the wild-type amino acid.

[0203] In some embodiments, the PK moiety is any of the Fc variants described herein.

[0204] In some embodiments, the PK moiety is wild-type HST. In other embodiments, the PK moiety is HST with mutations at N413 and / or N611 and / or S12 (S12 is a potential O-linked glycosylation site), resulting in an HST with altered glycosylation (i.e., HST N413S, HST N611S, HST N413S / N611S and HST S12A / N413S / N611S).

[0205] Linker Domain In some embodiments, the RNase-Fc fusion protein includes a linker domain. In some embodiments, the RNase-Fc fusion protein includes multiple linker domains. In some embodiments, the linker domain is a polypeptide linker. In certain embodiments, it is desirable to use a polypeptide linker to fuse Fc or a variant or fragment thereof to one or more nuclease domains to form an RNase-Fc fusion protein.

[0206] In one embodiment, the polypeptide linker is synthetic. As used herein, the term "synthetic" with respect to polypeptide linker includes a peptide (or polypeptide) that contains an amino acid sequence (which may or may not be naturally present) linked in a linear sequence of amino acids to a sequence (which may or may not be naturally present) that is not naturally linked in nature (which may or may not be naturally present) (e.g., an Fc sequence). For example, the polypeptide linker may be a modified form of a naturally present polypeptide (including mutations such as addition, substitution, or deletion), or it may include a naturally non-natural polypeptide containing a first amino acid sequence (which may or may not be naturally present). Using the polypeptide linker of the present invention, for example, it is possible to ensure that Fc or its variant or fragment is juxtaposed, thereby ensuring proper folding and formation of a functional Fc or its variant or fragment. Preferably, a polypeptide linker compatible with the present invention will be relatively non-immunogenic and will not inhibit any non-covalent association between monomeric subunits of the binding protein.

[0207] In certain embodiments, the RNase-Fc fusion protein uses the NLG linker shown in SEQ ID NO: 37.

[0208] In certain embodiments, the RNase-Fc fusion protein of this disclosure uses a polypeptide linker to link any two or more domains in frame within a single polypeptide chain. In one embodiment, the two or more domains may be independently selected from any of the Fc domains or their variants or fragments or nuclease domains discussed herein. In one embodiment, the RNase domain of the RNase-Fc fusion protein is operably linked to the Fc domain via a linker domain. In one embodiment, the polypeptide linker may be used to fuse identical Fc fragments, thereby forming a homodimeric Fc region. In other embodiments, the polypeptide linker may be used to fuse different Fc fragments, thereby forming a heterodimeric Fc region. In other embodiments, the polypeptide linker of the present invention may be used to genetically fuse the C-terminus of a first Fc fragment to the N-terminus of a second Fc fragment to form a complete Fc domain.

[0209] In one embodiment, the polypeptide linker includes a portion of the Fc domain or a variant or fragment thereof. For example, in one embodiment, the polypeptide linker may include an Fc fragment (e.g., a C or N domain), or a different portion of the Fc domain or a variant thereof.

[0210] In another embodiment, the polypeptide linker comprises or comprises a gly-ser linker. As used herein, the term “gly-ser linker” refers to a peptide comprising glycine and serine residues. An exemplary gly / ser linker comprises the amino acid sequence of formula (Gly4Ser)n, where n is a positive integer (e.g., 1, 2, 3, 4, or 5). A preferred gly / ser linker is (Gly4Ser)4. Another preferred gly / ser linker is (Gly4Ser)3. Another preferred gly / ser linker is (Gly4Ser)5. In certain embodiments, a gly-ser linker can be inserted between two other sequences of a polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In other embodiments, a gly-ser linker is attached to one or both ends of another sequence of a polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In yet another embodiment, two or more gly-ser linkers are sequentially incorporated into the polypeptide linker.

[0211] In other embodiments, the polypeptide linker of the present invention includes a biologically relevant peptide sequence or a portion of its sequence. For example, the biologically relevant peptide sequence may be, but is not limited to, a sequence derived from an anti-rejection or anti-inflammatory peptide. The anti-rejection or anti-inflammatory peptide may be selected from the group consisting of cytokine inhibitory peptides, cell adhesion inhibitory peptides, thrombin inhibitory peptides, and platelet inhibitory peptides. In preferred embodiments, the polypeptide linker includes a peptide sequence selected from the group consisting of IL-1 inhibitory or antagonist peptide sequences, erythropoietin (EPO) mimetic peptide sequences, thrombopoietin (TPO) mimetic peptide sequences, G-CSF mimetic peptide sequences, TNF antagonist peptide sequences, integrin-binding peptide sequences, selectin antagonist peptide sequences, antipathogenic peptide sequences, vasoactive intestinal peptide (VIP) mimetic peptide sequences, calmodulin antagonist peptide sequences, mast cell antagonists, SH3 antagonist peptide sequences, urokinase receptor (UKR) antagonist peptide sequences, somatostatin or cortistatin mimetic peptide sequences, and macrophage and / or T cell inhibitory peptide sequences. Exemplary peptide sequences, any one of which can be used as a polypeptide linker, are disclosed in U.S. Patent No. 6,660,843, incorporated herein by reference.

[0212] Other linkers suitable for use in RNase-Fc fusion proteins are known in the art and include, for example, the serine-rich linker disclosed in US5,525,491, the helix-forming peptide linker (e.g., A(EAAAK)nA(n=2~5)) disclosed in Arai et al., Protein Eng 2001;14:529-32, and the stable linker disclosed in Chen et al., Mol Pharm 2011;8:457-65, namely the dipeptide linker LE, the thrombin-sensitive disulfide cyclopeptide linker, and the alpha-helix-forming linker LEA(EAAAK)4ALEA(EAAAK)4ALE (SEQ ID NO: 39).

[0213] Other exemplary linkers include the GS linker (i.e., (GS)n), the GGSG (SEQ ID NO: 40) linker (i.e., (GGSG)n), the GSAT linker (SEQ ID NO: 41), the SEG linker, and the GGS linker (i.e., (GGSGGS)n), where n is a positive integer (e.g., 1, 2, 3, 4, or 5). Other suitable linkers for use in RNase-Fc fusion proteins can be found using publicly available databases such as the Linker Database (ibi.vu.nl / programs / linkerdbwww). The Linker Database is a database of interdomain linkers in multifunctional enzymes that function as potential linkers in novel fusion proteins (see, e.g., George et al., Protein Engineering 2002;15:871-9).

[0214] It will be understood that variant forms of these exemplary polypeptide linkers can be constructed by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence encoding the polypeptide linker, so that one or more amino acid substitutions, additions, or deletions are introduced into the polypeptide linker. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0215] The polypeptide linkers of this disclosure are at least one amino acid in length and may vary in length. In one embodiment, the polypeptide linker of the present invention is about 1 to about 50 amino acids in length. In this context, the term "about" indicates + / - 2 amino acid residues. Since the linker length must be a positive integer, a length of about 1 to about 50 amino acids means a length from 1 to 48 to 52 amino acids. In another embodiment, the polypeptide linker of this disclosure is about 10 to 20 amino acids in length. In yet another embodiment, the polypeptide linker of this disclosure is about 15 to about 50 amino acids in length.

[0216] In another embodiment, the polypeptide linker of the Disclosure is about 20 to about 45 amino acids long. In another embodiment, the polypeptide linker of the Disclosure is about 15 to about 25 amino acids long. In another embodiment, the polypeptide linker of the Disclosure is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 or more amino acids long.

[0217] Polypeptide linkers can be introduced into polypeptide sequences using techniques known in the art. Modifications can be confirmed by DNA sequence analysis. Host cells can be transformed using plasmid DNA for stable production of the polypeptide.

[0218] RNase-containing nuclease fusion protein with modified glycosylation Glycosylation (e.g., O-linked (O-lined) or N-linked glycosylation) can affect the serum half-life of RNase-containing nuclease fusion proteins of the present disclosure, including RNase-Fc fusion proteins, by minimizing clearance from circulation, for example by mannose and asialoglycoprotein receptors and other lectin-like receptors. Accordingly, in some embodiments, the soluble interferon receptor of the present disclosure is prepared in an aglycosylated, deglycosylated, or underglycosylated form. Preferably, N-linked glycosylation is altered, and the RNase-Fc fusion protein is aglycosylated (aglycosyated).

[0219] In some embodiments, all asparagine residues in an RNase-Fc fusion protein that match the Asn-X-Ser / Thr consensus (wherein X can be any naturally occurring amino acid other than proline) are mutated to residues that do not function as acceptors for N-linked glycosylation (e.g., serine, glutamine), thereby eliminating glycosylation of the RNase-Fc fusion protein when synthesized in cells that glycosylate proteins.

[0220] In some embodiments, an RNase-Fc fusion protein lacking an N-linked glycosylation site is produced in mammalian cells. In one embodiment, the mammalian cell is a CHO cell. Accordingly, in a specific embodiment, the aglycosylated RNase-Fc fusion protein is produced in CHO cells.

[0221] In other embodiments, reduced or absent N-glycosylation is achieved, for example, by producing the RNase-Fc fusion protein in a host (e.g., a bacterium such as E. coli), a mammalian cell engineered to lack one or more enzymes important for glycosylation, or a mammalian cell treated with an agent that prevents glycosylation such as tunicamycin, an inhibitor of Dol-PP-GlcNAc formation.

[0222] In some embodiments, the RNase-Fc fusion protein is produced in lower eukaryotes that have been engineered to produce glycoproteins containing complex N-glycans rather than high-mannose sugars (see, for example, US2007 / 0105127).

[0223] In some embodiments, glycosylated RNase-Fc fusion proteins (e.g., receptors produced in mammalian cells such as CHO cells) are treated chemically or enzymatically to remove or modify or shield one or more carbohydrate residues (e.g., one or more mannose, fucose, and / or N-acetylglucosamine residues). Such modification or shielding can reduce the binding of the RNase-Fc fusion protein to mannose receptors and / or asialoglycoprotein receptors and / or other lectin-like receptors. Chemical deglycosylation can be achieved, for example, by treating the RNase-Fc fusion protein with trifluoromethanesulfonic acid (TFMS) as disclosed in Sojar et al., JBC 1989;264:2552-9 and Sojar et al., Methods Enzymol 1987;138:341-50, or by treating it with hydrogen fluoride as disclosed in Sojar et al. (1987, see above). Enzymatic removal of N-linked carbohydrates from RNase-Fc fusion proteins was described by Thotakura et al. (Methods Enzymol As disclosed in 1987;138:350-9), this can be achieved by treating the RNase-Fc fusion protein with protein N-glycosidase (PNGase) A or F. Other commercially available deglycosylating enzymes recognized in the art and suitable for use include endo-alpha-N-acetyl-galactosaminidase, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3, and endoglycosidase H. In some embodiments, one or more of these enzymes can be used to deglycosylate the RNase-Fc fusion protein of this disclosure. Alternative methods for deglycosylation are disclosed, for example, in US8,198,063.

[0224] In some embodiments, the RNase-Fc fusion protein is partially deglycosylated. Partial deglycosylation can be achieved by treating the RNase-Fc fusion protein with an endoglycosidase that cleaves N-linked high-mannose carbohydrates rather than complex carbohydrates (e.g., endoglycosidase H), leaving a single GlcNAc residue linked to asparagine. The RNase-Fc fusion protein treated with endoglycosidase H will lack the high-mannose carbohydrate and will result in reduced interaction with the hepatic mannose receptor. While this receptor recognizes the terminal GlcNAc, the probability of productive interaction with a single GlcNAc on the protein surface is not as high as the probability of productive interaction with the intact high-mannose structure.

[0225] In other embodiments, glycosylation of the RNase-Fc fusion protein is modified, for example, by oxidation, reduction, dehydration, substitution, esterification, alkylation, sialylation, carbon-carbon bond cleavage, or other means to reduce the clearance of the RNase-Fc fusion protein from blood. In some embodiments, the RNase-Fc fusion protein is treated with periodate and sodium borohydride to modify its carbohydrate structure. Periodate treatment oxidizes adjacent diols, cleaving carbon-carbon bonds and replacing hydroxyl groups with aldehyde groups; borohydride reduces aldehydes to hydroxyls. Many sugar residues contain adjacent diols and are therefore cleaved by this treatment. The extended serum half-lives with periodates and sodium borohydride are illustrated by sequential treatment of the lysosomal enzyme β-glucuronidase with these agents (e.g., Houba et al. (1996) Bioconjug Chem 1996:7:606-11; Stahl et al. PNAS 1976;73:4045-9; Achord et al. Pediat. Res 1977;11:816-22; Achord et al. (See Cell 1978;15:269-78). Methods for treatment with periodate and sodium borohydride are disclosed in Hickman et al., BBRC 1974;57:55-61. Methods for treatment with periodate and cyanoboron hydride to increase the serum half-life and tissue distribution of lysine are disclosed in Thorpe et al. Eur J Biochem 1985;147:197-206.

[0226] In one embodiment, the carbohydrate structure of the RNase-Fc fusion protein can be shielded by the addition of one or more additional moieties (e.g., carbohydrate groups, phosphate groups, alkyl groups, etc.) that interfere with structural recognition by mannose or asialoglycoprotein receptors or other lectin-like receptors.

[0227] In some embodiments, one or more potential glycosylation sites are removed by mutations in the nucleic acid encoding the RNase-Fc fusion protein, thereby reducing the glycosylation of the RNase-Fc fusion protein when synthesized in cells that glycosylate the protein, such as mammalian cells like CHO cells (resulting in insufficient glycosylation). In some embodiments, for example, if an insufficiently glycosylated RNase-Fc fusion protein exhibits increased activity or contributes to an increased serum half-life, it may be desirable to selectively insufficiently glycosylate the RNase-Fc fusion protein by mutating its potential N-linked glycosylation site. In other embodiments, for example, if such modification improves the serum half-life of the RNase-Fc fusion protein, it may be desirable to insufficiently glycosylate a portion of the RNase-Fc fusion protein such that a particular domain lacks N-glycosylation. Alternatively, the recognition motif for the glycosylation enzyme can be disrupted by modifying other amino acids near the glycosylation acceptor, without necessarily altering the amino acids that would otherwise be normally glycosylated.

[0228] In some embodiments, glycosylation of the RNase-Fc fusion protein can be modified by introducing a glycosylation site. For example, the amino acid sequence of the RNase-Fc fusion protein can be modified to introduce a consensus sequence for N-linked glycosylation of Asn-X-Ser / Thr (where X is any amino acid other than proline). The additional N-linked glycosylation site may be added at any location in the amino acid sequence of the RNase-Fc fusion protein. Preferably, the glycosylation site is introduced at a position in the amino acid sequence that does not substantially reduce the activity of the RNase-Fc fusion protein.

[0229] The addition of an O-linked glycosylation site has been reported to alter the serum half-life of many proteins, including growth hormone, follicle-stimulating hormone, IGFBP-6, factor IX, and many others (as disclosed in, e.g., Okada et al., Endocr Rev 2011;32:2-342; Weenen et al., J Clin Endocrinol Metab 2004;89:5204-12; Marinaro et al., European Journal of Endocrinology 2000;142:512-6;US2011 / 0154516). Therefore, in some embodiments, the O-linked glycosylation (at serine / threonine residues) of RNase-Fc fusion proteins is altered. Methods for altering O-linked glycosylation are common in the art, for example, by beta-efflux (e.g., Huang et al.). See also: al., Rapid Communications in Mass Spectrometry 2002;16:1199-204;Conrad, Curr Protoc Mol Biol 2001; Chapter 17:Unit 17.15A;Fukuda, Curr Protoc Mol Biol 2001;Chapter 17;Unit 17.15B;Zachara et al., Curr Protoc Mol Biol 2011;Unit 17.6; by using commercially available kits (e.g., GlycoProfile® Beta-Emission Kit, Sigma); or Gal β1-3GalNAc and / or GlcNAc This can be achieved by attaching an RNase-Fc fusion protein to a treatment with a series of exoglycosidases, including but not limited to β1-4 galactosidase and β-N-acetylglucosaminidase, followed by treatment with endo-α-N-acetylgalactosaminidase (i.e., O-glycosidase), until only β1-3GalNAc remains. Such enzymes are commercially available, for example, from New England Biolabs. In further embodiments, the RNase-Fc fusion protein is modified to introduce O-linked glycosylation, as disclosed, for example, in Okada et al. (see above), Weenen et al. (see above), US2008 / 0274958; and US2011 / 0171218. In some embodiments, one or more O-linked glycosylation consensus sites, such as CXXGGT / SC (SEQ ID NO: 59) (van den Steen et al., In Critical Reviews in Biochemistry and Molecular Biology, Michael Cox, ed., 1998;33:151-208), NST-E / DA (SEQ ID NO: 60), NITQS (SEQ ID NO: 61), QSTQS (SEQ ID NO: 62), D / E-FT-R / KV (SEQ ID NO: 63), CE / D-SN (SEQ ID NO: 64), and GGSC-K / R (SEQ ID NO: 65), are introduced into the RNase-Fc fusion protein.The additional O-linked glycosylation site may be added to any location in the amino acid sequence of the RNase-Fc fusion protein. Preferably, the glycosylation site is introduced at a position in the amino acid sequence that does not substantially reduce the activity of the RNase-Fc fusion protein. Alternatively, the O-linked sugar moiety can be found, for example, in WO87 / 05330 and Aplin et al., CRC. As described in Crit Rev Biochem 1981;259-306), it is introduced by chemically modifying amino acids in the RNase-Fc fusion protein.

[0230] In some embodiments, both N-linked and O-linked glycosylation sites are introduced into the RNase-Fc fusion protein, preferably at amino acid sequences that do not substantially reduce the activity of the RNase-Fc fusion protein.

[0231] It is well within the capabilities of those skilled in the art to introduce, reduce, or eliminate glycosylation (e.g., N-linked or O-linked glycosylation) in RNase-Fc fusion proteins and to determine, using methods common in the art, whether such modifications to the glycosylation state increase or decrease the activity or serum half-life of the RNase-Fc fusion protein.

[0232] In some embodiments, the RNase-Fc fusion protein may include a modified glycoform (e.g., underfucosylated or fucose-free glycan).

[0233] In some embodiments, an RNase-Fc fusion protein having altered glycosylation has a serum half-life that is at least about 1.5 times, for example, at least 3 times, at least 5 times, at least 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1000 times, or 1000 times or more increased compared to a corresponding glycosylated RNase-Fc fusion protein (e.g., an RNase-Fc fusion protein without mutations in the potential N-linked glycosylation site). The serum half-life of an RNase-Fc fusion protein having altered glycosylation can be determined using conventional methods recognized in the art.

[0234] In some embodiments, an RNase-Fc fusion protein having altered glycosylation (e.g., a non-glycosylated, deglycosylated, or insufficiently glycosylated RNase-Fc fusion protein) retains at least 50%, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the activity of the corresponding glycosylated RNase-Fc fusion protein (e.g., an RNase-Fc fusion protein in which the potential N-linked glycosylation site is not mutated).

[0235] In some embodiments, altering the glycosylation state of an RNase-Fc fusion protein can increase its activity either by directly increasing its activity or by increasing its bioavailability (e.g., serum half-life). Thus, in some embodiments, the activity of an RNase-Fc fusion protein having altered glycosylation is increased by at least 1.3 times, for example, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times or 10 times or more compared to the corresponding glycosylated RNase-Fc fusion protein (e.g., an RNase-Fc fusion protein with no mutation at the potential N-linked glycosylation site).

[0236] Those skilled in the art can easily determine the glycosylation status of an RNase-Fc fusion protein using methods recognized in the art. In preferred embodiments, the glycosylation status is determined using mass spectrometry. In other embodiments, the interaction with concanavalin A (Con A) can be evaluated to determine if the RNase-Fc fusion protein is insufficiently glycosylated. Insufficiently glycosylated RNase-Fc fusion proteins are expected to show reduced binding to Con A-Sepharose compared to the corresponding glycosylated RNase-Fc fusion protein. SDS-PAGE analysis can also be used to compare the mobility of insufficiently glycosylated proteins and the corresponding glycosylated proteins. Insufficiently glycosylated proteins are expected to have greater mobility on SDS-PAGE compared to the glycosylated proteins. Other suitable methods recognized in the art for analyzing protein glycosylation status are disclosed, for example, in Roth et al., International Journal of Carbohydrate Chemistry 2012;1-10.

[0237] The pharmacokinetics of RNase-Fc fusion proteins with different glycosylation states, such as their serum half-lives, can be assayed using conventional methods, for example, by introducing RNase-Fc fusion proteins into mice, for example, intravenously, collecting blood samples at predetermined time points, and assaying and comparing the levels and / or activity of RNase-Fc fusion proteins in the samples.

[0238] Method for producing RNase-Fc fusion proteins RNase-containing nuclease fusion proteins, including RNase-Fc fusion proteins, are produced in transformed or transfected host cells using recombinant DNA techniques. To do this, recombinant DNA molecules encoding RNase-Fc fusion proteins are prepared. Methods for preparing such DNA molecules are well known in the art. For example, the sequence encoding the RNase-Fc fusion protein can be excised from DNA using a suitable restriction enzyme. Alternatively, the DNA molecule can be synthesized using chemical synthesis techniques, such as the phosphoramido salt method. Combinations of these techniques can also be used.

[0239] The present invention also includes a vector capable of expressing an RNase-Fc fusion protein in a suitable host. The vector comprises a DNA molecule encoding an RNase-Fc fusion protein, which is operably ligated to a suitable expression regulatory sequence. Methods for influencing this operable ligation before or after the DNA molecule is inserted into the vector are well known. The expression regulatory sequence includes promoters, activators, enhancers, operators, ribosomal nuclease domains, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the regulation of transcription or translation.

[0240] The resulting vector containing the DNA molecule is used for the transformation or transfection of a suitable host. In some embodiments, the RNase-Fc fusion protein of this disclosure can be prepared by co-transfecting or co-transforming a suitable host with two or more expression vectors containing the DNA encoding the RNase-Fc fusion protein. This transformation or transfection can be carried out using methods well known in the art.

[0241] Any of the many available and well-known host cells can be used in carrying out the present invention. The selection of a particular host depends on a number of factors recognized in the art. Such factors include, for example, compatibility with the selected expression vector, toxicity of the RNase-Fc fusion protein encoded by the DNA molecule, rate of transformation or transfection, ease of recovery of the RNase-Fc fusion protein, expression characteristics, biosafety, and cost. These factors must be balanced with the understanding that not all hosts may be equally effective in expressing a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (e.g., E. coli), yeasts (e.g., Saccharomyces) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art. In some embodiments, the RNase-Fc fusion protein is produced in CHO cells.

[0242] Next, the transformed or transfected host is cultured and purified. The host cells can be cultured under conventional fermentation or culture conditions to express the desired compound. Such fermentation and culture conditions are well known in the art. Finally, the RNase-Fc fusion protein is purified from the culture by methods well known in the art.

[0243] The compounds can also be prepared by synthetic methods. For example, solid-phase synthesis techniques can be used. Suitable techniques are well known in the art, and refer to Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis This includes techniques described in et al., Biochem Intl 1985;10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Patent No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527. In some embodiments, compounds containing derivatized peptides or non-peptide groups can be synthesized by well-known organic chemical techniques.

[0244] Other methods of molecular expression / synthesis are generally known in the art to those skilled in the art.

[0245] Pharmaceutical composition In certain embodiments, the RNase-Fc fusion protein of the Disclosure, comprising the RNase-Fc fusion protein, is administered alone. In certain embodiments, the RNase-Fc fusion protein is administered before the administration of at least one other therapeutic agent. In certain embodiments, the RNase-Fc fusion protein is administered concurrently with the administration of at least one other therapeutic agent. In certain embodiments, the RNase-Fc fusion protein is administered after the administration of at least one other therapeutic agent. In other embodiments, the RNase-Fc fusion protein is administered before the administration of at least one other therapeutic agent. As will be understood by those skilled in the art, in some embodiments, the RNase-Fc fusion protein is combined with other drugs / compounds. In some embodiments, the RNase-Fc fusion protein and other drugs are administered simultaneously. In some embodiments, the RNase-Fc fusion protein and other drugs are not administered simultaneously, but the RNase-Fc fusion protein is administered before or after the administration of the drugs. In some embodiments, the subject receives both the RNase-Fc fusion protein and other drugs during the same prevention period, onset of the disorder, and / or treatment period.

[0246] The pharmaceutical compositions of this disclosure can be administered in combination therapy, i.e., in combination with other agents. In certain embodiments, the combination therapy comprises a combination of an RNase-Fc fusion protein and at least one other agent. Agents include, but are not limited to, chemical compositions prepared by synthesis in vitro, antibodies, antigen-binding regions, and combinations and conjugates thereof. In certain embodiments, the agent may function as an agonist, antagonist, allosteric modulator, or toxin.

[0247] In certain embodiments, the disclosure provides a pharmaceutical composition comprising an RNase-Fc fusion protein together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0248] In certain embodiments, the present invention provides a pharmaceutical composition comprising an RNase-Fc fusion protein and at least one therapeutically effective amount of an additional therapeutic agent together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0249] In certain embodiments, the acceptable formulation materials are preferably non-toxic to the recipient at the dosage and concentration used. In some embodiments, the formulation materials (one or more) are for sc administration and / or IV administration. In certain embodiments, the pharmaceutical composition may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality by weight, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or permeation of the composition.In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antibacterial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., gelatin); colorants, flavorings, and diluents; and emulsifiers. Agents; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants. Other examples include wetting agents (e.g., polysorbates such as Pluronic®, PEG, sorbitan esters, polysorbate 20, polysorbate 80, etc., Triton, tromethamine, lecithin, cholesterol, tyroxapol, etc.); stability enhancers (e.g., sucrose or sorbitol, etc.); tonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company (1995)).In some embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and / or 10 mM NAOAC, pH 5.2, 9% sucrose.

[0250] In certain embodiments, an RNase-Fc fusion protein and / or therapeutic molecule is linked to a half-life extension vehicle known in the art. Such vehicles include, but are not limited to, polyethylene glycol, glycogen (e.g., glycosylation of the RNase-Fc fusion protein), and dextran. Such vehicles are described, for example, in U.S. Patent Application No. 09 / 428,082, currently U.S. Patent No. 6,660,843, and PCT Patent Publication No. WO99 / 25044.

[0251] In certain embodiments, the optimal pharmaceutical composition can be determined by those skilled in the art, for example, based on the intended route of administration, the form of delivery, and the desired dose. See, for example, Remington's Pharmaceutical Sciences, above. In certain embodiments, such a composition may affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the fusion protein of this disclosure.

[0252] In certain embodiments, the primary vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous. For example, in certain embodiments, a suitable vehicle or carrier may be water for injection, physiological saline, or artificial cerebrospinal fluid, optionally supplemented with other materials commonly found in compositions for parenteral administration. In some embodiments, the physiological saline may include isotonic phosphate-buffered saline. In certain embodiments, the pharmaceutical composition may include Tris buffer at approximately pH 7.0–8.5 or acetate buffer at approximately pH 4.0–5.5, and therefore may further include sorbitol or a suitable substitute. In certain embodiments, a composition comprising an RNase-Fc fusion protein with or without at least one additional therapeutic agent may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing a selected composition of a desired degree of purity with a formulation agent as needed (Remington's Pharmaceutical Sciences, above). Furthermore, in certain embodiments, compositions comprising an RNase-Fc fusion protein with or without at least one additional therapeutic agent can be formulated as lyophilized products using appropriate excipients such as sucrose.

[0253] In certain embodiments, a pharmaceutical composition may be selected for parenteral delivery. In certain embodiments, a composition may be selected for delivery via inhalation or through the gastrointestinal tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the scope of those skilled in the art.

[0254] In certain embodiments, the formulation components are present at the administration site at an acceptable concentration. In certain embodiments, a buffer is used to maintain the composition at a physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0255] In certain embodiments, when parenteral administration is intended, the therapeutic composition may be in the form of a parenterally acceptable aqueous solution, pyrogen-free, containing the desired RNase-Fc fusion protein with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water, properly stored, formulated as a sterile isotonic solution containing the RNase-Fc fusion protein with or without at least one additional therapeutic agent. In certain embodiments, the preparation involves formulating the desired molecule with a drug such as an injectable microsphere, bioerosive particles, a polymer (e.g., polylactic acid or polyglycolic acid), beads, or liposomes, which can result in controlled or sustained release of the product and can be delivered by depot injection. In certain embodiments, hyaluronic acid may also be used, which has the effect of promoting a long duration of action in circulation. In certain embodiments, the desired molecule can be introduced using an implantable drug delivery device.

[0256] In certain embodiments, a pharmaceutical composition can be formulated for inhalation. In certain embodiments, an RNase-Fc fusion protein can be formulated as a dry powder for inhalation, with or without at least one additional therapeutic agent. In certain embodiments, an inhalation solution containing an RNase-Fc fusion protein, with or without at least one additional therapeutic agent, can be formulated using a propellant for aerosol delivery. In certain embodiments, the solution can be sprayed. Transpulmonary delivery is further described in PCT application PCT / US94 / 001875, which describes transpulmonary delivery of chemically modified proteins.

[0257] In certain embodiments, the formulation is intended to be administered orally. In certain embodiments, the RNase-Fc fusion protein administered in this manner, with or without at least one additional therapeutic agent, can be formulated with or without carriers conventionally used in the formulation of solid dosage forms such as tablets and capsules. In certain embodiments, capsules can be designed so that the active portion of the formulation is released at a point in the gastrointestinal tract when its bioavailability is maximized and pre-systemic degradation is minimized. In certain embodiments, at least one additional agent may be included to facilitate the absorption of the RNase-Fc fusion protein and / or any additional therapeutic agent. In certain embodiments, diluents, flavoring agents, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0258] In certain embodiments, the pharmaceutical composition may include an effective quantity of RNase-Fc fusion protein as a mixture with a non-toxic excipient suitable for tablet preparation, with or without at least one additional therapeutic agent. In certain embodiments, the solution can be prepared in unit dose form by dissolving the tablets in sterile water or another suitable vehicle. In certain embodiments, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or gum arabic; or lubricants such as magnesium stearate, stearic acid or talc.

[0259] Additional pharmaceutical compositions, including formulations containing RNase-Fc fusion proteins with or without at least one additional therapeutic agent, as sustained-delivery or controlled-delivery formulations, will be apparent to those skilled in the art. In certain embodiments, techniques for formulating various other sustained-delivery or controlled-delivery means, such as liposome carriers, bio-erosive microparticles or porous beads and depot injections, are also known to those skilled in the art. See, for example, PCT application PCT / US93 / 00829, which describes the controlled release of porous polymer microparticles for delivering pharmaceutical compositions. In certain embodiments, the sustained-release preparation may include a semipermeable polymer matrix in the form of a molded article, e.g., a film or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels, polylactic acid (U.S. Patent No. 3,773,919 and EP058,481), copolymers of L-glutamic acid and gammaethyl-L-glutamic acid (Sidman et al, Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylic acid) (Langer et al., J Biomed Mater Res, 15: 167-277 (1981) and Langer, Chem Tech, 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., above), or poly-D(-)-3-hydroxybutyric acid (EP133,988). In certain embodiments, the sustained-release composition may also include liposomes, which can be prepared by any of several methods known in the art. For example, see Eppstein et al, PNAS, 82:3688-3692 (1985);EP036,676;EP088,046 and EP143,949.

[0260] Pharmaceutical compositions used for in vivo administration are typically sterilized. In certain embodiments, this can be achieved by filtration through a sterile filtration membrane. In certain embodiments, if the composition is lyophilized, sterilization using this method can be performed either before lyophilization or after lyophilization and reconstitution. In certain embodiments, compositions for parenteral administration can be stored in lyophilized form or as a solution. In certain embodiments, parenteral compositions are generally placed in sterile containers with access ports, such as intravenous solution bags or vials with stoppers that can be punctured with a subcutaneous needle.

[0261] In certain embodiments, once the pharmaceutical composition has been formulated, it is stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. In certain embodiments, such formulations may be stored in a ready-to-use form or in a form that is reconstituted before administration (e.g., in a lyophilized form).

[0262] In certain embodiments, a kit for producing single-dose dosage units is provided. In certain embodiments, the kit may include both a first container containing a dried protein and a second container containing an aqueous formulation. In certain embodiments, the kit includes single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and dispersion syringes (lyosyringes)).

[0263] In certain embodiments, the effective amount of a pharmaceutical composition containing an RNase-Fc fusion protein, with or without at least one additional therapeutic agent, for therapeutic use depends, for example, on the context and purpose of the treatment. Therefore, it will be understood by those skilled in the art that, according to certain embodiments, a reasonable dose level for treatment varies in part depending on the molecule being delivered, the indication for which the RNase-Fc fusion protein is used with or without at least one additional therapeutic agent, the route of administration, and the patient's size (body weight, surface or organ size) and / or condition (age and overall health). In certain embodiments, the clinician may dose-set and modify the route of administration to obtain the optimal therapeutic effect. In certain embodiments, a typical dose can range from about 0.5 μg / kg to a maximum of about 50 mg / kg or more, depending on the factors mentioned above. In certain embodiments, the dose can range from about 5–10 mg / kg, about 2–8 mg / kg, about 3–6 mg / kg, about 3 mg / kg, about 5 mg / kg, or about 10 mg / kg.

[0264] In certain embodiments, the frequency of administration takes into account the pharmacokinetic parameters of the RNase-Fc fusion protein and / or any additional therapeutic agents in the formulation used. In certain embodiments, the clinician administers the composition until a dose is reached in which the desired effect is achieved. In certain embodiments, the composition can therefore be administered as a single dose, or as two or more doses (containing the same or different amounts of the desired molecule), over time, or as a continuous infusion via an implantable device or catheter. Further refinement of a reasonable dose is routinely performed by those skilled in the art and falls within the scope of tasks routinely performed by those skilled in the art. In certain embodiments, a reasonable dose can be confirmed by using reasonable dose-response data.

[0265] In certain embodiments, the route of administration of the pharmaceutical composition is consistent with known methods, for example, by oral, intravenous, intraperitoneal, intracerebral (intraparum), intraventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal, or intrafocal route of injection; by a continuous-release system; or by an implantable device. In certain embodiments, the composition may be administered by bolus injection, or by infusion or by an implantable device.

[0266] In certain embodiments, the composition can be administered topically by implanting a membrane, sponge, or other suitable material that has absorbed or encapsulated the desired molecule. In certain embodiments using an implantable device, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule may be by diffusion, sustained bolus, or continuous administration.

[0267] In certain embodiments, it may be desirable to use a pharmaceutical composition containing an RNase-Fc fusion protein with or without at least one additional therapeutic agent in an ex vivo manner. In such examples, cells, tissues, and / or organs removed from a patient are exposed to a pharmaceutical composition containing an RNase-Fc fusion protein with or without at least one additional therapeutic agent, and subsequently, the cells, tissues, and / or organs are implanted back into the patient.

[0268] In certain embodiments, RNase-Fc fusion proteins and / or any additional therapeutic agents can be delivered by embedding certain genetically engineered cells using methods such as those described herein to express and secrete polypeptides. In certain embodiments, such cells may be animal or human cells and may be autologous, heterologous, or xenogeneic. In certain embodiments, the cells may be immortalized. In certain embodiments, the cells may be encapsulated to reduce the likelihood of an immunological response and prevent invasion by surrounding tissue. In certain embodiments, the encapsulation material is typically a biocompatible, semipermeable polymer encapsulant or membrane that allows for the release of the protein product(s) but prevents cell destruction by the patient's immune system or other harmful factors originating from surrounding tissue. in vitro assay

[0269] The efficacy of the RNase-Fc fusion proteins of this disclosure, including the RNase-Fc fusion protein, can be evaluated using various in vitro assays known in the art.

[0270] For example, cultured human PBMCs from normal subjects, lupus patient PBMCs, or Sjögren's PBMCs are isolated, cultured, and treated with various stimuli (e.g., TLR ligands, co-stimulatory antibodies, immune complexes, and normal or autoimmune serum) in or out of the presence of RNase-Fc fusion protein. Cytokine production by stimulated cells can be measured using commercially available reagents for various cytokines (e.g., IL-6, IL-8, IL-10, IL-4, IFN-γ, and TNF-α), such as antibody pairing kits from Biolegend (San Diego, CA). Culture supernatants are collected at various time points appropriate for the assay (e.g., 24 hours, 48 ​​hours, or later) to determine the effect of RNase-Fc fusion protein on cytokine production. IFN-α production is measured using, for example, anti-human IFN-α antibodies and standard curve reagents available from PBL interferon source (Piscataway, NJ). Similar assays are performed using purified human lymphocyte subpopulations (isolated monocytes, B cells, pDCs, T cells, etc.) with commercially available magnetic bead-based isolation kits, for example, from Miltenyi Biotech (Auburn, CA).

[0271] Multicolor flow cytometry may be used to evaluate the effect of RNase-Fc fusion proteins on immune cell activation by measuring the expression of lymphocyte-activating receptors, such as CD5, CD23, CD69, CD80, CD86, and CD25, in PBMCs or isolated cell subpopulations at various time points after stimulation, using conventional methods recognized in the art.

[0272] The efficacy of RNase-Fc fusion proteins has also been demonstrated, for example, by Ahlin et al., Lupus 2012:21:586-95; Mathsson et al., Clin Expt Immunol 2007;147:513-20; and Chiang et al., J Immunol As described in 2011;186:1279-1288, SLE or Sjögren's patient serum can be tested by incubating it with normal human pDCs to activate IFN output. While not bound by theory, circulating nucleic acid-containing immune complexes in SLE or Sjögren's patient serum facilitate nucleic acid antigen entry into pDC endosomes via Fc receptor-mediated endocytosis, followed by the binding of nucleic acids to endosomal TLR7, 8, and 9 and their activation. To assess the effect of RNase-Fc fusion proteins, SLE or Sjögren's patient serum or plasma is pretreated with RNase-Fc fusion proteins and then added to cultures of pDC cells isolated from healthy volunteers. The levels of IFN-α produced are then determined at multiple time points. By degrading nucleic acid-containing immune complexes, effective RNase-Fc fusion proteins are expected to reduce the amount of IFN-α produced.

[0273] The efficacy of RNase-Fc fusion proteins is demonstrated by comparing the results of assays from cells treated with the RNase-Fc fusion proteins disclosed herein with the results of assays from cells treated with a control formulation. After treatment, the levels of the various markers (e.g., cytokines, cell surface receptors, proliferation) are generally improved in the group treated with the effective RNase-Fc fusion protein compared to the marker levels present before treatment or compared to the levels measured in the control group.

[0274] Treatment method The RNase-Fc fusion proteins of the Disclosure, including the RNase-Fc fusion protein of the Disclosure, are particularly effective in treating autoimmune disorders or abnormal immune responses. In this regard, it is understood that the RNase-Fc fusion proteins of the Disclosure, including the RNase-Fc fusion protein of the Disclosure, are used to control, suppress, modulate, treat, or eliminate unwanted immune responses to both external and autoantigens. In some embodiments, the RNase-Fc fusion proteins of the Disclosure, including the RNase-Fc fusion protein of the Disclosure, are used to treat or reduce fatigue in patients with autoimmune disorders. In some embodiments, the fatigue is Sjögren's syndrome-related fatigue.

[0275] In some embodiments, the RNase-containing nuclease fusion proteins of the Disclosure, including RNase-Fc fusion proteins, are useful for treating autoimmune diseases in human patients by administering an effective or sufficient amount of the RNase-containing nuclease fusion proteins of the Disclosure, such as RNase-Fc fusion proteins, to human patients in need, thereby treating the disease. Any appropriate route of administration to achieve the desired effect is intended by the Disclosure (e.g., intravenous, intramuscular, subcutaneous). Treatment of a disease condition can result in a reduction of symptoms associated with the condition, which may be long-term, short-term, or even transient beneficial effects. In some embodiments, treatment of Sjögren's disease results in a reduction of fatigue associated with the disease or condition.

[0276] Biochemical assay In some embodiments, the RNase-containing nuclease fusion proteins of this disclosure, including an RNase-Fc fusion protein, are administered to human patients in need to treat Sjögren's syndrome. In some embodiments, the effect of the RNase-Fc fusion protein is demonstrated by comparing IFN-alpha levels, IFN-alpha response gene levels, autoantibody titers, renal function and pathology, and / or circulating immune complex levels in human patients treated with the RNase-Fc fusion protein disclosed herein, compared to placebo.

[0277] For example, select or identify human subjects requiring treatment (e.g., patients meeting the American-European Consensus Sjogren's Classification Criteria). Subjects may need to reduce the cause or symptoms of Sjogren's syndrome, such as pSS. In some embodiments, patients have Sjogren's syndrome and require fatigue reduction. Subject identification may be performed in a clinical setting or elsewhere, for example, at the subject's home, by the subject using a self-testing kit.

[0278] At baseline (Day 1), an appropriate first dose of the RNase-containing nuclease fusion protein of this disclosure, including the RNase-Fc fusion protein, is administered to patients in need. The RNase-Fc fusion protein is formulated as described herein. The patient's condition is assessed at baseline (Day 1) and after a period following the first dose, for example, on days 8, 15, 29, 43, 57, 71, 85, 99, or at the end of the study, by measuring, for example, IFN-alpha levels, IFN-alpha response gene levels, autoantibody titers, renal function and pathology, and / or circulating immune complex levels. Other relevant criteria may also be measured. The number and concentration of doses are adjusted according to the needs of the subject. Following treatment, the subject's IFN-alpha levels, IFN-alpha response gene levels, autoantibody titers, renal function and pathology, and / or circulating immune complex levels are reduced and / or improved compared to levels present before treatment or compared to levels measured in similarly affected but untreated / control subjects.

[0279] Fatigue assay Various patient-reported performance (PRO) measurement methods have been used and validated in measuring fatigue in subjects with chronic diseases. Such PROs are known in the art and can be used to evaluate the efficacy of RNase-containing nuclease fusion proteins of this disclosure, including RNase-Fc fusion proteins.

[0280] EULAR Sjögren's Syndrome Patient Reporting Index (ESSPRI) The European League Against Rheumatism (EULAR) developed the Sjögren's Syndrome (SS) Patient-Reported Index (ESSPRI) to assess symptoms in patients with primary Sjögren's syndrome (Seror et al., Ann. Rheum. Dis. 2011;70:968-972). The ESSPRI was developed as a comprehensive score to measure all important and functionally debilitating symptoms of primary Sjögren's syndrome: dryness, limb pain, and fatigue. The ESSPRI has been shown to be sufficient for measuring each symptom without loss of content validity, and the score is easy to calculate.

[0281] ESSPRI is a patient-administered questionnaire used to assess symptoms in patients with primary Sjögren's syndrome. The questionnaire includes three scales, one for each of the following symptoms: (1) dryness, (2) limb pain, and (3) fatigue. Each component of ESSPRI is measured on a single numerical scale from 0 to 10, and the overall ESSPRI score is the mean of the three scales: (dryness + limb pain + fatigue) / 3. A decrease of at least one point in the ESSPRI score is clinically significant.

[0282] In some embodiments, the efficacy of the RNase-containing nuclease fusion protein of the present disclosure, including the RNase-Fc fusion protein, or a pharmaceutical composition thereof, is demonstrated by evaluating the improvement in fatigue in patients after treatment with the RNase-containing nuclease fusion protein of the present disclosure, including the RNase-Fc fusion protein, or a pharmaceutical composition thereof. After treatment, fatigue is generally reduced in patients as measured by ESSPRI, compared to the level of fatigue in patients before treatment and / or compared to patients treated with a control formulation.

[0283] FACIT - Fatigue The Fatigue Assessment Scale for Chronic Disease Treatment (FACIT-Fatigue) is used to assess an individual's fatigue level during normal daily activities over the past week. The FACIT-Fatigue Questionnaire and Scoring & Interpretation Materials are available from FACIT.org (Elmhurst, Ill., USA). The FACIT-Fatigue Questionnaire provides a number of general and targeted scales. The FACIT-Fatigue scale offers many advantages, including high internal validity, high test-retest reliability, reliability and sensitivity to changes in patients with various chronic health conditions, ease of use, and use in various settings (KF Tennant, Try This: best Practices in Nursing Care to Older Adults, Issue 30, 2012; Chandran et al., Ann. Rheum. Dis. 2007; 66: 936-939).

[0284] FACIT-Fatigue is a 13-item questionnaire originally developed to measure fatigue in cancer patients and is now used to measure fatigue in Sjögren's disease patients. Patients are asked to answer 13 questions scored on a scale of 0 to 4 (0 = strongly disagree, 1 = slightly agree, 2 = somewhat agree, 3 = strongly agree, 4 = very agree). The fatigue scale has 13 items, with 52 being the highest possible score. Higher scores on the fatigue scale correspond to lower levels of fatigue and indicate a better quality of life.

[0285] To calculate the FACIT fatigue score, response scores for negatively worded questions are inverted, and then the responses for the 13 items are added together. For the 11 items with responses, their scores are inverted (item score = 4 - response if no response is missing), and for the 2 items (items 7-8), their responses are not altered. All items are added together so that higher scores correspond to lower fatigue. If an individual question is skipped, the score is apportioned using the mean of the other answers on the scale. FACIT = 13 * [Sum (inverted items) + Sum (items 7-8)] / Number of items answered

[0286] In some embodiments, the efficacy of the RNase-containing nuclease fusion protein of the present disclosure, including the RNase-Fc fusion protein, or a pharmaceutical composition thereof, is demonstrated by evaluating the improvement in fatigue in patients treated with the RNase-containing nuclease fusion protein of the present disclosure, including the RNase-Fc fusion protein, or a pharmaceutical composition thereof. After treatment, fatigue is generally reduced in patients as measured by the FACIT fatigue scale, compared to the level of fatigue in patients before treatment and / or compared to patients treated with a control formulation.

[0287] Fatigue profile We developed the Fatigue Profile (ProF) to establish an effective assessment tool for characterizing fatigue associated with primary Sjögren's syndrome. Therefore, the term "patient with primary Sjögren's syndrome," used to describe patients' complaints of fatigue, discomfort, and pain, was used in the ProF questionnaire. ProF has been shown to be a reliable and valid measure for assessing the severity of fatigue and general discomfort in patients with primary Sjögren's syndrome.

[0288] ProF is a 16-item self-administered questionnaire divided into two domains: one relating to physical fatigue and the other to mental fatigue. The physical fatigue domain includes 12 items divided into four facets: (a) need for rest (4 items), (b) poor starting (3 items), (c) low stamina (3 items), and (d) weak muscles (2 items). The mental fatigue domain includes 4 items divided into two facets: (a) poor concentration (2 items) and (b) poor memory (2 items). The patient's score for each item on a scale of 0-7 (0 = "no problem at all" and 7 = "worst possible") is based on how the patient felt about their worst condition over the past two weeks. The score for each facet can be obtained by summing the item scores within each facet and dividing the sum by the number of items in each facet. The score for each domain (e.g., physical, mental) can be obtained by summing the facet scores within each domain and dividing the sum by the number of facets within each domain. A higher score indicates greater fatigue (Bowman). et al., Rheumatology, 2004; 43: 758-764; Strombeck et al., Scand. J. Rheumatol. 2005;34:455-459; Segal et al., Arthritis Rheum. 2008 Dec. 15; 59(12):1780-1787).

[0289] In some embodiments, the efficacy of the RNase-containing nuclease fusion protein of the present disclosure, including the RNase-Fc fusion protein, or a pharmaceutical composition thereof, is demonstrated by evaluating the improvement in fatigue in patients treated with the RNase-containing nuclease fusion protein of the present disclosure, including the RNase-Fc fusion protein, or a pharmaceutical composition thereof. After treatment, fatigue is generally reduced in patients as measured by ProF, compared to the level of fatigue in patients before treatment and / or compared to patients treated with a control formulation.

[0290] Assessment of reduction in Sjögren's syndrome-related (associated) fatigue In some embodiments, the efficacy of the RNase-containing nuclease fusion proteins of this disclosure, including the RNase-Fc fusion protein, is demonstrated by evaluating the reduction in fatigue in patients treated with the RNase-containing nuclease fusion protein, including the RNase-Fc fusion protein. In some embodiments, patients treated with the RNase-Fc fusion protein will demonstrate a reduction in fatigue compared to the level of fatigue in patients before treatment and / or compared to patients treated with a control formulation. In some embodiments, the fatigue is Sjögren's syndrome-related fatigue.

[0291] In some embodiments, the patient's condition is assessed by measuring fatigue in the patient using one or more patient-reported indices (e.g., ESSPRI, ProF, FACIT) compared to the fatigue level in the patient before treatment, or compared to the fatigue level in similarly affected untreated or control patients. In some embodiments, the effectiveness of the RNase-Fc fusion protein is demonstrated by evaluating the EULAR SS Patient-Reported Indices (ESSPRI), Fatigue Profile (ProF), and / or Assessment of Functional Treatment for Chronic Disease (FACIT) fatigue scales in patients treated with the RNase-Fc fusion protein disclosed herein, compared to patients treated with a control formulation. In some embodiments, patients treated with the RNase-Fc fusion protein will demonstrate improvements in the ESSPRI index, ProF, and / or FACIT fatigue scales compared to the ESSPRI index, ProF, and / or FACIT fatigue scales in patients before treatment, or compared to patients treated with a control formulation.

[0292] For example, selecting or identifying human subjects who require treatment (e.g., American Patients who meet the College of Rheumatology criteria for SLE, or the American-European Consensus Sjogren's Classification Criteria. Participants may need to reduce the causes or symptoms of SLE or Sjogren's syndrome, such as fatigue. Participant identification can be done in a clinical setting or elsewhere, for example, at the participant's home, using a self-test kit.

[0293] At baseline (day 1), an appropriate first dose of the RNase-Fc fusion is administered to the subject. The RNase-Fc fusion protein is formulated as described herein. The patient's condition is assessed at baseline (day 1) and after a certain period from the first dose, for example, on days 8, 15, 29, 43, 57, 71, 85, 99, or at the end of the study, using the ESSPRI index, ProF, and / or FACIT fatigue scale. Other relevant criteria may also be measured. The number and intensity of doses are adjusted according to the subject's needs. After treatment, one or more of the following outcomes may be observed: (1) improvement in the ESSPRI index compared to the pre-treatment ESSPRI index, or compared to similarly affected but untreated / control subjects; (2) improvement in PROF compared to the pre-treatment PROF, or compared to similarly affected but untreated / control subjects; (3) improvement in the FACIT fatigue scale compared to the pre-treatment FACIT fatigue scale, or compared to similarly affected but untreated / control subjects. In some embodiments, improvement in the ESSPRI index is a clinically meaningful improvement. A clinically meaningful improvement in the ESSPRI index is a decrease of at least 1 point in the ESSPRI score.

[0294] Neuropsychological analysis of fatigue assays The efficacy of the RNase-Fc fusion proteins of this disclosure, including the RNase-Fc fusion protein, can be evaluated using various neuropsychological assays known in the art.

[0295] Numerical code substitution check The Digital Substitution Test (DSST) provides a valid and highly sensitive test for measuring cognitive impairment influenced by multiple domains. The DSST is highly sensitive to both the presence of cognitive impairment and changes in cognitive function across a range of clinical populations, including those with Sjögren's syndrome. This neuropsychological test is widely used, highly validated, and extremely sensitive, readings from executive function-related inputs.

[0296] The DSST is a timed, written cognitive test conducted on a single sheet of paper. The test requires the patient to match symbols to numbers according to a key written at the beginning of the paper. The patient copies the symbols into the space below the row of numbers, and the number of correct symbols within a set time (e.g., 90 or 120 seconds) is calculated. The test provides data on the accuracy and speed of task performance. Patient performance in the DSST correlates with real-world functional outcomes, such as the ability to perform everyday tasks and recovery from functional impairments in a range of psychiatric conditions. The DSST test can be used to assess a patient's attention and / or concentration.

[0297] The DSST is a polyfactorial test that measures a range of cognitive operations and provides a practical and effective method for monitoring cognitive function over time. To perform well on the DSST, patients should demonstrate intact motor skills, including scanning and the ability to write or draw (i.e., basic mental dexterity). The test requires the ability to perform tasks such as speed, attention, and visual perception. The DSST offers high sensitivity in detecting cognitive impairment and has many advantages, including its brevity, reliability, sensitivity to change, and minimal influence of language, culture, and education on test performance (Jaeger, J., Journal of Clinical Psycopharmacology, 38(5), 513-518, October 2018).

[0298] Furthermore, DSST is used in clinical development to define pharmacokinetic / pharmacodynamic (PK / PD) relationships. This test is also used as a PD biomarker in CNS studies and can distinguish between two doses of selective serotonin reuptake inhibitors (SSRIs).

[0299] In some embodiments, the efficacy of the RNase-containing nuclease fusion protein of the Disclosure, comprising an RNase-Fc fusion protein, or a pharmaceutical composition thereof, is demonstrated by evaluating the improvement in cognitive function in patients treated with the RNase-containing nuclease fusion protein of the Disclosure, comprising an RNase-Fc fusion protein, or a pharmaceutical composition thereof. After treatment, cognitive function is generally improved in patients as measured by DSST testing, compared to the level of cognitive function in patients before treatment and / or compared to patients treated with a control formulation.

[0300] Evaluation of cognitive function improvement related to Sjögren's syndrome In some embodiments, the efficacy of the RNase-containing nuclease fusion proteins of the Disclosure, including the RNase-Fc fusion protein, is demonstrated by evaluating the improvement in cognitive function in patients treated with the RNase-containing nuclease fusion proteins of the Disclosure, including the RNase-Fc fusion protein. In some embodiments, patients treated with the RNase-Fc fusion protein demonstrate an improvement in cognitive function compared to the level of cognitive function in patients before treatment and / or compared to patients treated with a control formulation. In some embodiments, the patients have Sjögren's syndrome.

[0301] In some embodiments, the patient's condition is assessed by measuring cognitive function in the patient using one or more neuropsychological assays (e.g., DSST) compared to the level of cognitive function in the patient before treatment, or compared to the level of cognitive function in similarly affected untreated or control patients. In some embodiments, the effectiveness of the RNase-Fc fusion protein is demonstrated by evaluating the results of DSST tests in patients treated with the RNase-Fc fusion protein disclosed herein compared to patients treated with a control formulation. In some embodiments, patients treated with the RNase-Fc fusion protein will demonstrate an improvement in DSST test scores compared to the DSST test scores of patients before treatment, or compared to patients treated with a control formulation.

[0302] For example, human subjects requiring treatment are selected or identified (e.g., patients meeting the American-European Consensus Sjogren's Classification Criteria). Subjects may require reduction of the cause or symptoms of Sjogren's syndrome, such as cognitive impairment. Subject identification may occur in a clinical setting or elsewhere, for example, at the subject's home, through the use of a self-testing kit by the subject themselves.

[0303] At baseline (day 1), an appropriate first dose of the RNase-Fc fusion is administered to the subject. The RNase-Fc fusion protein is formulated as described herein. The patient's condition is assessed, for example, by DSST at baseline (day 1) and after a certain period from the first dose, for example, on days 8, 15, 29, 43, 57, 71, 85, 99, or at the end of the study. Other relevant criteria may also be measured. The number and concentration of doses are adjusted according to the subject's needs. After treatment, an improvement in DSST test score is obtained compared to the pre-treatment DSST test score, or compared to similarly affected but untreated / control subjects.

[0304] Medication regimen The RNase-Fc fusion proteins of the Disclosure, including the RNase-Fc fusion protein of the Disclosure, and / or the pharmaceutical compositions of the Disclosure, are formulated into pharmaceutically acceptable dosage forms for human subjects by conventional methods known to those skilled in the art. In some embodiments, the actual dose level of the active ingredient (i.e., the RNase-Fc fusion) in the pharmaceutical compositions of the Disclosure is varied to obtain an amount of the active ingredient effective in achieving a desired therapeutic response, composition, and / or mechanism of administration for a particular human patient without becoming unacceptably toxic to the patient.

[0305] The selected dosage level will depend on various factors, including the activity of the specific RNase-Fc fusion protein, the route of administration, the timing of administration, the rate of excretion or metabolism of the specific RNase-Fc fusion protein being administered, the rate and extent of absorption, the treatment used in combination with the specific RNase-Fc fusion protein being administered, the duration of other drugs, compounds, and / or materials, the age, sex, weight, condition, overall health, and medical history of the patient being treated, and similar factors well known in the medical field.

[0306] Generally, an appropriate dose of the RNase-Fc fusion protein or composition of this disclosure is the amount of the active ingredient that is the minimum effective dose to produce a therapeutic effect in a human subject. Such an effective dose will generally depend on the factors described above. Generally, intravenous, oral, and subcutaneous doses of the RNase-Fc fusion protein or composition of this disclosure for human patients, when used for the indicated effect, range from about 0.5 mg to about 50 mg per kilogram of body weight per week. In some embodiments, the RNase-Fc fusion protein or pharmaceutical composition of this disclosure is administered by injection (e.g., by intravenous injection, e.g., by infusion) to a human patient in need at a dose of about 0.5 mg to about 50 mg per kilogram of body weight per week.

[0307] In some embodiments, the RNase-Fc fusion protein or composition of this disclosure is generally administered to human patients at doses of approximately 1 to 20 mg / kg per week, 2 to 10 mg / kg per week, 5 to 15 mg / kg per week, 5 to 10 mg / kg per week, or 2 to 5 mg / kg per week. In some embodiments, doses exceeding 10 mg / kg, 15 mg / kg, or 20 mg / kg per week may be required. In some embodiments, doses less than 20 mg / kg, 15 mg / kg, or 10 mg / kg per week may be required. In some embodiments, parenteral doses, such as intravenous administration to human patients, are approximately 5 to 10 mg / kg per week. In some embodiments, the RNase-Fc fusion protein is administered to human patients in weekly, bi-weekly, monthly, or semi-monthly doses (e.g., every two or three months) of approximately 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, or 25 mg / kg. In some embodiments, the RNase-Fc fusion protein is administered to human patients in weekly, bi-weekly, monthly, or semi-monthly doses of 1 mg / kg. In some embodiments, the RNase-Fc fusion protein is administered to human patients at a dose of 2 mg / kg weekly, bi-weekly, monthly, or bi-monthly. In some embodiments, the RNase-Fc fusion protein is administered to human patients at a dose of 3 mg / kg weekly, bi-weekly, monthly, or bi-monthly. In some embodiments, the RNase-Fc fusion protein is administered to human patients at a dose of 4 mg / kg weekly, bi-weekly, monthly, or bi-monthly. In some embodiments, the RNase-Fc fusion protein is administered to human patients at a dose of 5 mg / kg weekly, bi-weekly, monthly, or bi-monthly. In some embodiments, the RNase-Fc fusion protein is administered to human patients at a dose of 6 mg / kg weekly, bi-weekly, monthly, or bi-monthly.In some embodiments, the RNase-Fc fusion protein is administered to human patients at a dose of 7 mg / kg weekly, bi-weekly, monthly, or bi-monthly. In some embodiments, the RNase-Fc fusion protein is administered to human patients at a dose of 8 mg / kg weekly, bi-weekly, monthly, or bi-monthly. In some embodiments, the RNase-Fc fusion protein is administered to human patients at a dose of 9 mg / kg weekly, bi-weekly, monthly, or bi-monthly. In some embodiments, the RNase-Fc fusion protein is administered to human patients at a dose of 10 mg / kg weekly, bi-weekly, monthly, or bi-monthly. In some embodiments, the RNase-Fc fusion protein is administered to human patients at a dose of 12 mg / kg weekly, bi-weekly, monthly, or bi-monthly. In some embodiments, the RNase-Fc fusion protein is administered to human patients at a dose of 15 mg / kg weekly, bi-weekly, monthly, or bi-monthly. In some embodiments, the aforementioned doses are formulated for intravenous injection.

[0308] If desired, effective weekly, bi-weekly, monthly, or bi-monthly doses of the RNase-Fc fusion protein or composition of this disclosure are administered to a human patient as 2, 3, 4, 5, 6 or more sub-doses, separately at appropriate intervals throughout the day (e.g., as intravenous injection or infusion), in unit dosage forms as needed. In some embodiments, the drug is administered once per day. In some embodiments, the drug is administered once or more times, as appropriate, every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, to obtain a therapeutic effect (e.g., sufficient for digestion of circulating RNA complexed with autoantibodies and / or RNA-containing immune complexes). In some embodiments, the medication is administered once a week as a single dose (e.g., intravenous injection or infusion). In some embodiments, the medication is administered once or multiple times every two weeks. In some embodiments, the medication is administered once every two weeks as a single dose. In some embodiments, the medication is administered once or multiple times every month. In some embodiments, the medication is administered once every month as a single dose. In some embodiments, the medication is administered once or multiple times every two weeks (e.g., every two or three months). In some embodiments, the medication is administered once every two or three months. In some embodiments, the aforementioned doses are formulated for intravenous injection.

[0309] In some embodiments, the medication is administered once every week for two weeks to achieve or maintain a therapeutic effect (e.g., intravenous injection or infusion), followed by one dose every two weeks thereafter. In some embodiments, the medication is administered once every week for three weeks to achieve or maintain a therapeutic effect, followed by one dose every two weeks thereafter. In some embodiments, the medication is administered once every week for four weeks to achieve or maintain a therapeutic effect, followed by one dose every two weeks thereafter. In some embodiments, the medication is administered once every week for two weeks to achieve or maintain a therapeutic effect, followed by one dose every month thereafter. In some embodiments, the medication is administered once every week for three weeks to achieve or maintain a therapeutic effect, followed by one dose every month thereafter. In some embodiments, the medication is administered once every week for four weeks to achieve or maintain a therapeutic effect, followed by one dose every month thereafter. In some embodiments, the aforementioned doses are formulated for intravenous injection. As used herein, the initial weekly dose and subsequent bi-weekly, monthly, or bi-weekly doses are referred to as “loading doses.”

[0310] In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 1 mg / kg (e.g., by intravenous injection or infusion). In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 2 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 3 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 4 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 5 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 6 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 7 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 8 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 9 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 10 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 12 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 15 mg / kg. In some embodiments, the aforementioned doses are formulated for intravenous injection. As used herein, "weekly" is understood to have the meaning of "every week" as accepted in the Art.

[0311] In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of approximately 1 mg / kg (e.g., by intravenous injection or infusion). In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of approximately 2 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of approximately 3 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of approximately 4 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of approximately 5 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of approximately 6 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of approximately 7 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of approximately 8 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of about 9 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of about 10 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of about 12 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every other week at a dose of about 15 mg / kg. In some embodiments, the aforementioned doses are formulated for intravenous injection. As used herein, every other week is understood to have the same meaning as every two weeks as accepted in the Art.

[0312] In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every three weeks at a dose of approximately 1 mg / kg (e.g., by intravenous injection or infusion). In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every three weeks at a dose of approximately 2 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every three weeks at a dose of approximately 3 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every three weeks at a dose of approximately 4 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every three weeks at a dose of approximately 5 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every three weeks at a dose of approximately 6 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every three weeks at a dose of approximately 7 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every three weeks at a dose of approximately 8 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every three weeks at a dose of approximately 9 mg / kg. In some embodiments, the RNase-Fc fusion protein is administered every three weeks at a dose of approximately 10 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every three weeks at a dose of approximately 12 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered every three weeks at a dose of approximately 15 mg / kg. In some embodiments, the aforementioned doses are formulated for intravenous injection. As used herein, every three weeks is understood to have the meaning of once every three weeks as is accepted in the art.

[0313] In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 1 mg / kg (e.g., by intravenous injection or infusion). In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 2 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 3 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 4 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 5 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 6 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 7 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 8 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 9 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 10 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 12 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered monthly at a dose of approximately 15 mg / kg. In some embodiments, the aforementioned doses are formulated for intravenous injection. As used herein, "monthly" is understood to have the meaning of "once a month" as accepted in the art.

[0314] In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 2 mg / kg over two weeks, and then every other week at a dose of approximately 2 mg / kg (e.g., by intravenous injection or infusion). In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 2 mg / kg over three weeks, and then every other week at a dose of approximately 2 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 2 mg / kg over four weeks, and then every other week at a dose of approximately 2 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 2 mg / kg over two weeks, and then every month at a dose of approximately 2 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 2 mg / kg over three weeks, and then every other week at a dose of approximately 2 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of this disclosure is administered weekly at a dose of approximately 2 mg / kg over a period of 4 weeks, and then monthly at a dose of approximately 2 mg / kg. In some embodiments, the aforementioned doses are formulated for intravenous injection.

[0315] In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 5 mg / kg over two weeks, and then every other week at a dose of approximately 5 mg / kg (e.g., by intravenous injection or infusion). In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 5 mg / kg over three weeks, and then every other week at a dose of approximately 5 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 5 mg / kg over four weeks, and then every other week at a dose of approximately 5 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 5 mg / kg over two weeks, and then every month at a dose of approximately 5 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 5 mg / kg over three weeks, and then every other week at a dose of approximately 5 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the present disclosure is administered weekly at a dose of approximately 5 mg / kg over a period of 4 weeks, and then monthly at a dose of approximately 5 mg / kg. In some embodiments, the aforementioned doses are formulated for intravenous injection.

[0316] In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 10 mg / kg over two weeks, and then every other week at a dose of approximately 10 mg / kg (e.g., by intravenous injection or infusion). In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 10 mg / kg over three weeks, and then every other week at a dose of approximately 10 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 10 mg / kg over four weeks, and then every other week at a dose of approximately 10 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 10 mg / kg over two weeks, and then every month at a dose of approximately 10 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of the Disclosure is administered weekly at a dose of approximately 10 mg / kg over three weeks, and then every other week at a dose of approximately 10 mg / kg. In some embodiments, the RNase-Fc fusion protein or composition of this disclosure is administered weekly at a dose of approximately 10 mg / kg over a period of 4 weeks, and then monthly at a dose of approximately 10 mg / kg. In some embodiments, the aforementioned doses are formulated for intravenous injection.

[0317] As is understood in the art, weekly, bi-weekly, quarterly, or monthly administration may be performed as described above, either once or multiple times, or in secondary doses.

[0318] In one embodiment, the effective amount of RNase-Fc fusion protein is approximately 2 mg / kg per human subject per week. In one embodiment, the effective amount of RNase-Fc fusion protein is approximately 3 mg / kg per human subject per week. In one embodiment, the effective amount of RNase-Fc fusion protein is approximately 4 mg / kg per human subject per week. In one embodiment, the effective amount of RNase-Fc fusion protein is approximately 5 mg / kg per human subject per week. In one embodiment, the effective amount of RNase-Fc fusion protein is approximately 6 mg / kg per human subject per week. In one embodiment, the effective amount of RNase-Fc fusion protein is approximately 7 mg / kg per human subject per week. In one embodiment, the effective amount of RNase-Fc fusion protein is approximately 8 mg / kg per human subject per week. In one embodiment, the effective amount of RNase-Fc fusion protein is approximately 9 mg / kg per human subject per week. In one embodiment, the effective amount of RNase-Fc fusion protein is approximately 10 mg / kg per human subject per week. In some embodiments, the aforementioned doses are formulated for intravenous injection.

[0319] In one embodiment, the effective dose of RNase-Fc fusion protein is approximately 2 mg / kg per human subject every two weeks. In one embodiment, the effective dose of RNase-Fc fusion protein is approximately 3 mg / kg per human subject every two weeks. In one embodiment, the effective dose of RNase-Fc fusion protein is approximately 4 mg / kg per human subject every two weeks. In one embodiment, the effective dose of RNase-Fc fusion protein is approximately 5 mg / kg per human subject every two weeks. In one embodiment, the effective dose of RNase-Fc fusion protein is approximately 6 mg / kg per human subject every two weeks. In one embodiment, the effective dose of RNase-Fc fusion protein is approximately 7 mg / kg per human subject every two weeks. In one embodiment, the effective dose of RNase-Fc fusion protein is approximately 8 mg / kg per human subject every two weeks. In one embodiment, the effective dose of RNase-Fc fusion protein is approximately 9 mg / kg per human subject every two weeks. In one embodiment, the effective dose of RNase-Fc fusion protein is approximately 10 mg / kg per human subject every two weeks. In some embodiments, the aforementioned doses are formulated for intravenous injection.

[0320] Methods and Uses of Inflammation-Related Molecules Methods and uses for the diagnosis and treatment of inflammation-related molecules (e.g., inflammation-related genes, inflammation-related proteins, pro-inflammatory molecules) described herein are provided herein. Methods for identifying subjects with Sjögren's disease who may respond to treatment with RNA nuclease agents described herein by detecting the presence of one or more inflammation-related molecules in a sample obtained from the subject, or by determining the amount or expression level (e.g., amount or expression level) thereof, wherein the presence or amount or expression level of one or more inflammation-related molecules indicates that the subject may respond to treatment with RNA nuclease agents.

[0321] Inflammation-related molecules In some embodiments, the Disclosure provides a method for detecting the presence of inflammation-related molecules (e.g., inflammation-related genes) in a sample derived from a subject (e.g., a sample derived from a subject with Sjögren's syndrome), or for determining the amount or expression level thereof. In some embodiments, the Disclosure provides a method for identifying a subject with Sjögren's disease as a candidate for treatment with an RNase nuclease (e.g., RSLV-132) by determining an inflammation-related gene expression profile in a sample obtained from a subject, and comparing the inflammation-related gene expression profile determined in the sample obtained from the subject with an inflammation-related gene expression profile in a sample obtained from a suitable control subject, wherein the inflammation-related gene expression profile indicates that the subject is a candidate for treatment with an RNA nuclease (e.g., RSLV-132).

[0322] As used herein, the term “inflammation-related molecule” refers to a molecule that functions in inflammation or an inflammatory response. In some embodiments, the inflammation-related molecule is a pro-inflammatory molecule. In some embodiments, the inflammation-related molecule is an anti-inflammatory molecule. In some embodiments, the inflammation-related molecule is an inflammation mediator. In some embodiments, the inflammation-related molecule is an inflammation-related protein. In some embodiments, the inflammation-related molecule is an inflammation-related cytokine. In some embodiments, the inflammation-related molecule is an inflammation-related gene.

[0323] In some embodiments, inflammation-related genes include IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4, STAT5B, CXCL10 (IP-10), CD163, RIPK2, and / or CCR2.

[0324] In some embodiments, inflammation-related genes include IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4, and / or STAT5B.

[0325] In some embodiments, the inflammation-related genes are CXCL10(IP-10), CD163, RIPK2, and / or CCR2.

[0326] In some embodiments, the inflammation-related gene is IL-5.

[0327] In some embodiments, the inflammation-related gene is the TNF receptor.

[0328] In some embodiments, the inflammation-related gene is the IL-6 receptor.

[0329] In some embodiments, the inflammation-related gene is an IL-1 accessory protein.

[0330] In some embodiments, the inflammation-related gene is CXCL1.

[0331] In some embodiments, the inflammation-related gene is the IL-17 receptor A.

[0332] In some embodiments, the inflammation-related gene is LTBR4.

[0333] In some embodiments, the inflammation-related gene is STAT5B.

[0334] In some embodiments, the inflammation-related gene is CXCL10(IP-10).

[0335] In some embodiments, the inflammation-related gene is CD163.

[0336] In some embodiments, the inflammation-related gene is RIPK2.

[0337] In some embodiments, the inflammation-related gene is CCR2. In some embodiments, the inflammation-related gene is IL5, which is the gene encoding the protein "interleukin 5 (IL-5)". In some embodiments, the inflammation-related gene is TNFRSF1A, which encodes the protein "TNF receptor superfamily member 1A". In some embodiments, the inflammation-related gene is IL6R, a gene that codes for the protein "interleukin-6 receptor (IL-6 receptor)". In some embodiments, the inflammation-related gene is IL1RAP, which encodes the protein "interleukin-1 receptor accessory protein".

[0338] In some embodiments, the inflammation-related gene is CXCL1, which is the gene encoding the protein "CXC motif chemocacin ligand 1 (CXCL1)". In some embodiments, the inflammation-related gene is IL17RA, which encodes the protein "interleukin-17 receptor A". In some embodiments, the inflammation-related gene is LTB4R, a gene that encodes the protein "leukotriene B4 receptor". In some embodiments, the inflammation-related gene is STAT5B, a gene that encodes the protein "signaling and transcriptional activator 5B (transcription factor STAT 5B)". In some embodiments, the inflammation-related gene is CXCL10, which is the gene encoding the protein "CXC motif chemocacin ligand 10 (IP-10)". In some embodiments, the inflammation-related gene is CD163, which is the gene encoding the protein "CD163". In some embodiments, the inflammation-related gene is RIPK2, a gene that encodes the protein "receptor-interacting serine / threonine kinase 2". In some embodiments, the inflammation-related gene is CCR2, the gene that encodes the protein "CC motif chemocakine receptor 2 (CCR2)".

[0339] In some embodiments, the inflammation-related genes are IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R, STAT5B, CXCL10, CD163, RIPK2, and / or CCR2.

[0340] In some embodiments, the inflammation-related genes are IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R, and / or STAT5B.

[0341] In some embodiments, the inflammation-related genes are CXCL10, CD163, RIPK2, and / or CCR2.

[0342] In some embodiments, inflammation-related genes include APOL3, HGF, TBC1D23, SETD6, CCR2, CD47, CD163, CD36, CYBB, PLA2G7, IDO1, RIPK2, ACER3, CXCL10, AIMP1, BIRC3, SNX4, PTPN2, VAMP7, APPL1, CSF1, GBA, GPS2, AKT1, MAPKAPK2, PGLYRP1, NUPR1, TNFRSF1A, MAPK13, ORM 2, CCN3, F11R, NFAM1, IL17RA, MMP25, ADAM8, NDST1, FOS, NLRP12, PIK3CD, IL1RAP, IL1R2, STAT5B, TREM1, SIRPA, IL6R, SLC11A1, LTB4R, BCL6, MMP9, FPR1, FPR2, TBXA2R, NOD2, IL1RN, IL5, CXCL1, TPST1, ZC3H12A, TYROBP, and / or CDK19.

[0343] In some embodiments, the inflammation-related gene is APOL3. In some embodiments, the inflammation-related gene is HGF. In some embodiments, the inflammation-related gene is TBC1D23. In some embodiments, the inflammation-related gene is SETD6. In some embodiments, the inflammation-related gene is CCR2. In some embodiments, the inflammation-related gene is CD47. In some embodiments, the inflammation-related gene is CD163. In some embodiments, the inflammation-related gene is CD36. In some embodiments, the inflammation-related gene is CYBB. In some embodiments, the inflammation-related gene is PLA2G7. In some embodiments, the inflammation-related gene is IDO1. In some embodiments, the inflammation-related gene is RIPK2. In some embodiments, the inflammation-related gene is ACER3. In some embodiments, the inflammation-related gene is CXCL10. In some embodiments, the inflammation-related gene is AIMP1. In some embodiments, the inflammation-related gene is BIRC3. In some embodiments, the inflammation-related gene is SNX4. In some embodiments, the inflammation-related gene is PTPN2. In some embodiments, the inflammation-related gene is VAMP7. In some embodiments, the inflammation-related gene is APPL1. In some embodiments, the inflammation-related gene is CSF1. In some embodiments, the inflammation-related gene is GBA. In some embodiments, the inflammation-related gene is GPS2. In some embodiments, the inflammation-related gene is AKT1. In some embodiments, the inflammation-related gene is MAPKAPK2. In some embodiments, the inflammation-related gene is PGLYRP1. In some embodiments, the inflammation-related gene is NUPR1. In some embodiments, the inflammation-related gene is TNFRSF1A. In some embodiments, the inflammation-related gene is MAPK13. In some embodiments, the inflammation-related gene is ORM2. In some embodiments, the inflammation-related gene is CCN3. In some embodiments, the inflammation-related gene is F11R. In some embodiments, the inflammation-related gene is NFAM1. In some embodiments, the inflammation-related gene is IL17RA. In some embodiments, the inflammation-related gene is MMP25.In some embodiments, the inflammation-related gene is ADAM8. In some embodiments, the inflammation-related gene is NDST1. In some embodiments, the inflammation-related gene is FOS. In some embodiments, the inflammation-related gene is NLRP12. In some embodiments, the inflammation-related gene is PIK3CD. In some embodiments, the inflammation-related gene is IL1RAP. In some embodiments, the inflammation-related gene is IL1R2. In some embodiments, the inflammation-related gene is STAT5B. In some embodiments, the inflammation-related gene is TREM1. In some embodiments, the inflammation-related gene is SIRPA. In some embodiments, the inflammation-related gene is IL6R. In some embodiments, the inflammation-related gene is SLC11A1. In some embodiments, the inflammation-related gene is LTB4R. In some embodiments, the inflammation-related gene is BCL6. In some embodiments, the inflammation-related gene is MMP9. In some embodiments, the inflammation-related gene is FPR1. In some embodiments, the inflammation-related gene is FPR2. In some embodiments, the inflammation-related gene is TBXA2R. In some embodiments, the inflammation-related gene is NOD2. In some embodiments, the inflammation-related gene is IL1RN. In some embodiments, the inflammation-related gene is IL5. In some embodiments, the inflammation-related gene is CXCL1. In some embodiments, the inflammation-related gene is TPST1. In some embodiments, the inflammation-related gene is ZC3H12A. In some embodiments, the inflammation-related gene is TYROBP. In some embodiments, the inflammation-related gene is CDK19.

[0344] In some embodiments, inflammation-related genes include STAT1, STAT2, ZNF606, TRIM37, ACKR3, and / or MAP3K8.

[0345] In some embodiments, the inflammation-related genes are STAT1 and STAT2.

[0346] In some embodiments, the inflammation-related gene is STAT1.

[0347] In some embodiments, the inflammation-related gene is STAT2.

[0348] In some embodiments, the inflammation-related genes are ZNF606 and TRIM37.

[0349] In some embodiments, the inflammation-related gene is ZNF606.

[0350] In some embodiments, the inflammation-related gene is TRIM37.

[0351] In some embodiments, the inflammation-related genes are ACKR3 and MAP3K8.

[0352] In some embodiments, the inflammation-related gene is ACKR3.

[0353] In some embodiments, the inflammation-related gene is MAP3K8.

[0354] In some embodiments, the inflammation-related gene is "STAT1," which is the gene encoding the protein "signaling factor and transcriptional activator 1," a key mediator in the cytokine signaling pathway.

[0355] In some embodiments, the inflammation-related gene is "STAT2," which is the gene encoding the protein "signaling factor and transcriptional activator 2," a key mediator in the cytokine signaling pathway.

[0356] In some embodiments, the inflammation-related gene is "ZNF606," which is the gene encoding "zinc finger protein 606," a protein that functions in the host response to viral infection.

[0357] In some embodiments, the inflammation-related gene is "TRIM37," which is a gene encoding "TRIM37," a protein that functions in the class I MHC-mediated antigen presentation pathway.

[0358] In some embodiments, the inflammation-related gene is "MAP3K8," which is the gene encoding "mitogen-activated protein kinase kinase 8," a protein that induces NFκB, TNF, IL-2, and TLR4 signaling.

[0359] In some embodiments, "ACKR3" refers to the gene encoding the protein "atypical chemokine receptor 3," which functions as a receptor for CXCL11 and CXCL12.

[0360] In some embodiments, inflammation-related genes include CRELD1, PARVG, ACAP1, RXRB, COX19, CERS4, B4GALT7, ZNF329, ZFAND2B, NELFB, EMD, UBTF, PYCR2, RNF216, SEC24C, NUMA1, CARD11, EMG1, ZNF576, TRAF2, MAP2K7, CDK4, KHDC4, GIPC1, ILF3, GBP4, FCER1G, STAT1, STAT2, DTX3L, EPSTI1, PARP9, TRIM22, SP140, TRIM5, PSMB9, MAP3K8, ACOT9, XRCC2, KLF5, NBPF10, PRUNE2, LACTB, FAM241A, CCDC169, KLHL33, KDM1B, FANCL, MR1, and / or TRIM13.

[0361] In some embodiments, inflammation-related genes include PLCB1, EFHC2, RING1, REV1, HIBADH, C2ORF68, PPP2R2A, HADHA, ENY2, ZNF671, ERP29, TOB1, NUDT16L1, ZNF329, ZFAND2B, YIPF2, SNUPN, ZNF606, ELAC1, ECI1, HAX1, PFDN6, COQ8B, GOLGA8N, T These are OMM7, PIK3C2B, LOXHD1, FAM122C, IGHD, SYS1, OR2A42, OR2A1, IL4R, GRB10, RAB20, MOB3C, KLHL33, USF3, PFFIBP1, CD40, PLEKHA2, ABL2, PI3, TIMELESS, CLHC1, KMT5A, BCL7A, HACE1, TRIM37, and / or C5ORF22.

[0362] In some embodiments, inflammation-related genes include KHDC4, PMS2, GIMAP1-GIMAP5, SLC25A25, EML2, ZNF790, VSIG1, AXIN2, DHRS3, TESPA1, RGPD5, SPOUT1, TRAF3IP3, RPL13A, NUDT16L1, ACKR3, TFPT, SPAG7, TOB1, ZFAND2B, ZNF329, UBTF, HIC2, TR These are MT61A, ZNF324B, PRKCE, PLEKHA2, BCL7A, ZNF608, TIMELESS, FCHSD2, SMG7, ATXN1, CNNM2, SIPA1L2, CDKL5, TSKU, GGA3, TESK2, BTN2A2, UBXN7, CHP2, MAP3K8, POU5F2, NF1, XRCC2, NME9, KLHL33, MR1, and / or USF3.

[0363] In some embodiments, inflammation-related genes include CRELD1, PARVG, ACAP1, RXRB, COX19, CERS4, B4GALT7, ZNF329, ZFAND2B, NELFB, EMD, UBTF, PYCR2, RNF216, SEC24C, NUMA1, CARD11, EMG1, ZNF576, TRAF2, MAP2K7, CDK4, KHDC4, GIPC1, ILF3, GBP4, FCER1G, STAT1, STAT2, DTX3L, EPSTI1, PARP9, TRIM22, SP140, TRIM5, PSMB9, MAP3K 8, ACOT9, PPP2R2A, HADHA, ENY2, ZNF671, ERP29, TOB1, NUDT16L1, ZNF329, ZFAND2B, YIPF2, SNUPN, ZNF606, ELAC1, ECI1, HAX1, PFDN6, COQ8B, GOLGA8N, TOMM7, P IK3C2B, LOXHD1, FAM122C, IGHD, SYS1, OR2A42, OR2A1, IL4R, GRB10, RAB20, MOB3C, KLHL33, USF3, PFFIBP1, CD40, PLEKHA2, ABL2, PI3, TIMELESS, CLHC 1, KMT5A, BCL7A, HACE1, TRIM37, C5ORF22, KHDC4, PMS2, GIMAP1-GIMAP5, SLC25A25, EML2, ZNF790, VSIG1, AXIN2, DHRS3, ESPA1, RGPD5, SPOUT1, TRAF3 These are IP3, RPL13A, NUDT16L1, ACKR3, TFPT, SPAG7, TOB1, ZFAND2B, ZNF329, UBTF, HIC2, TRMT61A, ZNF324B, PRKCE, PLEKHA2, BCL7A, ZNF608, TIMELESS, FCHSD2, SMG7, ATXN1, CNNM2, SIPA1L2, CDKL5, TSKU, GGA3, TESK2, BTN2A2, UBXN7, CHP2, MAP3K8, POU5F2, NF1, XRCC2, NME9, KLHL33, MR1, and / or USF3.

[0364] In some embodiments, the inflammation-related gene is CRELD1. In some embodiments, the inflammation-related gene is PARVG. In some embodiments, the inflammation-related gene is ACAP1. In some embodiments, the inflammation-related gene is RXRB. In some embodiments, the inflammation-related gene is COX19. In some embodiments, the inflammation-related gene is CERS4. In some embodiments, the inflammation-related gene is B4GALT7. In some embodiments, the inflammation-related gene is ZNF329. In some embodiments, the inflammation-related gene is ZFAND2B. In some embodiments, the inflammation-related gene is NELFB. In some embodiments, the inflammation-related gene is EMD. In some embodiments, the inflammation-related gene is UBTF. In some embodiments, the inflammation-related gene is PYCR2. In some embodiments, the inflammation-related gene is RNF216. In some embodiments, the inflammation-related gene is SEC24C. In some embodiments, the inflammation-related gene is NUMA1. In some embodiments, the inflammation-related gene is CARD11. In some embodiments, the inflammation-related gene is EMG1. In some embodiments, the inflammation-related gene is ZNF576. In some embodiments, the inflammation-related gene is TRAF2. In some embodiments, the inflammation-related gene is MAP2K7. In some embodiments, the inflammation-related gene is CDK4. In some embodiments, the inflammation-related gene is KHDC4. In some embodiments, the inflammation-related gene is GIPC1. In some embodiments, the inflammation-related gene is ILF3. In some embodiments, the inflammation-related gene is GBP4. In some embodiments, the inflammation-related gene is FCER1G. In some embodiments, the inflammation-related gene is STAT1. In some embodiments, the inflammation-related gene is STAT2. In some embodiments, the inflammation-related gene is DTX3L. In some embodiments, the inflammation-related gene is EPSTI1. In some embodiments, the inflammation-related gene is PARP9. In some embodiments, the inflammation-related gene is TRIM22. In some embodiments, the inflammation-related gene is SP140. In some embodiments, the inflammation-related gene is TRIM5.In some embodiments, the inflammation-related gene is PSMB9. In some embodiments, the inflammation-related gene is MAP3K8. In some embodiments, the inflammation-related gene is ACOT9. In some embodiments, the inflammation-related gene is XRCC2. In some embodiments, the inflammation-related gene is KLF5. In some embodiments, the inflammation-related gene is NBPF10. In some embodiments, the inflammation-related gene is PRUNE2. In some embodiments, the inflammation-related gene is LACTB. In some embodiments, the inflammation-related gene is FAM241A. In some embodiments, the inflammation-related gene is CCDC169. In some embodiments, the inflammation-related gene is KLHL33. In some embodiments, the inflammation-related gene is KDM1B. In some embodiments, the inflammation-related gene is FANCL. In some embodiments, the inflammation-related gene is MR1. In some embodiments, the inflammation-related gene is TRIM13. In some embodiments, the inflammation-related gene is PLCB1. In some embodiments, the inflammation-related gene is EFHC2. In some embodiments, the inflammation-related gene is RING1. In some embodiments, the inflammation-related gene is REV1. In some embodiments, the inflammation-related gene is HIBADH. In some embodiments, the inflammation-related gene is C2ORF68. In some embodiments, the inflammation-related gene is PPP2R2A. In some embodiments, the inflammation-related gene is HADHA. In some embodiments, the inflammation-related gene is ENY2. In some embodiments, the inflammation-related gene is ZNF671. In some embodiments, the inflammation-related gene is ERP29. In some embodiments, the inflammation-related gene is TOB1. In some embodiments, the inflammation-related gene is NUDT16L1. In some embodiments, the inflammation-related gene is ZNF329. In some embodiments, the inflammation-related gene is ZFAND2B. In some embodiments, the inflammation-related gene is YIPF2. In some embodiments, the inflammation-related gene is SNUPN. In some embodiments, the inflammation-related gene is ZNF606. In some embodiments, the inflammation-related gene is ELAC1. In some embodiments, the inflammation-related gene is ECI1.In some embodiments, the inflammation-related gene is HAX1. In some embodiments, the inflammation-related gene is PFDN6. In some embodiments, the inflammation-related gene is COQ8B. In some embodiments, the inflammation-related gene is GOLGA8N. In some embodiments, the inflammation-related gene is TOMM7. In some embodiments, the inflammation-related gene is PIK3C2B. In some embodiments, the inflammation-related gene is LOXHD1. In some embodiments, the inflammation-related gene is FAM122C. In some embodiments, the inflammation-related gene is IGHD. In some embodiments, the inflammation-related gene is SYS1. In some embodiments, the inflammation-related gene is OR2A42. In some embodiments, the inflammation-related gene is OR2A1. In some embodiments, the inflammation-related gene is IL4R. In some embodiments, the inflammation-related gene is GRB10. In some embodiments, the inflammation-related gene is RAB20. In some embodiments, the inflammation-related gene is MOB3C. In some embodiments, the inflammation-related gene is KLHL33. In some embodiments, the inflammation-related gene is USF3. In some embodiments, the inflammation-related gene is PFFIBP1. In some embodiments, the inflammation-related gene is CD40. In some embodiments, the inflammation-related gene is PLEKHA2. In some embodiments, the inflammation-related gene is ABL2. In some embodiments, the inflammation-related gene is PI3. In some embodiments, the inflammation-related gene is TIMELESS. In some embodiments, the inflammation-related gene is CLHC1. In some embodiments, the inflammation-related gene is KMT5A. In some embodiments, the inflammation-related gene is BCL7A. In some embodiments, the inflammation-related gene is HACE1. In some embodiments, the inflammation-related gene is TRIM37. In some embodiments, the inflammation-related gene is C5ORF22. In some embodiments, the inflammation-related gene is KHDC4. In some embodiments, the inflammation-related gene is PMS2. In some embodiments, the inflammation-related gene is GIMAP1-GIMAP5. In some embodiments, the inflammation-related gene is SLC25A25. In some embodiments, the inflammation-related gene is EML2.In some embodiments, the inflammation-related gene is ZNF790. In some embodiments, the inflammation-related gene is VSIG1. In some embodiments, the inflammation-related gene is AXIN2. In some embodiments, the inflammation-related gene is DHRS3. In some embodiments, the inflammation-related gene is ESPA1. In some embodiments, the inflammation-related gene is RGPD5. In some embodiments, the inflammation-related gene is SPOUT1. In some embodiments, the inflammation-related gene is TRAF3IP3. In some embodiments, the inflammation-related gene is RPL13A. In some embodiments, the inflammation-related gene is NUDT16L1. In some embodiments, the inflammation-related gene is ACKR3. In some embodiments, the inflammation-related gene is TFPT. In some embodiments, the inflammation-related gene is SPAG7. In some embodiments, the inflammation-related gene is TOB1. In some embodiments, the inflammation-related gene is ZFAND2B. In some embodiments, the inflammation-related gene is ZNF329. In some embodiments, the inflammation-related gene is UBTF. In some embodiments, the inflammation-related gene is HIC2. In some embodiments, the inflammation-related gene is TRMT61A. In some embodiments, the inflammation-related gene is ZNF324B. In some embodiments, the inflammation-related gene is PRKCE. In some embodiments, the inflammation-related gene is PLEKHA2. In some embodiments, the inflammation-related gene is BCL7A. In some embodiments, the inflammation-related gene is ZNF608. In some embodiments, the inflammation-related gene is TIMELESS. In some embodiments, the inflammation-related gene is FCHSD2. In some embodiments, the inflammation-related gene is SMG7. In some embodiments, the inflammation-related gene is ATXN1. In some embodiments, the inflammation-related gene is CNNM2. In some embodiments, the inflammation-related gene is SIPA1L2. In some embodiments, the inflammation-related gene is CDKL5. In some embodiments, the inflammation-related gene is TSKU. In some embodiments, the inflammation-related gene is GGA3. In some embodiments, the inflammation-related gene is TESK2. In some embodiments, the inflammation-related gene is BTN2A2.In some embodiments, the inflammation-related gene is UBXN7. In some embodiments, the inflammation-related gene is CHP2. In some embodiments, the inflammation-related gene is MAP3K8. In some embodiments, the inflammation-related gene is POU5F2. In some embodiments, the inflammation-related gene is NF1. In some embodiments, the inflammation-related gene is XRCC2. In some embodiments, the inflammation-related gene is NME9. In some embodiments, the inflammation-related gene is KLHL33. In some embodiments, the inflammation-related gene is MR1. In some embodiments, the inflammation-related gene is USF3.

[0365] Accordingly, in some embodiments, the present disclosure provides a method for treating Sjögren's disease in a patient in need thereof, comprising the step of administering an effective amount of an RNA nuclease agent (e.g., RSLV-132) to the patient, wherein the treatment results in a reduction of one or more inflammation-related genes. In some embodiments, the inflammation-related genes are IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4 and / or STAT5B. In some embodiments, the inflammation-related genes are IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R and / or STAT5B.

[0366] Accordingly, in some embodiments, the present disclosure provides a method for treating Sjögren's disease in a patient in need thereof, comprising the step of administering an effective amount of an RNA nuclease agent (e.g., RSLV-132) to the patient, wherein the treatment results in a reduction of one or more inflammation-related genes and an improvement in fatigue. In some embodiments, the inflammation-related genes are IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4 and / or STAT5B. In some embodiments, the inflammation-related genes are IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R and / or STAT5B.

[0367] In some embodiments, the present disclosure provides a method for treating Sjögren's disease in a patient in need thereof, comprising the step of administering an effective amount of an RNA nuclease agent (e.g., RSLV-132) to the patient, wherein the treatment results in an increase in one or more inflammation-related genes. In some embodiments, the inflammation-related genes are CXCL10 (IP-10), CD163, RIPK2 and / or CCR2.

[0368] In some embodiments, the present disclosure provides a method for treating Sjögren's disease in a patient in need, comprising the step of administering an effective amount of an RNA nuclease agent (e.g., RSLV-132) to the patient, wherein the treatment results in an increase in one or more inflammation-related genes and an improvement in fatigue. In some embodiments, the inflammation-related genes are CXCL10 (IP-10), CD163, RIPK2 and / or CCR2.

[0369] In some embodiments, the present disclosure provides a method for treating Sjögren's disease in a patient in need, comprising the step of administering an effective amount of an RNA nuclease agent (e.g., RSLV-132) to the patient, wherein the treatment results in an increase of one or more cytokines. In some embodiments, the cytokine is CXCL10 (IP-10).

[0370] In some embodiments, the present disclosure provides a method for treating Sjögren's disease in a patient in need, comprising the step of administering an effective amount of an RNA nuclease agent (e.g., RSLV-132) to the patient, wherein the treatment results in an increase of one or more cytokines and an improvement in fatigue. In some embodiments, the cytokine is CXCL10 (IP-10).

[0371] In some embodiments, the Disclosure provides a method for identifying a subject with Sjögren's disease as a candidate for RNA nuclease treatment by determining an inflammation-related gene expression profile in a sample obtained from a subject and comparing the inflammation-related gene expression profile from a subject with Sjögren's disease with an inflammation-related gene expression profile from a sample obtained from a suitable control subject, wherein the inflammation-related gene expression profile indicates that the subject is a candidate for RNA nuclease treatment. In some embodiments, the inflammation-related genes include MAP3K8, ACKR3, STAT1, STAT2, TRIM37, and / or ZNF606.

[0372] Accordingly, in some embodiments, the Disclosure provides a method for identifying subjects with Sjögren's syndrome who may respond to treatment with an RNA nuclease agent (e.g., RSLV-132) described herein by detecting the presence of inflammation-related molecules (e.g., inflammation-related genes) in a sample obtained from a subject, wherein the presence of inflammation-related molecules (e.g., inflammation-related genes) indicates that the subject may respond to treatment with the agent. In some embodiments, the amount or expression level of inflammation-related molecules (e.g., inflammation-related genes) in the sample is determined and compared to a reference amount or reference expression level of inflammation-related molecules (e.g., inflammation-related genes). In some embodiments, if the amount or expression level of inflammation-related molecules (e.g., inflammation-related genes) in the sample is increased compared to a reference amount or reference expression level of inflammation-related molecules (e.g., inflammation-related genes), the patient may respond to treatment with an RNA nuclease agent disclosed herein. In some embodiments, if the amount or expression level of inflammation-related molecules (e.g., inflammation-related genes) in a sample is reduced compared to a reference amount or reference expression level of inflammation-related molecules (e.g., inflammation-related genes), the patient may respond to treatment with the RNA nuclease agents disclosed herein.

[0373] In some embodiments, this disclosure provides a method for identifying patients who may respond to treatment with an RNA nuclease agent (e.g., RSLV-132) described herein, wherein a patient-derived sample is contacted with a nucleic acid probe that hybridizes to a complementary target sequence in the DNA or RNA of an inflammation-related molecule (e.g., an inflammation-related gene), thereby forming a hybridization complex between the nucleic acid probe and the DNA or RNA of the inflammation-related molecule (e.g., an inflammation-related gene). To detect the hybridization of the probe to the target DNA or RNA sequence of the inflammation-related molecule (e.g., an inflammation-related gene), the probe is labeled with a molecular marker; for example, a radiomarker, a fluorescent marker, an enzyme marker, or digoxigenin. In some embodiments, the presence of a probe-target complex indicates that the patient may respond to treatment. In some embodiments, the amount of the probe-target complex in the sample is compared to a control. In some embodiments, if the amount of the probe-target complex in the sample is increased compared to a control, the patient may respond to treatment with an RNA nuclease agent (e.g., RSLV-132). In some embodiments, if the amount of probe-target complex in the sample is reduced compared to the control, the patient may respond to treatment with an RNA nuclease agent (e.g., RSLV-132).

[0374] In some embodiments, the Disclosure provides a method for identifying patients with Sjögren's syndrome who may respond to treatment with an RNA nuclease agent (e.g., RSLV-132) described herein, wherein a patient-derived sample is contacted with an antibody or its antigen-binding fragment that specifically binds to inflammation-related molecules (e.g., inflammation-related proteins), thereby forming a complex with the inflammation-related molecules, the presence of which is detected, and the presence of the complex indicates that the patient may respond to treatment. In some embodiments, the amount of antibody-inflammation-related molecule complex in the sample is compared to a control. In some embodiments, if the amount of antibody-inflammation-related molecule complex in the sample is increased compared to a control, the patient may respond to treatment with an RNA nuclease agent (e.g., RSLV-132). In some embodiments, if the amount of antibody-inflammation-related molecule complex in the sample is decreased compared to a control, the patient may respond to treatment with an RNA nuclease agent (e.g., RSLV-132).

[0375] Diagnostic methods This disclosure provides a method relating to the step of detecting and / or quantifying one or more inflammation-related molecules (e.g., inflammation-related genes) (e.g., STAT1, STAT2, ZNF606, TRIM37, ACKR3, and / or MAP3K8) described herein in one or more samples, wherein the detection and / or quantification of one or more individual or combined inflammation-related molecules would indicate that an RNA nuclease agent (e.g., RSLV-132) is likely to provide therapeutic effects or benefits to patients with Sjögren's disease.

[0376] A method is provided herein for identifying subjects with Sjögren's disease as candidates for treatment with an RNA nuclease, by determining the inflammation-related gene expression profile in a sample obtained from a subject and comparing the inflammation-related gene expression profile from a subject with Sjögren's disease with the inflammation-related gene expression profile from a sample obtained from a suitable control subject, wherein the inflammation-related gene expression profile indicates that the subject is a candidate for treatment with an RNA nuclease (e.g., RSLV-132). In some embodiments, the inflammation-related genes in the gene expression profile include MAP3K8, ACKR3, STAT1, STAT2, TRIM37, and / or ZNF606.

[0377] A method for identifying a subject with Sjögren's disease as a candidate for treatment with an RNA nuclease is provided herein, by determining the inflammation-related gene expression profile in a sample obtained from the subject and comparing the inflammation-related gene expression profile from the subject with an inflammation-related gene expression profile from a sample obtained from a suitable control subject, wherein if the amount of one or more inflammation-related genes in the sample obtained from the subject with Sjögren's disease is equal to or greater than the amount of one or more inflammation-related genes in the sample obtained from a suitable control subject, the inflammation-related gene expression profile indicates that the subject is a candidate for treatment with an RNA nuclease (e.g., RSLV-132). In some embodiments, the inflammation-related genes in the gene expression profile include ZNF606 and / or ACKR3.

[0378] A method for identifying a subject with Sjögren's disease as a candidate for RNA nuclease treatment is provided herein, by determining the inflammation-related gene expression profile in a sample obtained from the subject and comparing the inflammation-related gene expression profile from the subject with the inflammation-related gene expression profile from a sample obtained from a suitable control subject, wherein if the amount of one or more inflammation-related genes in the sample obtained from the subject with Sjögren's disease is less than the amount of one or more inflammation-related genes in the sample obtained from a suitable control subject, the inflammation-related gene expression profile indicates that the subject is a candidate for RNA nuclease treatment. In some embodiments, the inflammation-related genes in the gene expression profile include STAT1, STAT2, TRIM37 and / or MAP3K8.

[0379] A method for identifying subjects with Sjögren's disease as candidates for RNA nuclease treatment, wherein the inflammation-related gene expression profiles in the profiles include MAP3K8, ACKR3, STAT1, STAT2, TRIM37 and / or ZNF606, and a) Methods are further provided herein that involve any combination of (a), (b), (c), (d), (e), and (f): a) an increase in the expression level of ZNF606 in the sample compared to the control; b) an increase in the expression level of ACKR3 in the sample compared to the control; c) a decrease in the expression level of STAT1 in the sample compared to the control; d) a decrease in the expression level of STAT2 in the sample compared to the control; e) a decrease in the expression level of TRIM37 in the sample compared to the control; f) a decrease in the expression level of MAP3K8 in the sample compared to the control; or g) any combination of (a), (b), (c), (d), (e), and (f).

[0380] A method for identifying patients with Sjögren's disease who are likely to respond to treatment with an RNA nuclease agent (e.g., RSLV-132), comprising the step of determining the amount of one or more inflammation-related molecules (e.g., inflammation-related genes) (e.g., STAT1, STAT2, ZNF606, TRIM37, ACKR3 and / or MAP3K8) in a sample obtained from the patient compared to a reference amount of inflammation-related molecules (e.g., inflammation-related genes), wherein the amount of inflammation-related molecules (e.g., inflammation-related genes) in the sample compared to a reference amount indicates the likelihood that the patient will respond to treatment, is further provided herein.

[0381] A method for identifying patients with Sjögren's disease who may respond to treatment with an RNA nuclease agent (e.g., RSLV-132), comprising the steps of: determining the amount of one or more inflammation-related genes in a sample obtained from the patient; and comparing the amount of one or more inflammation-related genes in the sample to a reference amount of one or more inflammation-related genes, wherein if the amount of one or more inflammation-related genes in the sample is equal to or greater than the reference amount of one or more inflammation-related genes, the patient may respond to treatment, is further provided herein. In some embodiments, the inflammation-related genes are ZNF606 and / or ACKR3.

[0382] A method for identifying patients with Sjögren's disease who may respond to treatment with an RNA nuclease agent (e.g., RSLV-132), comprising the steps of: determining the amount of one or more inflammation-related genes in a sample obtained from the patient; and comparing the amount of one or more inflammation-related genes in the sample to a reference amount of one or more inflammation-related genes, wherein if the amount of one or more inflammation-related genes in the sample is less than the reference amount of one or more inflammation-related genes, the patient may respond to treatment, is further provided herein. In some embodiments, the inflammation-related genes are STAT1, STAT2, TRIM37 and / or MAP3K8.

[0383] A method for identifying patients with Sjögren's disease who may respond to treatment with an RNA nuclease agent (e.g., RSLV-132), comprising the steps of: determining the expression levels of a panel of inflammation-related genes in a patient-derived sample, wherein the panel comprises STAT1, STAT2, ZNF606, TRIM37, ACKR3 and / or MAP3K8; and comparing the expression levels of the panel in the sample to the expression levels of the panel in a control, wherein the amount of inflammation-related genes in the sample compared to the amount of inflammation-related genes in the control indicates the likelihood that the patient will respond to treatment.

[0384] A method for identifying patients with Sjögren's disease who may respond to treatment with an RNA nuclease agent (e.g., RSLV-132), comprising the steps of: determining the expression levels of a panel of inflammation-related genes in a patient-derived sample, wherein the panel comprises STAT1, STAT2, ZNF606, TRIM37, ACKR3 and / or MAP3K8; comparing the expression levels of the panel in the sample to the expression levels of the panel in a control; and identifying patients who may respond to treatment with an RNA nuclease agent, wherein a) in the sample A method comprising the steps of (a), (b), (c), (d), (e), and (f) is further provided herein.

[0385] A method for identifying a patient with Sjögren's disease who may respond to treatment with an RNA nuclease (e.g., RSLV-132), comprising the steps of: contacting a sample with a nucleic acid probe that hybridizes to a complementary target sequence in the DNA or RNA of an inflammation-related molecule (e.g., an inflammation-related gene), thereby forming a hybridization complex between the nucleic acid probe and the DNA or RNA of the inflammation-related molecule (e.g., an inflammation-related gene); and determining the amount of the complex in the sample compared to the amount of the complex in a reference sample, wherein the amount of the complex in the sample compared to the amount of the complex in the reference sample indicates the likelihood that the patient is susceptible to treatment with an RNA nuclease (e.g., RSLV-132).

[0386] A method for identifying a patient with Sjögren's disease who may respond to treatment with an RNA nuclease (e.g., RSLV-132), comprising the steps of: contacting a sample with at least one diagnostic antibody or its antigen-binding fragment that specifically binds to an inflammation-related molecule (e.g., an inflammation-related gene), thereby forming a diagnostic antibody-inflammation-related molecule complex; and determining the amount of the complex in the sample compared to the amount of the complex in a reference sample, wherein the amount of the complex in the sample compared to the amount of the complex in the reference sample indicates the likelihood that the patient is susceptible to treatment with an RNA nuclease (e.g., RSLV-132).

[0387] A method for monitoring the response of a patient with Sjögren's disease treated with an RNA nuclease (e.g., RSLV-132), comprising the steps of: determining the expression level and / or activity of one or more inflammation-related molecules (e.g., inflammation-related genes) in a first patient-derived sample obtained before treatment with the RNA nuclease; determining the expression level and / or activity of one or more inflammation-related molecules (e.g., inflammation-related genes) in a second patient-derived sample obtained after treatment with the RNA nuclease; and determining the expression level and / or activity of one or more inflammation-related molecules in the second sample. A method is further provided herein that includes the step of comparing the expression level and / or activity of a child (e.g., inflammation-related gene) with the expression level and / or activity of one or more inflammation-related molecules (e.g., inflammation-related genes) in a first sample, wherein the change in the expression level and / or activity of one or more inflammation-related molecules (e.g., inflammation-related genes) in a second sample compared with the expression level and / or activity of one or more inflammation-related molecules (e.g., inflammation-related genes) in a first sample indicates a response in a patient treated with an RNA nuclease agent. In some embodiments, one or more inflammation-related molecules are inflammation-related genes. In some embodiments, inflammation-related genes are IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4, STAT5B, CXCL10 (IP-10), CD163, RIPK2 and / or CCR2. In some embodiments, the inflammation-related genes are IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R, STAT5B, CXCL10, CD163, RIPK2, and / or CCR2.

[0388] A method for monitoring the response of a patient with Sjögren's disease treated with an RNA nuclease (e.g., RSLV-132), comprising the steps of: determining the expression level and / or activity of one or more inflammation-related molecules (e.g., inflammation-related genes) in a first patient-derived sample obtained before treatment with the RNA nuclease; determining the expression level and / or activity of one or more inflammation-related molecules (e.g., inflammation-related genes) in a second patient-derived sample obtained after treatment with the RNA nuclease; and determining the expression level and / or activity of one or more inflammation-related molecules in the second sample. A method is further provided herein that includes the step of comparing the expression level and / or activity of a child (e.g., inflammation-related gene) with the expression level and / or activity of one or more inflammation-related molecules (e.g., inflammation-related genes) in a first sample, wherein a decrease in the expression level and / or activity of one or more inflammation-related molecules (e.g., inflammation-related genes) in a second sample compared to the expression level and / or activity of one or more inflammation-related molecules (e.g., inflammation-related genes) in a first sample indicates a response in a patient treated with an RNA nuclease agent. In some embodiments, one or more inflammation-related molecules are inflammation-related genes. In some embodiments, the inflammation-related genes are IL-5, TNF receptor, IL-6 receptor, IL-1 accessory protein, CXCL1, IL-17 receptor A, LTBR4 and / or STAT5B. In some embodiments, the inflammation-related genes are IL5, TNFRSF1A, IL6R, IL1RAP, CXCL1, IL17RA, LTB4R, and / or STAT5B.

[0389] A method for monitoring the response of a patient with Sjögren's disease treated with an RNA nuclease (e.g., RSLV-132), comprising the steps of: determining the expression level and / or activity of one or more inflammation-related molecules (e.g., inflammation-related genes) in a first patient-derived sample obtained before treatment with the RNA nuclease; determining the expression level and / or activ...

Claims

[Claim 1] A method for treating Sjögren's disease in a human patient who requires treatment for Sjögren's disease by reducing fatigue, comprising the step of administering an effective amount of RNase-Fc fusion protein to the patient, thereby treating Sjögren's disease in the patient by reducing fatigue.

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