Therapeutic nuclease composition and method

Abstract

To provide therapeutic nuclease compositions and methods.SOLUTION: The invention provides: hybrid nuclease molecules and methods for treating an immune-related disease or disorder in a mammal; a pharmaceutical composition for treating an immune-related disease in a mammal; and polypeptides comprising a first nuclease domain and a modified Fc domain and having specific sequences.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 61 / 480,961, filed on April 29, 2011, and U.S. Provisional Application No. 61 / 617,241, filed on March 29, 2012. This application also relates to International Patent Application No. PCT / US2010 / 055131, filed on November 2, 2010; U.S. Provisional Application No. 61 / 257,458, filed on November 2, 2009; and U.S. Provisional Application No. 61 / 370,752, filed on August 4, 2010. The entire disclosure of each of the above - mentioned applications is hereby incorporated by reference into this specification for all purposes.

[0002] Statement Regarding Federally Sponsored Research or Development The present invention was made with government support under grants AI44257, NS065933, and AR048796 from the National Institutes of Health, the Alliance for Lupus Research, and the Washington State Life Science Discovery Fund (2087750). The government has certain rights in the invention.

Background Art

[0003] Background Excessive release of (ribo)nucleoprotein particles from dead and dying cells can cause lupus pathogenesis through two mechanisms: (i) deposition or in-situ formation of chromatin / anti-chromatin complexes leads to nephritis and impaired renal function; and (ii) nucleoproteins activate innate immunity via Toll-like receptors (TLRs) 7, 8, and 9, as well as TLR-independent pathways. The release of nucleoproteins may act as potent antigens for autoantibodies in SLE, potentially leading to amplification of B cell and DC activation through antigen receptor and TLR co-engagement. Therefore, there is a demand for means to remove the inducing antigen and / or to mitigate immune stimulation, immune amplification, and immune complex-mediated diseases in those who require it. [Overview of the project]

[0004] overview Disclosed herein are hybrid nuclease molecules comprising a first nuclease domain and a modified Fc domain, wherein the first nuclease domain is functionally connected to the Fc domain. The Fc domain is modified such that the cytotoxic effect of the molecule is reduced compared to a hybrid nuclease molecule having an unmodified Fc domain. In some embodiments, the hybrid nuclease molecule has an Fc domain modified to have reduced binding affinity to an Fcγ receptor, a complement protein, or both. In some embodiments, the cytotoxic effect of the hybrid nuclease molecule is reduced by at least 1 / 2, 1 / 3, 1 / 4, or 1 / 5 compared to a control molecule, e.g., a hybrid nuclease molecule without a modified Fc domain.

[0005] In some embodiments, the hybrid nuclease molecule further comprises a first linker domain, the first nuclease domain functionally connected to the modified Fc domain by the first linker domain.

[0006] In some aspects, the hybrid nuclease molecule contains a modified Fc domain, which is a mutant IgG1 Fc domain. In some aspects, the mutant Fc domain contains one or more mutations within the hinge domain, CH2 domain, and / or CH3 domain. In some aspects, the Fc domain contains an amino acid sequence having one or more of the mutations P238S, P331S, SCC, SSS (residues 220, 226, and 229), G236R, L328R, L234A, and L235A. In some aspects, the mutant Fc domain contains the P238S mutation. In some aspects, the mutant Fc domain contains the P331S mutation. In some aspects, the mutant Fc domain contains both the P238S mutation and the P331S mutation. In some aspects, the mutant Fc domain may include P238S and / or P331S, and also include mutations in one or more of the three cysteines in the hinge region. In some aspects, the mutant Fc domain includes P238S and / or P331S, and / or mutations in one or more of the three cysteines in the hinge region. In some aspects, the mutant Fc domain includes mutations to SCC (where CCC refers to the three cysteines present in the wild-type hinge domain) in P238S and / or P331S, and / or one of the three cysteines in the hinge region (located at residue 220 according to the EU numbering scheme). In some aspects, the mutant Fc domain includes mutations to SSS (located at residues 220, 226, and 229 according to the EU numbering scheme). In some aspects, the mutant Fc domain includes mutations in P238S and P331S, as well as in three cysteine ​​groups in the hinge region. In some aspects, the mutant Fc domain includes P238S, P331S, and SCC. In some aspects, the mutant Fc domain includes P238S, P331S, and SSS.In some aspects, the mutant Fc domain contains P238S and SCC. In some aspects, the mutant Fc domain contains P238S and SSS. In some aspects, the mutant Fc domain contains P331S and SCC. In some aspects, the mutant Fc domain contains P331S and SSS. In some aspects, the mutant Fc domain contains mutations in one or more of the three cysteine ​​molecules in the hinge region. In some aspects, the mutant Fc domain contains mutations in the three cysteine ​​molecules in the hinge region. In some aspects, the mutant Fc domain contains one mutation to SCC in the three cysteine ​​molecules in the hinge region. In some aspects, the mutant Fc domain contains mutations to SSS in the three cysteine ​​molecules in the hinge region. In some aspects, the mutant Fc domain contains SCC. In some aspects, the mutant Fc domain contains SSS.

[0007] In some cases, the nucleic acid encoding the mutant Fc domain is as shown in SEQ ID NO: 59. In some cases, the mutant Fc domain is as shown in SEQ ID NO: 60. In some cases, the nucleic acid encoding the mutant Fc domain is as shown in SEQ ID NO: 71. In some cases, the mutant Fc domain is as shown in SEQ ID NO: 72. In some cases, the nucleic acid encoding the mutant Fc domain is as shown in SEQ ID NO: 73. In some cases, the mutant Fc domain is as shown in SEQ ID NO: 74. In some cases, the nucleic acid encoding the mutant Fc domain is shown in SEQ ID NO: 75. In some cases, the mutant Fc domain is as shown in SEQ ID NO: 76. In some cases, the nucleic acid encoding the mutant Fc domain is as shown in SEQ ID NO: 87. In some cases, the mutant Fc domain is as shown in SEQ ID NO:88. In some cases, the nucleic acid encoding the mutant Fc domain is as shown in SEQ ID NO:89. In some cases, the mutant Fc domain is as shown in SEQ ID NO:90.

[0008] In some aspects, the hybrid nuclease molecule contains a wild-type human RNase1 domain ligated with mutant human IgG1 Fc domains containing SCC, P238S, and P331S, or mutant human IgG1 Fc domains containing SSS, P238S, and P331S. In some aspects, the nucleic acid encoding the hybrid nuclease molecule is as shown in SEQ ID NO: 61, 77, or 91. In some aspects, the hybrid nuclease molecule is as shown in SEQ ID NO: 209, 62, 78, 92, or 94.

[0009] In some aspects, the hybrid nuclease molecule contains a wild-type human RNase1 domain linked via a (Gly4Ser)4 linker domain to mutant human IgG1 Fc domains containing SCC, P238S, and P331S, or to mutant human IgG1 Fc domains containing SSS, P238S, and P331S. In some aspects, the nucleic acid encoding the hybrid nuclease molecule is as shown in SEQ ID NO: 63 or 79. In some aspects, the hybrid nuclease molecule is as shown in SEQ ID NO: 64 or 79.

[0010] In some aspects, the hybrid nuclease molecule includes a human DNase1 G105R A114F domain linked to mutant human IgG1 Fc domains containing SCC, P238S, and P331S via a (Gly4Ser)4 linker domain, and that Fc domain linked to a wild-type human RNase1 domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule includes a human DNase1 G105R A114F domain linked to mutant human IgG1 Fc domains containing SSS, P238S, and P331S via a (Gly4Ser)4 linker domain, and that Fc domain linked to a wild-type human RNase1 domain via an NLG linker domain. In some aspects, the nucleic acid encoding the hybrid nuclease molecule is as shown in SEQ ID NO: 65 or 81. In some aspects, the hybrid nuclease molecule is as shown in SEQ ID NO: 66 or 82.

[0011] In other embodiments, the hybrid nuclease molecule contains the amino acid sequence described in SEQ ID NO: 62, 64, 78, 80, 92, or 96, or contains an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 62, 64, 78, 80, 92, or 96. In some aspects, the hybrid nuclease molecule contains the amino acid sequence described in SEQ ID NO: 96. In other aspects, the hybrid nuclease molecule contains the amino acid sequence described in SEQ ID NO: 66, 68, 70, 82, 84, 86, 94, or 98, or contains an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 66, 68, 70, 82, 84, 86, 94, or 98. In other aspects, the hybrid nuclease molecule contains the amino acid sequence described in SEQ ID NO: 98.

[0012] In some aspects, the hybrid nuclease molecule includes a wild-type human RNase1 domain linked to mutant human IgG1 Fc domains containing SCC, P238S, and P331S via a (Gly4Ser)4 linker domain, and that Fc domain linked to a human DNase1 G105R A114F domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule includes a wild-type human RNase1 domain linked to mutant human IgG1 Fc domains containing SSS, P238S, and P331S via a (Gly4Ser)4 linker domain, and that Fc domain linked to a human DNase1 G105R A114F domain via an NLG linker domain. In some aspects, the nucleic acid encoding the hybrid nuclease molecule is as shown in SEQ ID NO: 67 or 83. In some aspects, the hybrid nuclease molecule is shown in SEQ ID NO: 68 or 84.

[0013] In some aspects, the hybrid nuclease molecule includes a wild-type human RNase1 domain ligated to mutant human IgG1 Fc domains containing SCC, P238S, and P331S, with the Fc domain ligated to the human DNase1 G105R A114F domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule includes a wild-type human RNase1 domain ligated to mutant human IgG1 Fc domains containing SSS, P238S, and P331S, with the Fc domain ligated to the human DNase1 G105R A114F domain via an NLG linker domain. In some aspects, the nucleic acids encoding the hybrid nuclease molecule are shown in SEQ ID NO: 69, 85, or 93. In some aspects, the hybrid nuclease molecule is shown in SEQ ID NO: 70, 86, 94, or 98.

[0014] In some aspects, the cytotoxic effect induced by hybrid nuclease molecules is reduced compared to the control molecule. In some aspects, the cytotoxic effect induced by hybrid nuclease molecules is reduced compared to the control molecule by approximately 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, The cytotoxic effect of hybrid nuclease molecules is reduced by 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%. In some aspects, the cytotoxic effect of hybrid nuclease molecules is reduced to about 1 / 3 to 1 / 5, or at least about 1 / 3, compared to hybrid nuclease molecules with an unmodified Fc domain (e.g., wild-type Fc domain).

[0015] In several aspects, the activity of the DNase-containing hybrid nuclease molecule is approximately 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 11, 1 / 12, 1 / 13, 1 / 14, 1 / 15, 1 / 16, 1 / 17, 1 / 18, 1 / 19, 1 / 20, 1 / 21, 1 / 22, 1 / 23, 1 / 24, 1 / 25, 1 / 26, 1 / 27, 1 / 28, 1 / 29, 1 / 30, or approximately 1 / 30 or more of the activity of the control DNase molecule. In some aspects, the activity of hybrid nuclease molecules containing DNase is approximately equal to that of control DNase molecules. In several aspects, the activity of the RNase-containing hybrid nuclease molecule is approximately 1 / 1, 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 11, 1 / 12, 1 / 13, 1 / 14, 1 / 15, 1 / 16, 1 / 17, 1 / 18, 1 / 19, 1 / 20, 1 / 21, 1 / 22, 1 / 23, 1 / 24, 1 / 25, 1 / 26, 1 / 27, 1 / 28, 1 / 29, 1 / 30, or approximately 1 / 30 or more of the activity of the control RNase molecule. In some aspects, the activity of hybrid nuclease molecules containing RNase is approximately equal to that of control RNase molecules.

[0016] In some embodiments, the hybrid nuclease molecule is a polypeptide in which the amino acid sequence of the first nuclease domain comprises the amino acid sequence of human wild-type RNase, the first linker domain is (Gly4Ser)n, where n is 0, 1, 2, 3, 4, or 5, the amino acid sequence of the Fc domain comprises the amino acid sequence of human mutant IgG1 Fc domain, and the first linker domain is connected to the C-terminus of the first nuclease domain and the N-terminus of the Fc domain. In some embodiments, the hybrid nuclease molecule is a polypeptide comprising or consisting of the sequences shown in Table 1.

[0017] In some embodiments, the hybrid nuclease molecule contains wild-type human DNase1 linked to a mutant human IgG1 Fc domain. In some embodiments, the hybrid nuclease molecule contains (Gly4Ser) nThe hybrid nuclease molecule comprises human DNase1 G105R A114F linked to a mutant human IgG1 Fc domain by a linker domain, where n=0, 1, 2, 3, 4, or 5. In some embodiments, the hybrid nuclease molecule comprises wild-type human RNase1 linked to a mutant human IgG1 Fc domain linked to wild-type human DNase1. In some embodiments, the hybrid nuclease molecule comprises wild-type human RNase1 linked to a mutant human IgG1 Fc domain linked to human DNase1 G105R A114F. In some embodiments, the hybrid nuclease molecule is a polypeptide in which the amino acid sequence of the first nuclease domain comprises the amino acid sequence of RNase, the first linker domain is 5-32 amino acids long, the amino acid sequence of the Fc domain comprises the amino acid sequence of a human Fc domain, and the first linker domain is linked to the C-terminus of the first nuclease domain and the N-terminus of the Fc domain. In some embodiments, the linker domain comprises (Gly4Ser)5 and restriction sites BglII, AgeI, and XhoI. In some embodiments, the hybrid nuclease molecule is a polypeptide in which the amino acid sequence of the first nuclease domain comprises the amino acid sequence of a human RNase, the first linker domain is a 5-32 amino acid length NLG peptide, the amino acid sequence of the Fc domain comprises the amino acid sequence of a human mutant Fc domain, and the first linker domain is connected to the C-terminus of the first nuclease domain and the N-terminus of the Fc domain.

[0018] In some embodiments, the Fc domain does not substantially bind to the Fc receptor on human cells. In some embodiments, the Fc domain is modified to reduce its binding affinity to the Fcγ receptor, complement protein, or both. In some aspects, the binding affinity to the Fc receptor is approximately 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, The binding affinity of hybrid nuclease molecules to Fc receptors is reduced by 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%. In some aspects, the binding affinity of hybrid nuclease molecules to Fc receptors is reduced to 1 / 3 to 1 / 5, or at least about 1 / 3, compared to hybrid nuclease molecules with an unmodified Fc domain (e.g., wild-type Fc domain).

[0019] In some embodiments, the serum half-life of the molecule is significantly longer than the serum half-life of the first nuclease domain alone. In some embodiments, the nuclease activity of the molecule's first nuclease domain is the same as or higher than that of the nuclease domain alone. In some embodiments, administration of the molecule to mice increases mouse survival as measured by assays of a mouse lupus model. In some aspects, hybrid nuclease molecules degrade RNA, DNA, or both in circulating blood. In other aspects, hybrid nuclease molecules degrade RNA, DNA, or both in immune complexes. In some embodiments, hybrid nuclease molecules inhibit interferon-α production. In several aspects, interferon-α production was approximately 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, 4 It has decreased by 8, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%.

[0020] In some embodiments, the hybrid nuclease molecule includes a leader sequence. In some embodiments, the leader sequence is a human VK3LP peptide derived from the human κ light chain family, and the leader sequence is connected to the N-terminus of the first nuclease domain. In some embodiments, VK3LP has the sequence described in SEQ ID NO:100.

[0021] In some embodiments, the molecule is a polypeptide. In some embodiments, the molecule is a polynucleotide.

[0022] In some embodiments, the first nuclease domain comprises an RNase. In some embodiments, the RNase is a human RNase. In some embodiments, the RNase is a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of the RNase set forth in Table 1. In some embodiments, the RNase is a member of the human RNase A family. In some embodiments, the RNase is human pancreatic RNase1.

[0023] In some embodiments, the first nuclease domain comprises a DNase. In some embodiments, the DNase is a human DNase. In some embodiments, the DNase is a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of the DNase set forth in Table 1. In some embodiments, the DNase is selected from the group consisting of human DNase I, TREX1, and human DNase 1L3.

[0024] In some embodiments, the Fc domain is a human Fc domain. In some embodiments, the Fc domain is a mutant Fc domain. In some embodiments, the Fc domain is a mutant Fc domain comprising SSS, P238S, and / or P331S. In some embodiments, the Fc domain is a human IgG1 Fc domain. In some embodiments, the Fc domain is a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of the Fc domain set forth in Table 1.

[0025] In some embodiments, the first linker domain has a length of approximately 1 to approximately 50 amino acids. In some embodiments, the first linker domain has a length of approximately 5 to approximately 31 amino acids. In some embodiments, the first linker domain has a length of approximately 15 to approximately 25 amino acids. In some embodiments, the first linker domain has a length of approximately 20 to approximately 32 amino acids. In some embodiments, the first linker domain has a length of approximately 20 amino acids. In some embodiments, the first linker domain has a length of approximately 25 amino acids. In some embodiments, the first linker domain has a length of approximately 18 amino acids. In some embodiments, the first linker domain contains a gly / ser peptide. In some embodiments, the gly / ser peptide is of the formula (Gly4Ser) n The formula is such that n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the gly / ser peptide contains (Gly4Ser)3. In some embodiments, the gly / ser peptide contains (Gly4Ser)4. In some embodiments, the gly / ser peptide contains (Gly4Ser)5. In some embodiments, the first linker domain contains at least one restriction site. In some embodiments, the first linker domain contains about 12 or more nucleotides containing at least one restriction site. In some embodiments, the first linker domain contains two or more restriction sites. In some embodiments, the first linker domain contains a number of restriction sites. In some embodiments, the first linker domain contains an NLG peptide. The NLG peptide contains an N-linked glycosylation consensus sequence. In some embodiments, the NLG peptide has the sequence described in SEQ ID NO:99. In some embodiments, the first linker domain contains an N-linked glycosylation site.

[0026] In some embodiments, the first nuclease domain is ligated to the N-terminus of the Fc domain. In some embodiments, the first nuclease domain is ligated to the C-terminus of the Fc domain.

[0027] In some embodiments, the hybrid nuclease molecule further comprises a second nuclease domain. In some embodiments, the first and second nuclease domains are different nuclease domains. In some embodiments, the first and second nuclease domains are the same nuclease domain. In some embodiments, the second nuclease domain is linked to the C-terminus of the Fc domain. In some embodiments, the second nuclease domain is linked to the N-terminus of the Fc domain. In some embodiments, the second nuclease domain is linked to the C-terminus of the first nuclease domain. In some embodiments, the second nuclease domain is linked to the N-terminus of the first nuclease domain.

[0028] Also disclosed herein is a dimeric polypeptide comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a first nuclease domain and an Fc domain, and the first nuclease domain is functionally connected to the Fc domain. In some embodiments, the second polypeptide is a second hybrid nuclease molecule comprising a second nuclease domain and a second Fc domain, and the second nuclease domain is functionally connected to the second Fc domain.

[0029] Also disclosed herein is a pharmaceutical composition comprising at least one hybrid nuclease molecule and / or at least one dimeric polypeptide described herein and a pharmaceutically acceptable excipient.

[0030] Also disclosed herein are nucleic acid molecules encoding the hybrid nuclease molecules disclosed herein. Also disclosed herein are recombinant expression vectors containing the nucleic acid molecules disclosed herein. Also disclosed herein are host cells transformed by the recombinant expression vectors disclosed herein.

[0031] Also disclosed herein is a method for producing a hybrid nuclease disclosed herein, comprising the steps of: preparing a host cell containing a nucleic acid sequence encoding a hybrid nuclease molecule; and maintaining the host cell under conditions in which the hybrid nuclease molecule is expressed.

[0032] Also disclosed herein are methods for treating or preventing a medical condition associated with an abnormal immune response, the method comprising the step of administering an effective amount of an isolated hybrid nuclease molecule disclosed herein to a patient in need thereof. In some embodiments, the medical condition is an autoimmune disease. In some embodiments, the autoimmune disease is insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyroidopathy, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes mellitus, Goodpasture syndrome, and other conditions. The group is selected from pemphigus purpura, bullous pemphigoid, sympathetic ophthalmitis, lens-induced uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-VE, idiopathic cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus erythematosus, systemic lupus erythematosus (SLE), and connective tissue disease. In some embodiments, the autoimmune disease is SLE.

[0033] Also disclosed herein are methods for treating SLE, comprising the step of administering a composition containing a nuclease to a subject in an amount effective to degrade RNA, DNA, or an immune complex containing both RNA and DNA. In some aspects, the composition comprises a pharmaceutically acceptable carrier and a hybrid nuclease molecule as described herein. In other aspects, the composition comprises a hybrid nuclease molecule having an amino acid sequence as described in SEQ ID NO: 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, or 98. [Invention 1001] A hybrid nuclease molecule comprising a first nuclease domain and a modified Fc domain, The first nuclease domain is functionally connected to the Fc domain, and the Fc domain is modified so that the molecule exhibits reduced cytotoxicity compared to a hybrid nuclease molecule having an unmodified Fc domain. The aforementioned hybrid nuclease molecule. [Invention 1002] A hybrid nuclease molecule containing the amino acid sequence described in SEQ ID NO: 62, 64, 78, 80, 92, or 96, or a hybrid nuclease molecule containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 62, 64, 78, 80, 92, or 96. [Invention 1003] A hybrid nuclease molecule containing the amino acid sequence described in SEQ ID NO:96. [Invention 1004] A hybrid nuclease molecule containing the amino acid sequence described in SEQ ID NO: 66, 68, 70, 82, 84, 86, 94, or 98, or a hybrid nuclease molecule containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 66, 68, 70, 82, 84, 86, 94, or 98. [Invention 1005] A hybrid nuclease molecule containing the amino acid sequence described in SEQ ID NO:98. [Invention 1006] A hybrid nuclease molecule according to the present invention 1001, wherein the Fc domain is modified to reduce its binding affinity to the Fcγ receptor, complement protein, or both. [Invention 1007] A hybrid nuclease molecule of the present invention 1001 having a cytotoxic effect reduced to at least 1 / 1, 1 / 2, 1 / 3, 1 / 4, or 1 / 5. [Invention 1008] A hybrid nuclease molecule according to the present invention 1001, further comprising a second nuclease domain functionally connected to an Fc domain. [Invention 1009] A hybrid nuclease molecule according to the present invention 1001, wherein the Fc domain contains a human immunoglobulin Fc domain such as the human IgG1 Fc domain. [Invention 1010] A hybrid nuclease molecule according to Invention 1009, wherein the Fc domain includes a hinge domain, a CH2 domain, and a CH3 domain. [Invention 1011] A hybrid nuclease molecule of the present invention 1009, wherein the Fc domain contains an amino acid sequence having one or more of the mutants P238S, P331S, SCC, SSS (residues 220, 226, and 229), G236R, L328R, L234A, and L235A. [Invention 1012] A hybrid nuclease molecule of the present invention 1009, wherein the modified Fc domain contains an amino acid sequence having mutant SCC or SSS, P238S, and P331S. [Invention 1013] A hybrid nuclease molecule of the present invention 1001, having a longer serum half-life compared to the first nuclease domain alone. [Invention 1014] A hybrid nuclease molecule according to the present invention 1001, wherein the first nuclease domain is functionally connected to the Fc domain via the first linker domain. [Invention 1015] A hybrid nuclease molecule according to the present invention 1014, wherein the first linker domain is a polypeptide linker such as a gly-ser linker. [Invention 1016] A hybrid nuclease molecule according to the present invention 1008, wherein a second nuclease domain is functionally connected to an Fc domain via a second linker domain. [Invention 1017] A hybrid nuclease molecule according to the present invention 1016, wherein the second linker domain is a polypeptide linker such as an NLG peptide. [Invention 1018] A hybrid nuclease molecule according to the present invention, wherein the first nuclease domain comprises RNase or DNase. [Invention 1019] A hybrid nuclease molecule according to the present invention 1018, wherein the RNase is a human RNase such as human pancreatic RNase A. [Invention 1020] A hybrid nuclease molecule according to the present invention 1019, which degrades RNA in circulating blood and RNA in immune complexes, or inhibits interferon-α production, or both. [Invention 1021] A hybrid nuclease molecule according to the present invention 1019, wherein the RNase activity is approximately one-ninth or more of the activity of the control RNase molecule. [Invention 1022] A hybrid nuclease molecule according to the present invention 1019, in which the activity of the RNase molecule is approximately equal to that of the control RNase molecule. [Invention 1023] A hybrid nuclease molecule according to the present invention 1008, wherein the second nuclease domain contains DNase or RNase. [Invention 1024] A hybrid nuclease molecule according to the present invention 1023, wherein the DNase is selected from the group consisting of type I human DNase, human DNase 1L3, or human TREX1. [Invention 1025] A hybrid nuclease molecule according to the present invention 1024, wherein the DNase activity is approximately one-ninth or more of the activity of the control DNase molecule. [Invention 1026] A hybrid nuclease molecule according to the present invention 1024, in which the activity of the DNase molecule is approximately equal to that of the control DNase molecule. [Invention 1027] A composition comprising any of the hybrid nuclease molecules of the present invention described above and a pharmaceutically acceptable carrier. [Invention 1028] A nucleic acid molecule encoding the hybrid nuclease molecule of the present invention 1001. [Invention 1029] A recombinant expression vector containing the nucleic acid molecule of the present invention 1028. [Invention 1030] Host cells transformed with the recombinant expression vector of the present invention 1029. [Invention 1031] A method for producing a hybrid nuclease molecule according to the present invention 1001, comprising the following steps: The step of preparing host cells containing nucleic acid sequences encoding hybrid nuclease molecules; and A step of maintaining the host cell under conditions in which a hybrid nuclease molecule is expressed. [Invention 1032] A method for treating or preventing a medical condition associated with an abnormal immune response, comprising the step of administering an effective amount of the hybrid nuclease molecule of the present invention 1001 to a target. [Invention 1033] The method of the present invention 1032, wherein the medical condition is an autoimmune disease. [Invention 1034] Autoimmune diseases include insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyroidopathy, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes, Goodpasture syndrome, pemphigus vulgaris, bullous pemphigoid, and sympathetic thyroiditis. The method of the present invention 1033, selected from the group consisting of ophthalmitis, lens-induced uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-VE, idiopathic cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus erythematosus, systemic lupus erythematosus (SLE), and connective tissue disease. [Invention 1035] The method of the present invention 1034, wherein the autoimmune disease is SLE. [Invention 1036] A method for treating SLE, comprising the step of administering a composition containing a nuclease in an amount effective to degrade an immune complex containing RNA, DNA, or both RNA and DNA, The composition comprises a pharmaceutically acceptable carrier and a hybrid nuclease molecule having an amino acid sequence described in SEQ ID NO: 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, or 98. The aforementioned method. [Brief explanation of the drawing]

[0034] The above and other features, aspects and advantages of the present invention will be better understood in connection with the following description and accompanying drawings. [Figure 1] This shows prototype structures for creating various forms of hybrid nuclease molecules. [Figure 2] This shows the concentration of RSLV-124 recovered from mouse serum after a single intravenous injection. [Figure 3]The results of an RNase enzyme activity assay for RLSV-124 recovered from mouse serum, measured as relative fluorescence units (RFU) over time, are shown. [Figure 4] The concentration of RSLV-124 in mouse serum is shown, extrapolated from the molecular RNase enzyme activity. [Figure 5] This shows one-way radioactive diffusion (SRED) analysis of serum from two RNase transgenic (Tg) mice compared to normal B6 mice. [Figure 6] This chart shows the concentrations of RNase A in transgenic (Tg) mice and double transgenic (DTg) mice, as measured by ELISA. Each dot represents the concentration measured in an individual mouse. [Figure 7] The survival of TLR7.1 Tg mice and TLR7.1×RNaseA DTg mice is compared and shown. [Figure 8] This shows quantitative PCR of IRG in the spleen of Tg and DTg mice. [Figure 9] Western blots of COS transfection supernatants from RSLV 125-129 constructs (SEQ ID NO 208-217) are shown. [Figure 10] This shows an SRED analysis comparing aliquots of purified protein A derived from COS supernatant transfected with RSLV. [Figure 11a] The results of a DNase nuclease activity assay performed on purified protein A derived from COS7 supernatant transfected with an RSLV fusion plasmid are shown. [Figure 11b] The results of a DNase nuclease activity assay performed on purified protein A derived from COS7 supernatant transfected with an RSLV fusion plasmid are shown. [Figure 11c] The results of a DNase nuclease activity assay performed on purified protein A derived from COS7 supernatant transfected with an RSLV fusion plasmid are shown. [Figure 12] The RFU (relative fluorescence units) for each protein are shown as a function of time. [Figure 13-1] The Lineweaver Burk plots for various molecules tested are shown. [Figure 13-2] The Lineweaver-Burk plots for the various molecules tested are shown. [Figure 14] For RNaseIg molecules with wild-type or mutant Fc domains, the cytotoxicity data is shown as a graph of the percentage of dead cells as a function of fusion protein concentration. [Figure 15] This shows an overlay of histograms of THP-1 stained cells after 72 hours. [Figure 16] This demonstrates the ability of RSLV-132 to inhibit interferon-α production induced by immune complexes in SLE patients. [Figure 17] This demonstrates the in vivo ability of RSLV-132 to inhibit RNA-induced interferon-α production. [Figure 18] The RNase enzyme activity assays of two production lots of RSLV-132, stored at 4C for up to 8 weeks, are shown, compared to wild-type RNase and RSLV-124. [Figure 19] This shows the RNase enzyme activity assays of RSLV-133, RSLV-123, and RSLV-124 compared to RNase A, measured as RFU over time. [Figure 20] This shows the DNase enzyme activity assays of RSLV-133 and RSLV-123 compared to DNase 1, measured as RFU over time. [Figure 21] This shows the results of gel digestion experiments comparing the DNA digestion ability of RSLV-133 with that of RSLV-123 and wild-type DNase 1. [Figure 22]FACS analysis, which measures average fluorescence intensity, shows the binding of RSLV-124 and RSLV-132 to THP1 cells possessing the Fc receptor. [Modes for carrying out the invention]

[0035] Detailed explanation Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by the presence of high-titer autoantibodies targeting the body's own nucleoproteins. There is strong evidence that defects in the elimination or processing of dead and dying cells in SLE lead to disease primarily through the accumulation of ribonucleoproteins and deoxyribonucleoproteins (abbreviated as nucleoproteins). Nucleoproteins cause damage through three mechanisms: i) activating the innate immune system to produce inflammatory cytokines; ii) acting as antigens that generate immune complexes in the circulating blood; and iii) acting as antigens that cause in-situ complex formation at local sites such as the kidneys. This invention is based, at least in part, on the discovery that digestion of extracellular nucleic acids has an in vivo therapeutic effect.

[0036] Accordingly, the present invention provides a method for treating diseases characterized by defects in the elimination or processing of apoptotic cells and necrotic cell fragments, such as SLE, by administering an effective amount of nuclease activity for degrading complexes containing extracellular RNA and DNA. Such treatment can inhibit the production of type I interferon (IFN), a prominent cytokine in SLE that is strongly correlated with disease activity and nephritis.

[0037] In one embodiment, the subject is treated by administering nuclease activity, which is DNase or RNase activity, preferably in the form of a hybrid nuclease molecule. In one aspect, the nuclease activity is a first nuclease domain. In another aspect, the nuclease domain is connected to a modified Fc domain such that the cytotoxic effect of the molecule is reduced. In one aspect, the hybrid nuclease molecule contains a second nuclease domain.

[0038] In another aspect, the present invention provides a method for treating SLE, comprising administering an effective amount of a composition containing a nuclease to a subject. In one aspect, the treatment results in the degradation of an immune complex containing RNA, DNA, or both RNA and DNA. In another aspect, the treatment results in the inhibition of type I interferons, such as interferon-α, in the subject. In one aspect, the method for treating the subject comprises the step of administering an effective amount of a composition of a hybrid nuclease molecule containing the amino acid sequence described in SEQ ID NO: 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, or 98. In another aspect, the composition is a hybrid nuclease molecule containing the amino acid sequence described in SEQ ID NO: 96 or 98.

[0039] The claims and terms used herein are defined as follows, unless otherwise specified. In the event of any conflict between the terms used herein and those used in the parent provisional patent application, the terms used herein shall prevail.

[0040] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to natural amino acids. Natural amino acids include those encoded by the genetic code, as well as those modified after translation, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as natural amino acids, namely, an α-carbon bonded to a hydrogen, 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 natural amino acids. Amino acid mimes are compounds that have a different structure from the general chemical structure of amino acids, but function in a similar manner to natural amino acids.

[0041] In this specification, amino acids may be referred to by their commonly known three-letter symbols, or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly recognized single-letter codes.

[0042] "Amino acid substitution" refers to replacing 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 "replacement" amino acid residue. "Amino acid insertion" refers to incorporating at least one additional amino acid into a given amino acid sequence. Insertions are usually thought to consist of the insertion of one or two amino acid residues, but here larger-scale "peptide insertions," for example, the insertion of about 3 to 5 amino acid residues, or even up to about 10, 15, or 20 amino acid residues, are also possible. The inserted residues may be natural or non-natural as disclosed above. "Amino acid deletion" refers to removing at least one amino acid residue from a given amino acid sequence.

[0043] In this specification, "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acid residues. These terms apply to natural and non-natural amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are chemical mimics of corresponding natural amino acids.

[0044] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides, and polymers thereof in either single-stranded or double-stranded form. Unless otherwise specifically limited, this term includes nucleic acids, including known analogues of native nucleotides, that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to native nucleotides. Unless otherwise specified, individual nucleic acid sequences implicitly include their conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly specified sequences. Specifically, degenerate codon substitutions can be obtained by constructing sequences in which the third position of one or more (or all) selected codons is substituted with mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985); and Cassol et al., 1992; Rossolini et al., Mol. Cell. Probes 8:91-98, 1994). For arginine and leucine, modifications at the second base may also be conserved. The term nucleic acid is used interchangeably with gene, the cDNA and mRNA encoded by the gene.

[0045] The polynucleotides of the present invention may consist of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA. For example, polynucleotides may consist of single-stranded and double-stranded DNA, DNA which is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA which is a mixture of single-stranded and double-stranded regions, or hybrid molecules containing DNA and RNA which may be single-stranded but more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, polynucleotides may consist of RNA or DNA, or triple-stranded regions containing both RNA and DNA. Furthermore, polynucleotides may contain one or more modified bases or DNA or RNA backbone for stability or other reasons. "Modified" bases include, for example, tritylated bases and special bases such as inosine. Various modifications can be made to DNA and RNA; therefore, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms.

[0046] As used herein, the term “hybrid nuclease molecule” refers to a polynucleotide or polypeptide comprising at least one nuclease domain and at least one Fc domain. Hybrid nuclease molecules are also referred to as fusion proteins and fusion genes. For example, in one embodiment, a hybrid nuclease molecule may be a polypeptide comprising at least one Fc domain linked to a nuclease domain such as DNase and / or RNase. In another example, a hybrid nuclease molecule may comprise an RNase nuclease domain, a linker domain, and an Fc domain. Examples of hybrid nuclease molecules include SEQ ID NO: 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, and 98. Other examples are described in more detail below. In one embodiment, the hybrid nuclease molecule of the present invention may include further modifications. In another embodiment, the hybrid nuclease molecule may be modified to have a functional moisture (e.g., PEG, drug, or label) attached to it.

[0047] As used herein, the terms "hybrid bispecific nucleic acid molecule" or "binuclease molecule" refer to a hybrid nuclease molecule having two or more nuclease domains, such as a DNase domain and an RNase domain.

[0048] In certain aspects, the hybrid nuclease molecule of the present invention may be used with one or more "linker domains," such as polypeptide linkers. As used herein, the term "linker domain" refers to a sequence that links two or more domains in a linear sequence. As used herein, the term "polypeptide linker" refers to a peptide sequence or polypeptide sequence (e.g., a synthetic peptide sequence or polypeptide sequence) that links two or more domains in the linear amino acid sequence of a polypeptide chain. For example, a polypeptide linker may be used to link a nuclease domain and an Fc domain. Preferably, such a polypeptide linker can give flexibility to the polypeptide molecule. In certain embodiments, a polypeptide linker is used to link (e.g., genetically fuse) one or more Fc domains and / or one or more nuclease domains. The hybrid nuclease molecule of the present invention may contain multiple linker domains or peptide linkers.

[0049] As used herein, the term “gly-ser polypeptide linker” refers to a peptide consisting of a glycine residue and a serine residue. An exemplary gly / ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser)n. In one embodiment, n=1. In one embodiment, n=2. In another embodiment, n=3, i.e., Ser(Gly4Ser)3. In another embodiment, n=4, i.e., Ser(Gly4Ser)4. In another embodiment, n=5. In yet another embodiment, n=6. In another embodiment, n=7. In yet another embodiment, n=8. In another embodiment, n=9. In yet another embodiment, n=10. Another exemplary gly / ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser)n. In one embodiment, n=1. In one embodiment, n=2. In one preferred embodiment, n=3. In another embodiment, n=4. In another embodiment, n=5. In yet another embodiment, n=6.

[0050] As used herein, the terms “conjugated,” “fused,” and “fused” are interchangeable. These terms refer to the joining of two or more elements, components, or domains into one by any means, including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art.

[0051] As used herein, the term “Fc region” is defined as the portion of a native immunoglobulin formed by the Fc domains (or Fc moieties) of each of the two heavy chains of the native immunoglobulin.

[0052] As used herein, the term “Fc domain” refers to a portion of a single immunoglobulin (Ig) heavy chain, where the Fc domain does not include the Fv domain. Therefore, the Fc domain may also be referred to as “Ig” or “IgG”. In some embodiments, the Fc domain begins in the hinge region immediately upstream of the papain cleavage site of the antibody and ends at the C-terminus. Thus, a complete Fc domain includes at least a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, the Fc domain includes at least one of the following: a hinge domain (e.g., an upper, central, and / or lower hinge region), a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment 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 portion thereof) fused with a CH3 domain (or portion thereof). In another embodiment, the Fc domain includes a CH2 domain (or a portion thereof) fused with a CH3 domain (or a portion 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 the Fc molecule that is known in the art to be necessary for FcRn binding. In another embodiment, the Fc domain of the present invention includes at least a portion of the Fc molecule that is known in the art to be necessary for FcγR binding. In one embodiment, the Fc domain of the present invention includes at least a portion of an Fc molecule that is known in the art to be necessary for protein A binding.In one embodiment, the Fc domain of the present invention comprises at least a portion of an Fc molecule known in the art to be necessary for protein G binding. As used herein, Fc domain generally refers to a polypeptide comprising all or part of an Fc domain of an immunoglobulin heavy chain. This includes polypeptides comprising the entirety of the CH1 domain, hinge domain, CH2 domain, and / or CH3 domain, as well as fragments of such peptides comprising only, for example, the hinge domain, CH2 domain, and CH3 domain. The Fc domain may originate from any species and / or any subtype of immunoglobulin, including, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. The term Fc domain encompasses native Fc molecules 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 a whole antibody or otherwise generated. The assignment of amino acid residue numbers to the Fc domain follows Kabat's definition. See, for example, 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), each incorporated herein by reference for any purpose; Kabat et al., "Introduction" Sequences of Proteins of Immunological Interest, US Dept of 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).

[0053] As described herein, it will be understood by those skilled in the art that any Fc domain may be modified so that its amino acid sequence differs from that of the native Fc domain of a natural immunoglobulin molecule. In one exemplary embodiment, the Fc domain retains effector function (e.g., FcγR binding).

[0054] The Fc domain of the polypeptide of the present invention may be derived from different immunoglobulin molecules. For example, the Fc domain of the polypeptide 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 that is partly derived from the IgG1 molecule and partly from the IgG3 molecule. In yet another example, the Fc domain may include a chimeric hinge that is partly derived from the IgG1 molecule and partly from the IgG4 molecule.

[0055] A polypeptide sequence or amino acid sequence "derived from" a specified polypeptide or protein refers to the origin of the polypeptide. Preferably, a polypeptide sequence or amino acid sequence derived from a particular sequence has an amino acid sequence that is essentially identical to that particular sequence, or to a portion thereof 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 has an amino acid sequence in which its origin can be identified in a different way by those skilled in the art.

[0056] A polypeptide derived from another peptide may have one or more mutations compared to the starting polypeptide, such as one or more amino acid residues substituted by other amino acid residues, or one or more amino acid residues inserted or deleted.

[0057] Polypeptides may contain non-natural amino acid sequences. Such variants always have less than 100% sequence identity or similarity to the starting hybrid nuclease molecule. In one preferred embodiment, the variant is considered to have amino acid sequence identity or similarity of about 75% to less than 100%, more preferably about 80% to less than 100%, more preferably about 85% to less than 100%, more preferably about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably about 95% to less than 100%, to the amino acid sequence of the starting polypeptide, for example, across the entire length of the variant molecule.

[0058] In one embodiment, there is a one-amino acid difference between the starting polypeptide sequence and the sequence derived therefrom. This 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 to obtain the highest possible sequence identity and gaps have been introduced as necessary.

[0059] In one embodiment, the polypeptide of the present invention comprises, essentially comprises, or includes amino acid sequences and functionally active variants selected from Table 1. In one embodiment, the polypeptide comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences listed in Table 1. In one embodiment, the polypeptide comprises a sequence of amino acid sequences that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of amino acid sequences listed in Table 1. In one embodiment, the polypeptide comprises an amino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within this range) consecutive amino acids from the amino acid sequences listed in Table 1.

[0060] In one embodiment, the peptide of the present invention is encoded by a nucleotide sequence. The nucleotide sequences of the present invention are useful for a number of applications, including: cloning, gene therapy, protein expression and purification, mutagenesis, DNA vaccination of hosts requiring it, antibody production for, for example, passive immunization, PCR, primer and probe production, siRNA design and production (see, for example, the website of Dharmacon siDesign). In one embodiment, the nucleotide sequences of the present invention include, consist of, or essentially consist of nucleotide sequences selected from Table 1. In one embodiment, the nucleotide sequences include nucleotide sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequences listed in Table 1. In one embodiment, the nucleotide sequence includes a sequence of nucleotides that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of nucleotides listed in Table 1. In one embodiment, the nucleotide sequence includes a sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within this range) consecutive nucleotides from the sequence of nucleotides listed in Table 1.

[0061] A preferred hybrid nuclease molecule of the present invention comprises a sequence derived from a human immunoglobulin sequence (e.g., at least one Fc domain). However, the sequence may also comprise one or more sequences derived from another mammalian species. For example, a primate Fc domain or nuclease domain may be included in the sequence. Alternatively, one or more mouse amino acids may be present in the polypeptide. In some embodiments, the polypeptide sequence of the present invention is non-immunogenic and / or has low immunogenicity.

[0062] Furthermore, it will be understood by those skilled in the art that the hybrid nuclease molecules of the present invention can be modified so that their sequence differs from the natural or native sequence from which they originate, while preserving the desirable activity of the native sequence. For example, nucleotide or amino acid substitutions can be made that result in conservative substitutions or changes at "non-essential" amino acid residues. Isolated nucleic acid molecules encoding non-natural variants of hybrid nuclease molecules derived from immunoglobulins (e.g., Fc domains) can be produced by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of the immunoglobulin so that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard methods such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0063] The peptide hybrid nuclease molecule of the present invention may include a conserved amino acid substitution at the site of one or more amino acid residues, for example, at the site of an essential or non-essential amino acid residue. A "conservative amino acid substitution" is defined as the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-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 binding polypeptide are preferably replaced by other amino acid residues from the same side-chain family. In another embodiment, a chain of amino acids can be replaced by structurally similar chains with different order and / or composition of members of the side-chain family. Alternatively, in another embodiment, random mutations can be introduced across the entire or a portion of the coding sequence by means of saturated mutagenesis, and the resulting mutants can be incorporated into the binding polypeptide of the present invention and screened for their ability to bind to a desired target.

[0064] The term "improvement" refers to any therapeutically beneficial effect in the treatment of a disease condition, such as an autoimmune disease condition (e.g., SLE), including prevention, reduction of severity or progression, remission, or cure.

[0065] The term "in situ" refers to a process that occurs in living cells growing away from a living organism, such as in tissue culture.

[0066] The term "in vivo" refers to a process that occurs in a living organism.

[0067] As used herein, the terms “mammal,” “subject,” or “patient” include both human and non-human animals, including, but not limited to, humans, non-human primates, canids, felines, rodents, bovines, equids, and pigs.

[0068] In the context of two or more nucleic acid sequences or polypeptide sequences, the term "percent "identity"" refers to two or more sequences or subsequences that, when compared and aligned to obtain the greatest possible correspondence using one of the following sequence comparison algorithms (e.g., BLASTP and BLASTN, or other algorithms available to those skilled in the art), or by visual inspection, share a specified percentage of the same nucleotide or amino acid residues. Depending on the application, "identity" may exist across a region of the sequences being compared, such as a functional domain, or alternatively, across the entire length of both sequences being compared.

[0069] For sequence comparison, typically, one sequence is used as a reference sequence for comparison with the sequence under test. When using a sequence comparison algorithm, the sequence under test and the reference sequence are input into a computer, and the coordinates of subsequences are specified as needed, along with the parameters of the sequence algorithm program. The sequence comparison algorithm then calculates the degree of sequence match between the sequence under test and the reference sequence based on the specified program parameters.

[0070] Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see Ausubel et al., below for an overview).

[0071] One example of a suitable algorithm for determining sequence agreement and sequence similarity is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysis is available through the website of the National Center for Biotechnology Information.

[0072] The term "sufficient amount" means an amount sufficient to produce the desired effect, for example, an amount sufficient to alter protein aggregation within cells.

[0073] The term "therapeutic effective dose" refers to the amount of medication that is effective in improving the symptoms of a disease. Since prevention can be considered a treatment, the therapeutic effective dose may also be called the "preventive effective dose."

[0074] When used herein and in the appended claims, it should be noted that the singular forms “a,” “an,” and “the” include multiple references unless the context explicitly indicates otherwise.

[0075] composition Hybrid nuclease molecule In some embodiments, the composition of the present invention comprises a hybrid nuclease molecule. In some embodiments, the hybrid nuclease molecule comprises a nuclease domain functionally linked to an Fc domain. In some embodiments, the hybrid nuclease molecule comprises a nuclease domain linked to an Fc domain. In some embodiments, the hybrid nuclease molecule is a nuclease protein. In some embodiments, the hybrid nuclease molecule is a nuclease polynucleotide.

[0076] In some embodiments, the nuclease domain is linked to the Fc domain via a linker domain. In some embodiments, the linker domain is a linker peptide. In some embodiments, the linker domain is a linker nucleotide. In some embodiments, the hybrid nuclease molecule includes a leader molecule, 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 various embodiments, the hybrid nuclease molecule of the present invention contains a leader peptide at the N-terminus of the molecule, which is subsequently cleaved from the hybrid nuclease molecule. Methods for constructing nucleic acid sequences encoding a leader peptide fused to a recombinant protein are well known in the art. In various embodiments, any of the hybrid nuclease molecules of the present invention can be expressed with or without a leader fused to its N-terminus. The protein sequence of the hybrid nuclease molecule of the present invention after cleavage of the fused leader peptide can be predicted and / or estimated by those skilled in the art. Examples of hybrid nuclease molecules of the present invention, further comprising a VK3 leader peptide (VK3LP) fused to the N-terminus of the hybrid nuclease molecule, are shown in SEQ ID NO: 92 (RSLV-132) and 94 (RSLV-133). The corresponding nucleotide sequences are shown in SEQ ID NO: 91 and 93, respectively. In some embodiments, after cleavage of the VK3 leader, these hybrid nuclease molecules have the sequences described in SEQ ID NO: 96 (RSLV-132) and 98 (RSLV-133), respectively. The corresponding nucleotide sequences are shown in SEQ ID NO: 95 and 97, respectively. In some embodiments, the hybrid nuclease molecules of the present invention are expressed without the leader peptide fused to its N-terminus, and the resulting hybrid nuclease molecule has an N-terminal methionine.

[0077] In some embodiments, hybrid nuclease molecules are thought to contain a stop codon. In some embodiments, the stop codon is thought to be located at the C-terminus of the Fc domain.

[0078] In some embodiments, the hybrid nuclease molecule further comprises a second nuclease domain. In some embodiments, the second nuclease domain is linked to the Fc domain via a second linker domain. In some embodiments, the second linker domain is thought to be located at the C-terminus of the Fc domain. Figure 1 shows at least one embodiment of the hybrid nuclease molecule. In some embodiments, the hybrid nuclease molecule comprises the sequence shown in Table 1.

[0079] In some embodiments, the hybrid nuclease molecule is an RNase molecule or DNase molecule or a polyenzyme molecule (e.g., both RNase and DNase, or two RNA nucleases or DNA nucleases with different substrate specificities) attached 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 aspect, the hybrid nuclease molecule does not effectively bind to C1q. In other aspects, the hybrid nuclease molecule contains an in-frame Fc domain derived from IgG1. In other aspects, the hybrid nuclease molecule further contains mutations in the hinge domain, CH2 domain and / or CH3 domain. In other aspects, the mutations are P238S, P331S or N297S and may involve mutations in one or more of the three cysteine ​​molecules of the hinge region. In some such aspects, the mutations in one or more of the three cysteine ​​molecules of the hinge region may be SCC or SSS. In other aspects, the molecule contains an SCC hinge, but is otherwise wild-type with respect to the human IgG1 Fc CH2 and CH3 domains, effectively binding to the Fc receptor and promoting the uptake of hybrid nuclease molecules into the endocytotic compartment of the bound cell. In other aspects, the molecule has activity toward single-stranded and / or double-stranded RNA substrates.

[0080] In some aspects, the hybrid nuclease molecule contains a mutant Fc domain. In some aspects, the hybrid nuclease molecule contains a mutant IgG1 Fc domain. In some aspects, the mutant Fc domain contains one or more mutations in the hinge domain, the CH2 domain, and / or the CH3 domain. In some aspects, the mutant Fc domain contains the P238S mutation. In some aspects, the mutant Fc domain contains the P331S mutation. In some aspects, the mutant Fc domain contains the P238S mutation and the P331S mutation. In some aspects, the mutant Fc domain contains P238S and / or P331S, and may also contain a mutation in one or more of the three cysteines in the hinge region. In some aspects, the mutant Fc domain contains P238S and / or P331S, and / or one or more mutations in the three cysteines in the hinge region. In some aspects, the mutant Fc domain includes mutations to P238S and / or P331S, and / or to SSS in three cysteines of the hinge region, or to SCC in one cysteine ​​of the hinge region. In some aspects, the mutant Fc domain includes mutations to P238S and P331S, and to three cysteines of the hinge region. In some aspects, the mutant Fc domain includes P238S and P331S, and either SCC or SSS. In some aspects, the mutant Fc domain includes P238S and P331S and SCC. In some aspects, the mutant Fc domain includes P238S SSS. In some aspects, the mutant Fc domain includes P331S, and either SCC or SSS. In some aspects, the mutant Fc domain includes mutations in one or more of the three cysteines of the hinge region. In some aspects, the mutant Fc domain contains mutations in three cysteine ​​groups in the hinge region. In some aspects, the mutant Fc domain contains mutations to SSS groups in three cysteine ​​groups in the hinge region.In some aspects, the mutant Fc domain contains a mutation to SCC in one of the three cysteine ​​groups in the hinge region. In some aspects, the mutant Fc domain contains SCC or SSS. In some aspects, the mutant Fc domain is as shown in SEQ ID NO 59, 60, 71-76 or 87-90. In some aspects, the hybrid nuclease molecule is as shown in SEQ ID NO 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96 or 98. In some aspects, the hybrid nuclease molecule contains a wild-type human RNase1 domain ligated with a mutant human IgG1 Fc domain containing SCC, P238S, and P331S, or with a mutant human IgG1 Fc domain containing SSS, P238S, and P331S. In some cases, the nucleic acid sequences encoding the hybrid nuclease molecules are as shown in SEQ ID NO: 61, 77, or 91. In some cases, the hybrid nuclease molecules are as shown in SEQ ID NO: 62, 78, 92, or 96.

[0081] In some aspects, the hybrid nuclease molecule contains a wild-type human RNase1 domain linked via a (Gly4Ser)4 linker domain to mutant human IgG1 Fc domains containing SCC, P238S, and P331S, or to mutant human IgG1 Fc domains containing SSS, P238S, and P331S. In some aspects, the nucleic acid sequences encoding the hybrid nuclease molecule are shown in SEQ ID NO: 63 or 79. In some aspects, the hybrid nuclease molecule is shown in SEQ ID NO: 64 or 80.

[0082] In some aspects, the hybrid nuclease molecule includes a human DNase1 G105R A114F domain linked to a mutant human IgG1 Fc domain containing SCC, P238S, and P331S via a (Gly4Ser)4 linker domain, and that Fc domain linked to a wild-type human RNase1 domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule includes a human DNase1 G105R A114F domain linked to a mutant human IgG1 Fc domain containing SSS, P238S, and P331S via a (Gly4Ser)4 linker domain, and that Fc domain linked to a wild-type human RNase1 domain via an NLG linker domain. In some aspects, the nucleic acid sequence encoding the hybrid nuclease molecule is shown in SEQ ID NO: 65 or 81. In some aspects, the hybrid nuclease molecule is shown in SEQ ID NO: 66 or 82.

[0083] In some aspects, the hybrid nuclease molecule includes a wild-type human RNase1 domain linked to a mutant human IgG1 Fc domain containing SCC, P238S, and P331S via a (Gly4Ser)4 linker domain, and that Fc domain linked to a human DNase1 G105R A114F domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule includes a wild-type human RNase1 domain linked to a mutant human IgG1 Fc domain containing SSS, P238S, and P331S via a (Gly4Ser)4 linker domain, and that Fc domain linked to a human DNase1 G105R A114F domain via an NLG linker domain. In some aspects, the nucleic acid sequence encoding the hybrid nuclease molecule is shown in SEQ ID NO: 67 or 83. In some aspects, the hybrid nuclease molecule is shown in SEQ ID NO: 68 or 84.

[0084] In some aspects, the hybrid nuclease molecule contains a wild-type human RNase1 domain ligated to a mutant human IgG1 Fc domain containing SCC, P238S, and P331S, with the Fc domain ligated to a human DNase1 G105R A114F domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule contains a wild-type human RNase1 domain ligated to a mutant human IgG1 Fc domain containing SSS, P238S, and P331S, with the Fc domain ligated to a human DNase1 G105R A114F domain via an NLG linker domain. In some aspects, the nucleic acid sequences encoding the hybrid nuclease molecule are shown in SEQ ID NO: 69, 85, or 93. In some aspects, the hybrid nuclease molecule is shown in SEQ ID NO: 70, 86, 94, or 98.

[0085] In some cases, the activity of hybrid nuclease molecules is detectable in vitro and / or in vivo. In some cases, hybrid nuclease molecules bind to cells, malignant cells, or cancer cells and interfere with their biological activity.

[0086] In another context, a multifunctional RNase molecule is provided that is attached to another enzyme or antibody with binding specificity, such as an RNA-targeting scFv, or a second nuclease domain having the same or different specificity as the first domain.

[0087] In another context, a multifunctional DNase molecule is provided that is attached to another enzyme or antibody with binding specificity, such as an scFv that targets DNA, or a second nuclease domain having the same or different specificity as the first domain.

[0088] In another context, hybrid nuclease molecules have been adapted to prevent or treat diseases or disorders in mammals by administering hybrid nuclease molecules, attached to an Fc region, to mammals in need at a therapeutically effective dose in which the disease is prevented or treated. In yet another context, the disease or disorder may be an autoimmune disease or cancer. In some such contexts, autoimmune diseases include insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes mellitus, Goodpasture syndrome, pemphigus vulgaris, bullous pemphigoid, sympathetic ophthalmitis, lens-induced uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-VE, idiopathic cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus erythematosus, systemic lupus erythematosus, or connective tissue disease.

[0089] In some embodiments, the targets of the RNase enzymatic activity of RNase hybrid nuclease molecules are primarily extracellular, consisting of RNA contained within immune complexes with anti-RNP autoantibodies, for example, and RNA expressed on the surface of apoptotic cells. In some embodiments, RNase hybrid nuclease molecules are active within the acidic environment of endocytic vesicles. In some embodiments, RNase hybrid nuclease molecules contain a wild-type (wt) Fc domain to enable the molecule to bind to FcR and enter the endocytic compartment through entry pathways used by immune complexes. In some embodiments, RNase hybrid nuclease molecules containing a wt Fc domain are adapted to be active both extracellularly and within the endocytic environment (where TLR7 may be expressed). In some aspects, this allows RNase hybrid nuclease molecules containing a wt Fc domain to halt TLR7 signaling via previously phagocytosed immune complexes or by RNA that activates TLR7 after viral infection. In some embodiments, wt RNase, a hybrid RNase molecule, is not resistant to inhibition by cytoplasmic inhibitors of RNase. In some embodiments, wt RNase, a hybrid RNase molecule, is inactive in the cytoplasm of cells.

[0090] In some embodiments, hybrid nuclease molecules containing a wtFc domain are used for the treatment of autoimmune diseases, such as SLE.

[0091] In some embodiments, the binding affinity of an Fc domain to an Fc receptor (FcR) can be enhanced, for example, through modifications of glycosylation and / or changes in the amino acid sequence. In some embodiments, a hybrid nuclease molecule contains one or more Fc modifications that enhance FcR binding affinity.

[0092] Alternative methods for constructing hybrid nuclease molecules attached to an Fc domain are also conceivable. In some embodiments, the orientation of the domain can be changed to construct an Ig-RNase molecule, Ig-DNase molecule, RNase-Ig molecule, or RNase-Ig molecule that retains FcR binding ability and has an active nuclease domain.

[0093] In some embodiments, the DNase hybrid nuclease molecule includes a wt Fc domain that enables the molecule to undergo endocytosis after binding to FcR, for example. In some embodiments, the DNase hybrid nuclease molecule may have activity against extracellular immune complexes containing DNA, which are either in a soluble form or deposited as an insoluble complex, for example.

[0094] In some embodiments, hybrid nuclease molecules contain both DNase and RNase. In some embodiments, these hybrid nuclease molecules can improve the treatment of SLE because they can digest immune complexes containing, for example, RNA, DNA, or a combination of both RNA and DNA; and if they further contain a wtFc domain, they can be active both extracellularly and within the endocytotic compartment where TLR7 and TLR9 may localize.

[0095] In some embodiments, the linker domain includes (gly4ser)3,4, or5 variants that vary the linker length by 5 amino acids. In another embodiment, the linker domain is approximately 18 amino acids long and includes an N-linked glycosylation site, which may be sensitive to protease cleavage in vivo. In some embodiments, the N-linked glycosylation site can prevent the hybrid nuclease molecule from being cleaved at the linker domain. In some embodiments, the N-linked glycosylation site can assist in the compartmentalization of the folding of independent functional domains separated by the linker domain.

[0096] In some embodiments, the hybrid nuclease molecule may contain both mutant and / or wild-type human IgG1 Fc domains. In some embodiments, the hybrid nuclease molecule may be expressed from both transient transfection of COS and stable transfection of CHO. In some embodiments, both CD80 / 86 binding and RNase activity are retained in the hybrid nuclease molecule. In some embodiments, the hybrid nuclease molecule contains a DNase1L3-Ig-linker-RNase construct. In some embodiments, the hybrid nuclease molecule contains a DNase1-Ig-linker-RNase construct or an RNase-Ig-linker-DNase construct. In some embodiments, the fusion ligation between the enzyme domain and other domains of the hybrid nuclease molecule is optimized.

[0097] In some embodiments, hybrid nuclease molecules include DNase-Ig hybrid nuclease molecules and / or hybrid DNase-RNase hybrid nuclease molecules.

[0098] In some embodiments, the hybrid nuclease molecule contains TREX1. In some embodiments, the TREX1 hybrid nuclease molecule can digest chromatin. In some embodiments, the TREX1 hybrid nuclease molecule is expressed by cells. In some embodiments, the expressed hybrid nuclease molecule contains mouse TREX-1 and a mouse (wt or mutant) Fc domain. In some embodiments, a 20-25 amino acid (aa) linker domain may be required between TREX1 and the IgG hinge to enable DNase activity. In some embodiments, a hybrid nuclease molecule with a 15aa linker domain is inactive. In some embodiments, the use of 20-amino acid and 25-amino acid linker domains (plus two or more amino acids to incorporate restriction sites) results in functional activity as measured by chromatin digestion. In some embodiments, approximately 72aa of hydrophobic region can be removed from the COOH end of TREX-1 before fusing with the Fc domain via the linker domain. In some embodiments, the 20-amino acid linker domain version of the hybrid nuclease molecule exhibits higher expression levels compared to controls and / or other hybrid nuclease molecules. In some embodiments, kinetic enzyme assays are used to quantitatively compare the enzymatic activity of the hybrid nuclease molecule and the control.

[0099] In some embodiments, the expression of hybrid nuclease molecules can be improved by utilizing further optimization of the fusion ligation site selected for the shortening of the TREX1 enzyme.

[0100] In some embodiments, the hybrid nuclease molecule comprises a human TREX1-linker-Ig Fc domain hybrid nuclease molecule having 20aa and / or 25aa linker domains. In some embodiments, the linker domain is a variant of the (gly4ser)4 or (gly4ser)5 cassette having one or more restriction sites attached for incorporation into the hybrid nuclease molecule construct. In some embodiments, a more flexible and longer linker domain can be used to promote proper folding for head-tail dimerization useful for TREX1 enzyme activity.

[0101] In some embodiments, the hybrid nuclease molecule is a TREX1-tandem hybrid nuclease molecule. In some embodiments, an alternative method to promote head-tail folding of TREX1 is to construct a TREX1-TREX1-Ig hybrid nuclease molecule in which two TREX1 domains are incorporated in tandem, followed by a linker domain and an Ig Fc domain. In some embodiments, the head-tail configuration of the TREX1 cassette can be modified for head-tail folding on either arm of the immunoenzyme to introduce a single TREX1 functional domain to each arm of the molecule. In some embodiments, the hybrid nuclease molecule of each immunoenzyme has two functional TREX1 enzymes attached to a single IgG Fc domain.

[0102] In some embodiments, the hybrid nuclease molecule includes TREX1-linker1-Ig-linker2-RNase.

[0103] In some embodiments, the hybrid nuclease molecule contains the RNase-Ig linker TREX1. In some embodiments, the cassette is provided for the fusion of both the amino and carboxyl groups of each enzyme for incorporation into the hybrid nuclease molecule, in which the enzyme configuration is inverted. In some embodiments, the RNase enzyme exhibits equivalent functional activity regardless of its position within the hybrid nuclease molecule. In some embodiments, alternative hybrid nuclease molecules can also be designed to verify whether a particular configuration results in enhanced expression and / or function of the components of the hybrid nuclease molecule.

[0104] In some embodiments, the hybrid nuclease molecule contains 1L3-Ig. In some embodiments, 1L3 DNase is constructed and expressed from a mouse sequence. In some embodiments, the enzyme is active. In some embodiments, a mouse 1L3 DNase-Ig-RNase hybrid nuclease is constructed and expressed. In some embodiments, the molecule contains human 1L3-Ig, human 1L3-Ig-RNase, and / or human RNase-Ig-1L3.

[0105] In some embodiments, the hybrid nuclease molecule contains DNase1-Ig. In some embodiments, the DNase1-Ig hybrid nuclease molecule contains A114F, a native mutant allele exhibiting reduced sensitivity to actin. In some embodiments, this mutation is introduced into the hybrid nuclease molecule to produce a more stable derivative of human DNase1. In some embodiments, DNase1-linker-Ig is produced containing a 20aa or 25aa linker domain. In some embodiments, the hybrid nuclease molecule contains RNase-Ig-linker-DNase1, where the DNase1 domain is located on the COOH side of the Ig Fc domain. In some embodiments, hybrid nuclease molecules incorporating DNase1 are produced, including: DNase1-linker-Ig-linker2-RNase, and / or RNase-Ig-linker-DNase1.

[0106] Another aspect of the present invention is the use of gene therapy methods to treat or prevent disorders, diseases, and pathological conditions by one or more hybrid nuclease molecules. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences of the hybrid nuclease molecules into an animal to achieve the expression of one or more polypeptides of the present invention. The methods may include the introduction of one or more polynucleotides encoding the polypeptides of the hybrid nuclease molecules of the present invention, which are functionally linked to promoters and any other genetic elements necessary for polypeptide expression by the target tissue.

[0107] In gene therapy applications, genes for hybrid nuclease molecules are introduced into cells with the aim of achieving in vivo synthesis of therapeutically effective gene products. "Gene therapy" includes both conventional gene therapy, where a single treatment achieves a permanent effect, and gene therapy involving single or repeated administration of therapeutically effective DNA or mRNA. Oligonucleotides can be modified to enhance their uptake, for example, by substituting their negatively charged phosphodiester groups with uncharged groups.

[0108] FC Domain In some embodiments, the hybrid nuclease molecule includes an Fc domain. The Fc domain does not include a variable region that binds to an antigen. In some embodiments, the Fc domain does not include a variable region. Fc domains useful for constructing the hybrid nuclease molecule of the present invention can be obtained from several different sources. In a preferred embodiment, the Fc domain of the hybrid nuclease molecule is derived from human immunoglobulin. However, it will be understood that the Fc domain may be derived from immunoglobulin of another mammalian species, including, for example, rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species. Furthermore, the Fc domain or a portion of the hybrid nuclease molecule may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4. In one preferred embodiment, the human isotype IgG1 is used.

[0109] In some aspects, the hybrid nuclease molecule contains a mutant Fc domain. In some aspects, the hybrid nuclease molecule contains a mutant IgG1 Fc domain. In some aspects, the mutant Fc domain contains one or more mutations in the hinge domain, the CH2 domain, and / or the CH3 domain. In some aspects, the mutant Fc domain contains the P238S mutation. In some aspects, the mutant Fc domain contains the P331S mutation. In some aspects, the mutant Fc domain contains the P238S mutation and the P331S mutation. In some aspects, the mutant Fc domain contains P238S and / or P331S, and may also contain a mutation in one or more of the three cysteines in the hinge region. In some aspects, the mutant Fc domain contains P238S and / or P331S, and / or one or more mutations in the three cysteines in the hinge region. In some aspects, the mutant Fc domain includes mutations in P238S and / or P331S, and / or in one cysteine ​​of the hinge region to SCC, or in three cysteines of the hinge region to SSS. In some aspects, the mutant Fc domain includes mutations in P238S and P331S, and in at least one of the three cysteines of the hinge region. In some aspects, the mutant Fc domain includes P238S and P331S and SCC. In some aspects, the mutant Fc domain includes P238S and P331S and SSS. In some aspects, the mutant Fc domain includes P238S, and SCC or SSS. In some aspects, the mutant Fc domain includes P331S, and SCC or SSS. In some aspects, the mutant Fc domain includes mutations in one or more of the three cysteines of the hinge region. In some aspects, the mutant Fc domain contains mutations in three cysteine ​​molecules in the hinge region.In some aspects, the mutant Fc domain contains a mutation to SCC in one of the three cysteine ​​molecules in the hinge region. In some aspects, the mutant Fc domain contains SCC. In some aspects, the mutant Fc domain contains a mutation to SSS in one of the three cysteine ​​molecules in the hinge region. In some aspects, the mutant Fc domain contains SSS. In some aspects, the nucleic acid sequence encoding the mutant Fc domain is shown in SEQ ID NO: 59, 71, 73, 75, 87, or 89. In some aspects, the mutant Fc domain is as shown in SEQ ID NO: 60, 72, 74, 76, 88, or 90. In some aspects, the nucleic acid sequence encoding the hybrid nuclease molecule is as shown in SEQ ID NO: 61, 63, 65, 67, 69, 77, 79, 81, 83, 85, 91, 93, 95, or 97. In some aspects, the hybrid nuclease molecules are as shown in SEQ ID NO: 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, or 98.

[0110] Various Fc domain gene sequences (e.g., human constant region gene sequences) are available in the form of publicly available deposits. A constant region domain containing an Fc domain sequence can be selected that has (or lacks) a specific effector function, or has specific modifications to reduce immunogenicity. Many sequences of antibodies and antibody-coding genes are publicly available, and suitable Fc domain sequences (e.g., hinge sequences, CH2 sequences and / or CH3 sequences, or portions thereof) can be obtained from these sequences using techniques recognized in the art. Subsequently, the genetic material obtained using any of the above methods can be modified or synthesized to obtain the polypeptide of the present invention. Furthermore, it will be understood that the scope of the present invention also includes alleles, variants, and mutations of constant region DNA sequences.

[0111] The Fc domain sequence can be cloned, for example, using a polymerase chain reaction and primers selected to amplify the domain of interest. To clone the Fc domain sequence from an antibody, mRNA may be isolated from hybridoma cells, splenocytes, or lymphocytes, reverse transcribed to DNA, and then the antibody gene may be amplified by PCR. PCR amplification methods are described in detail in U.S. Patents 4,683,195; 4,683,202; 4,800,159; 4,965,188; and, for example, "PCR Protocols: A Guide to Methods and Applications" Innis et al. eds., Academic Press, San Diego, Calif. (1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol. 217:270). PCR can be initiated with consensus constant-region primers or with more specific primers based on published heavy and light chain DNA and amino acid sequences. As discussed above, PCR may also be used to isolate DNA clones encoding the antibody light and heavy chains. In this case, the library can be screened with consensus primers or larger homologous probes such as mouse constant-region probes. Numerous primer sets suitable for amplifying antibody genes are known in the art (e.g., 5' primers based on the N-terminal sequence of purified antibodies (Benhar and Pastan. 1994. Protein Engineering 7:1509); rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods 173:33); antibody leader sequences (Larrick et al. 1989 Biochem. Biophys. Res. Commun. 160:1250)). The cloning of antibody sequences is also described in U.S. Patent No. 5,658,570 by Newman et al., filed on January 25, 1995, which is incorporated herein by reference.

[0112] The hybrid nuclease molecule of the present invention may contain one or more Fc domains (for example, two, three, four, five, six, seven, eight, nine, ten or more Fc domains). In one embodiment, the Fc domains may be of different types. In one embodiment, at least one Fc domain present in the hybrid nuclease molecule includes a hinge domain or a portion thereof. In another embodiment, the hybrid nuclease molecule of the present invention contains at least one Fc domain including at least one CH2 domain or a portion thereof. In another embodiment, the hybrid nuclease molecule of the present invention contains at least one Fc domain including at least one CH3 domain or a portion thereof. In another embodiment, the hybrid nuclease molecule of the present invention contains at least one Fc domain including at least one CH4 domain or a portion thereof. In another embodiment, the hybrid nuclease molecule of the present invention comprises at least one Fc domain comprising at least one hinge domain or a portion thereof and at least one CH2 domain or a portion thereof (for example, in a hinge-CH2 orientation). In another embodiment, the hybrid nuclease molecule of the present invention comprises at least one Fc domain comprising at least one CH2 domain or a portion thereof and at least one CH3 domain or a portion thereof (for example, in a CH2-CH3 orientation). In another embodiment, the hybrid nuclease molecule of the present invention comprises at least one Fc domain comprising at least one hinge domain or a portion thereof, at least one CH2 domain or a portion thereof and at least one CH3 domain or a portion thereof, for example in a hinge-CH2-CH3, hinge-CH3-CH2 or CH2-CH3-hinge orientation.

[0113] In one embodiment, the hybrid nuclease molecule comprises at least one complete Fc region derived from one or more immunoglobulin heavy chains (e.g., an Fc domain including a hinge domain, a CH2 domain, and a CH3 domain, which do not necessarily have to be derived from the same antibody). In another embodiment, the hybrid nuclease molecule comprises at least two complete Fc domains derived from one or more immunoglobulin heavy chains. In a preferred embodiment, the complete Fc domains are derived from human IgG immunoglobulin heavy chains (e.g., human IgG1).

[0114] In another embodiment, the hybrid nuclease molecule of the present invention comprises at least one Fc domain containing a complete CH3 domain. In another embodiment, the hybrid nuclease molecule of the present invention comprises at least one Fc domain containing a complete CH2 domain. In another embodiment, the hybrid nuclease molecule of the present invention comprises at least one Fc domain containing at least a CH3 domain and at least one of a hinge region and a CH2 domain. In one embodiment, the hybrid nuclease molecule of the present invention comprises at least one Fc domain containing a hinge domain and a CH3 domain. In another embodiment, the hybrid nuclease molecule of the present invention comprises a hinge domain and at least one Fc domain containing a CH2 domain and a CH3 domain. In a preferred embodiment, the Fc domain is derived from a human IgG immunoglobulin heavy chain (e.g., human IgG1).

[0115] The constant region domains or portions thereof constituting the Fc domain of the hybrid nuclease molecule of the present invention may be derived from different immunoglobulin molecules. For example, the polypeptide of the present invention may include a CH2 domain or portion thereof derived from an IgG1 molecule, and a CH3 region or portion thereof derived from an IgG3 molecule. In another example, the hybrid nuclease molecule may include an Fc domain that includes a hinge domain that is partly derived from an IgG1 molecule and partly derived from an IgG3 molecule. As described herein, it will be understood by those skilled in the art that the Fc domain can be modified so that its amino acid sequence differs from that of a natural antibody molecule.

[0116] In another embodiment, the hybrid nuclease molecule of the present invention comprises one or more truncated Fc domains, which are still sufficient to confer Fc receptor (FcR) binding properties to the Fc region. Thus, the Fc domain of the hybrid nuclease molecule of the present invention may include or consist of an FcRn binding moiety. The FcRn binding moiety may originate from the heavy chain of any isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, an FcRn binding moiety derived from an antibody of human isotype IgG1 is used. In another embodiment, an FcRn binding moiety derived from an antibody of human isotype IgG4 is used.

[0117] In one embodiment, the hybrid nuclease molecule of the present invention lacks one or more constant domains of a complete Fc region, i.e., they are partially or entirely deleted. In one embodiment, the hybrid nuclease molecule of the present invention lacks the entire CH2 domain (ΔCH2 construct). Those skilled in the art will see that such constructs may be preferred due to the regulatory properties of the CH2 domain over the antibody catabolic rate. In one embodiment, the hybrid nuclease molecule of the present invention contains a CH2 domain deletion Fc region derived from a vector encoding the IgG1 human constant domain (e.g., IDEC Pharmaceuticals, San Diego) (see, e.g., WO02 / 060955A2 and WO02 / 096948A2). This exemplary vector is engineered to delete the CH2 domain, giving a synthetic vector expressing a domain-deleted IgG1 constant region. It should be noted that these exemplary constructs are preferably engineered so that the binding CH3 domain directly fuses with the hinge region of each Fc domain.

[0118] In other constructs, it may be desirable to provide peptide spacers between one or more constituent Fc domains. For example, peptide spacers may be placed between the hinge region and the CH2 domain and / or between the CH2 domain and the CH3 domain. For example, a compatible construct can be expressed in which the CH2 domain is deleted and the remaining CH3 domain (synthetic or non-synthetic) is linked to the hinge region using peptide spacers of 1-20, 1-10, or 1-5 amino acids. Such peptide spacers can be added, for example, to ensure that the regulatory elements of the constant region domain remain freely and easily accessible, or to ensure that the hinge region remains flexible and secure. Preferably, any linker peptide compatible with the present invention is considered to be relatively non-immunogenic and not interfere with the proper folding of Fc.

[0119] Changes in Fc amino acids In one embodiment, the Fc domain used in the hybrid nuclease molecule of the present invention 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 having at least one amino acid modification, e.g., 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) at the corresponding position of the human IgG1 Fc region compared to the wild-type amino acid.

[0120] The amino acid substitutions in the Fc variant may be located within the Fc domain, in a position that corresponds to a partial number that assigns that residue to the Fc region in the antibody.

[0121] In one embodiment, the Fc variant includes a substitution at an amino acid position located in the hinge domain or a portion thereof. In another embodiment, the Fc variant includes a substitution at an amino acid position located in the CH2 domain or a portion thereof. In another embodiment, the Fc variant includes a substitution at an amino acid position located in the CH3 domain or a portion thereof. In another embodiment, the Fc variant includes a substitution at an amino acid position located in the CH4 domain or a portion thereof.

[0122] In one embodiment, the hybrid nuclease molecule of the present invention comprises an Fc variant containing multiple amino acid substitutions. The hybrid nuclease molecule of the present invention may contain, for example, two, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions. Preferably, the amino acid substitutions are spatially arranged from each other at intervals of at least one amino acid position or more, for example, at intervals of at least two, three, four, five, six, seven, eight, nine or ten amino acid positions or more. More preferably, the manipulated amino acids are spatially arranged apart from each other at intervals of at least five, ten, fifteen, twenty or twenty-five amino acid positions or more.

[0123] In one embodiment, the Fc variant provides an improvement in at least one effector function provided by the Fc domain, including the wild-type Fc domain (e.g., an improvement in the Fc domain's ability to bind to an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII) or a complement protein (e.g., C1q), or an improvement in its ability to induce antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In another embodiment, the Fc variant provides an engineered cysteine ​​residue.

[0124] In some aspects, the Fc domain contains changes within the amino acid region 234-238, including the initial sequence LLGGP of the CH2 domain. In some aspects, Fc variants alter Fc-mediated effector function, particularly ADCC, and / or reduce binding avidity to the Fc receptor. In some aspects, sequence changes at positions closer to the CH2-CH3 junction, such as K322 or P331, eliminate complement-mediated cytotoxicity and / or alter avidity to FcR binding. In some aspects, the Fc domain incorporates changes at residues P238 and P331, for example, in which wild-type proline is replaced with serine at these positions. In some aspects, changes in one or more of the three cysteine ​​residues in the hinge region of a hinge area to encode CCC, SCC, SSC, SCS, or SSS can also affect FcR binding and molecular homogeneity, for example, by eliminating unpaired cysteine ​​that could destabilize the folded protein.

[0125] The hybrid nuclease molecules of the present invention may utilize Fc variants known in the art that result in improved effector function and / or FcR binding. Specifically, the hybrid nuclease molecules of the present invention may be, for example, those described in the International PCT Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, and WO02 / 060919, which are incorporated herein by reference, respectively. No. A2, No. WO03 / 074569A2, No. WO04 / 016750A2, No. WO04 / 029207A2, No. WO04 / 035752A2, No. WO04 / 063351A2, No. WO04 / 074455A2, No. WO04 / 099249A No. 2, WO05 / 040217A2, WO04 / 044859, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350A2 and WO06 / 085967A2; U.S. Patent Application Publications US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056; or U.S. Patent No. 5,648,260; No. 5,739,277 This may include changes (e.g., substitutions) at one or more amino acid positions disclosed in Patent Nos. 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 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 made to one or more of the disclosed amino acid positions. In another embodiment, a different change (a different substitution of one or more amino acids disclosed in the art) can be made to one or more of the disclosed amino acid positions.

[0126] Other amino acid mutations in the Fc domain are hypothesized to reduce binding affinity to the Fcγ receptor and Fcγ receptor subtypes. For example, at the Fc region positions 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, 33 Mutations at 5, 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 binding affinity, 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 altering Pro331 to Ser in IgG3 resulted in a 1 / 6 reduction in affinity compared to unmutated IgG3, indicating the involvement of Pro331 in FcγRI binding. In addition, U.S. Patent No. 5,624,821, issued on 29 April 1997, which is incorporated herein by reference in its entirety, discloses amino acid modifications at positions 234, 235, 236 and 237, 297, 318, 320 and 322 as potentially altering receptor binding affinity.

[0127] Further mutations intended for use include, for example, those described in U.S. Patent Application Publication No. 2006 / 0235208, published on 19 October 2006, which is incorporated herein by reference in its entirety. This publication includes 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, 330 This document describes Fc mutants that exhibit reduced binding to the Fcγ receptor, reduced antibody-dependent cell-mediated cytotoxicity, or reduced complement-dependent cytotoxicity, including at least one amino acid modification, including L, 330N, 330P, 330R, and 331L (numbering system conforms to the EU index), as well as the double mutants 236R / 237K, 236R / 325L, 236R / 328R, 325L / 328R, 235G / 236R, 267R / 269R, 234G / 235G, 236R / 237K / 325L, 236R / 325L / 328R, 235G / 236R / 237K, and 237K / 325L / 328R within the Fc region. Other mutations intended for use as described in this publication include 227G, 234D, 234E, 234G, 234I, 234Y, 235D, 235I, 235S, 236S, 239D, 246H, 255Y, 258H, 260H, 264I, 267D, 267E, 268D, 268E, 272H, 272I, 272R, 281D, 282G, 283H, 284E, 293R, 295E, 304T, 324G, 324I, 327D, 327A, 328A, 328D, 32 8E, 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, insertion of G between positions 297 and 298, position 29 This includes the insertion of A between 7 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 (the numbering scheme conforms to the EU index). Furthermore, the mutations described in U.S. Patent Application Publication No. 2006 / 0235208 include 227G / 332E, 234D / 332E, 234E / 332E, 234Y / 332E, 234I / 332E, 234G / 332E, 235I / 332E, 235S / 332E, 235D / 332E, 235E / 332E, 236S / 332E, 236A / 332E, and 236S / 332 D, 236A / 332D, 239D / 268E, 246H / 332E, 255Y / 332E, 258H / 332E, 260H / 332E, 264I / 332E, 267E / 332E, 2 67D / 332E, 268D / 332D, 268E / 332D, 268E / 332E, 268D / 332E, 268E / 330Y, 268D / 330Y, 272R / 332E, 272H / 332E, 283H / 332E, 284E / 332E, 293R / 332E, 295E / 332E, 304T / 332E, 324I / 332E, 324G / 332E, 324I / 332 D, 324G / 332D, 327D / 332E, 328A / 332E, 328T / 332E, 328V / 332E, 328I / 332E, 328F / 332E, 328Y / 332E, 32 8M / 332E, 328D / 332E, 328E / 332E, 328N / 332E, 328Q / 332E, 328A / 332D, 328T / 332D, 328V / 332D, 328I / 3 32D, 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 / 332E / 268E / 330Y, 239D / 332E / 3 27A, 239D / 332E / 268E / 327A, 239D / 332E / 330Y / 327A, 332E / 330Y / 268E / 327A, 239D / 332E / 268E / 330Y / 327A, Insertion of G to 297~298 / 332E, Insertion of A to 297~298 / 332E, Insertion of S to 297~298 / 332E, Insertion of D to 297~298 / 332E, Insertion of G to 326~327 / 332E, Insertion of A to 326~327 / 332 E, insertion of T into 326-327 / 332E, insertion of D into 326-327 / 332E, insertion of E into 326-327 / 332E, insertion of G into 235-236 / 332E, insertion of A into 235-236 / 332E, insertion of S into 235-236 / 332E, insertion of T into 235-236 / 332E, insertion of N into 235-236 / 332E, insertion of D into 235-236 / 332E, insertion of V into 235-236 / 332E, to 235-236 This includes insertions of L / 332E, G / 332D into 235-236, A / 332D into 235-236, S / 332D into 235-236, T / 332D into 235-236, N / 332D into 235-236, D / 332D into 235-236, V / 332D into 235-236, and L / 332D into 235-236 (numbering system conforms to EU index), and these are intended for use. Mutants 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 various embodiments, the modifications described are included either individually or in combination.

[0128] In one embodiment, the hybrid nuclease molecule of the present invention includes amino acid substitutions to the Fc domain that alter the antigen-independent effector function of the antibody, particularly the circulating half-life of the antibody. Such hybrid nuclease molecules exhibit either increased or decreased binding affinity to FcRn compared to hybrid nuclease molecules lacking these substitutions, resulting in either an extended or shortened serum half-life, respectively. Fc variants with improved affinity to FcRn are expected to have a longer serum half-life, and such molecules have useful applications in therapeutic methods for mammals where a long half-life of the administered polypeptide is desirable, such as for treating chronic diseases or chronic disorders. In contrast, Fc variants with decreased FcRn binding affinity are expected to have a shorter half-life, and such molecules are also useful for administration to mammals where a shortened circulating time is considered beneficial, such as for in vivo imaging, or in situations where prolonged presence of the starting polypeptide in the circulating blood would result in adverse side effects. Furthermore, Fc variants with reduced FcRn binding affinity are less likely to cross the placenta and are therefore useful in treating diseases or disorders in pregnant women. In addition, other applications where reduced FcRn binding affinity may be desirable include applications where localization to the brain, kidneys, and / or liver is desired. In one exemplary embodiment, the hybrid nuclease molecule of the present invention exhibits reduced transport from the vascular system through the epithelium of the renal glomeruli. In another embodiment, the hybrid nuclease molecule of the present invention exhibits reduced transport from the brain through the blood-brain barrier (BBB) ​​to the vascular lumen. In one embodiment, the hybrid nuclease molecule with altered FcRn binding affinity includes at least one Fc domain (e.g., one or two Fc domains) having one or more amino acid substitutions within the "FcRn binding loop" of the Fc domain. Exemplary amino acid substitutions that alter FcRn binding activity are disclosed in International PCT Publication WO05 / 047327, which is incorporated herein by reference.

[0129] In other embodiments, the hybrid nuclease molecules of the present invention include Fc variants that, compared to, for example, the wild-type Fc region, include amino acid substitutions that alter the antigen-dependent effector function of the polypeptide, particularly ADCC or complement activation. In exemplary embodiments, the hybrid nuclease molecule exhibits altered binding affinity to the Fcγ receptor (e.g., CD16). Such hybrid nuclease molecules exhibit either increased or decreased binding affinity to FcRγ compared to the wild-type polypeptide, thereby mediating enhancement or reduction of effector function, respectively. Fc variants with improved affinity to FcγR are expected to enhance effector function, and such molecules have useful applications in mammalian therapeutic methods where the destruction of the target molecule is desired. In contrast, Fc variants with decreased FcγR binding affinity are thought to reduce effector function, and such molecules are also useful in treating conditions where the destruction of target cells is undesirable, for example, when normal cells are thought to express the target molecule, or when long-term administration of polypeptides may lead to undesirable immune system activation. In one embodiment, a polypeptide containing Fc exhibits at least one modified antigen-dependent effector function, selected from the group consisting of opsonization, phagocytosis, complement-dependent cytotoxicity, antigen-dependent cell-mediated cytotoxicity (ADCC), or effector cell modulation, compared to a polypeptide containing a wild-type Fc region.

[0130] In one embodiment, the hybrid nuclease molecule exhibits modified binding affinity to activated FcγR (e.g., FcyI, FcγIIa, or FcγRIIIa). In another embodiment, the hybrid nuclease molecule exhibits modified binding affinity to inhibitory FcγR (e.g., FcγRIIb). Exemplary amino acid substitutions that alter FcR binding activity or complement binding activity are disclosed in International PCT Publication WO05 / 063815, which is incorporated herein by reference.

[0131] The hybrid nuclease molecules of the present invention may also include amino acid substitutions that alter the glycosylation of the hybrid nuclease molecule. For example, the Fc domain of the hybrid nuclease molecule may include an Fc domain having a mutation that leads to a reduction in glycosylation (e.g., N-linked or O-linked glycosylation), or it may include a modified glycoform of the wild-type Fc domain (e.g., a low-fucose glycan or a fucose-free glycan). In another embodiment, the hybrid nuclease molecule has amino acid substitutions near or within a glycosylation motif, such as an N-linked glycosylation motif containing the amino acid sequence NXT or NXS. Exemplary amino acid substitutions that reduce or alter glycosylation are disclosed in International PCT Publication WO05 / 018572 and U.S. Patent Application Publication 2007 / 0111281, which are incorporated herein by reference.

[0132] In other embodiments, the hybrid nuclease molecule of the present invention comprises at least one Fc domain having an engineered cysteine ​​residue or analogue located on a solvent-exposed surface. Preferably, the engineered cysteine ​​residue or analogue does not interfere with the effector function conferred by Fc. More preferably, the modification does not interfere with Fc's ability to bind to Fc receptors (e.g., FcγRI, FcγRII, or FcγRIII) or complement proteins (e.g., C1q), or to induce immunoeffector function (e.g., antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In a preferred embodiment, the hybrid nuclease molecule of the present invention comprises an Fc domain comprising at least one engineered free cysteine ​​residue or analogue substantially free of a disulfide bond with a second cysteine ​​residue. Any of the above-described manipulated cysteine ​​residues or their analogues can then be conjugated with a functional domain (for example, with a thiol-reactive heterobifunctional linker) using methods recognized in the art.

[0133] In one embodiment, the hybrid nuclease molecule of the present invention may include a genetically fused Fc domain having two or more of its constituent Fc domains independently selected from the Fc domains described herein. In one embodiment, the Fc domains are identical. In another embodiment, at least two of the Fc domains are different. For example, the Fc domains of the hybrid nuclease molecule of the present invention may contain the same number of amino acid residues, or they may differ in length by one or more amino acid residues (e.g., about 5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues), about 10 residues, about 15 residues, about 20 residues, about 30 residues, about 40 residues, or about 50 residues). In yet another embodiment, the Fc domains of the hybrid nuclease molecule of the present invention may differ at one or more amino acid positions. For example, at least two of the Fc domains may have different sequences at approximately five amino acid positions (e.g., positions 1, 2, 3, 4, or 5), approximately 10, approximately 15, approximately 20, approximately 30, approximately 40, or approximately 50).

[0134] Linker Domain In some embodiments, the hybrid nuclease molecule contains a linker domain. In some embodiments, the hybrid nuclease molecule contains multiple linker domains. In some embodiments, the linker domain is a polypeptide linker. In some aspects, it is desirable to use a polypeptide linker to fuse one or more Fc domains with one or more nuclease domains to form a hybrid nuclease molecule.

[0135] In one embodiment, the polypeptide linker is synthetic. As used herein, the term “synthetic” with respect to a polypeptide linker includes a peptide (or polypeptide) comprising a linear sequence of amino acids to which an amino acid sequence (which may or may not be naturally linked) is linked to a sequence (which may or may not be naturally linked) (e.g., an Fc domain sequence). For example, a polypeptide linker may include a non-natural polypeptide that is a modified form of a natural polypeptide (including mutations such as addition, substitution, or deletion), or a non-natural polypeptide comprising a first amino acid sequence (which may or may not be naturally linked). The polypeptide linkers of the present invention can be used, for example, to ensure that the Fc domain is juxtaposed so that proper folding and the formation of a functional Fc domain occur. Preferably, polypeptide linkers compatible with the present invention are considered to be relatively non-immunogenic and not inhibit the non-covalent association between monomeric subunits of binding proteins.

[0136] In one embodiment, the hybrid nuclease molecule of the present invention uses a polypeptide linker to link any two or more domains in frame into a single polypeptide chain. In one embodiment, the two or more domains may be independently selected from either the Fc domains or nuclease domains considered herein. For example, in one embodiment, identical Fc domains can be fused using the polypeptide linker to form a homomeric Fc region. In another embodiment, different Fc domains (e.g., a wild-type Fc domain and an Fc domain variant) can be fused using the polypeptide linker to form a heteromeric Fc region. In another embodiment, the polypeptide linker of the present invention can be used to genetically fuse the C-terminus of a first Fc domain (e.g., a hinge domain or a portion thereof, a CH2 domain or a portion thereof, a complete CH3 domain or a portion thereof, an FcRn binding portion, an FcγR binding portion, a complement binding portion, or a portion thereof) with the N-terminus of a second Fc domain (e.g., a complete Fc domain).

[0137] In one embodiment, the polypeptide linker includes a portion of the Fc domain. For example, in one embodiment, the polypeptide linker may include the immunoglobulin hinge domain of an IgG1, IgG2, IgG3, and / or IgG4 antibody. In another embodiment, the polypeptide linker may include the CH2 domain of an IgG1, IgG2, IgG3, and / or IgG4 antibody. In yet another embodiment, the polypeptide linker may include the CH3 domain of an IgG1, IgG2, IgG3, and / or IgG4 antibody. Other portions of immunoglobulins (e.g., human immunoglobulins) can also be used. For example, the polypeptide linker may include the CH1 domain or a portion thereof, the CL domain or a portion thereof, the VH domain or a portion thereof, or the VL domain or a portion thereof. The portions may be derived from any immunoglobulin, including, for example, an IgG1, IgG2, IgG3, and / or IgG4 antibody.

[0138] In exemplary embodiments, the polypeptide linker may include at least a portion of the immunoglobulin hinge region. In one embodiment, the polypeptide linker includes an upper hinge domain (e.g., the upper hinge domain of IgG1, IgG2, IgG3, or IgG4). In another embodiment, the polypeptide linker includes an intermediate hinge domain (e.g., the intermediate hinge domain of IgG1, IgG2, IgG3, or IgG4). In yet another embodiment, the polypeptide linker includes a lower hinge domain (e.g., the lower hinge domain of IgG1, IgG2, IgG3, or IgG4).

[0139] In other embodiments, the polypeptide linker may be constructed from hinge elements derived from the same or different antibody isotypes. In one embodiment, the polypeptide linker comprises a chimeric hinge comprising at least a portion of the IgG1 hinge region and at least a portion of the IgG2 hinge region. In one embodiment, the polypeptide linker comprises a chimeric hinge comprising at least a portion of the IgG1 hinge region and at least a portion of the IgG3 hinge region. In another embodiment, the polypeptide linker comprises a chimeric hinge comprising at least a portion of the IgG1 hinge region and at least a portion of the IgG4 hinge region. In one embodiment, the polypeptide linker comprises a chimeric hinge comprising at least a portion of the IgG2 hinge region and at least a portion of the IgG3 hinge region. In one embodiment, the polypeptide linker comprises a chimeric hinge comprising at least a portion of the IgG2 hinge region and at least a portion of the IgG4 hinge region. In one embodiment, the polypeptide linker includes a chimeric hinge comprising at least a portion of an IgG1 hinge region, at least a portion of an IgG2 hinge region, and at least a portion of an IgG4 hinge region. In another embodiment, the polypeptide linker may include an IgG1 upper and intermediate hinge, as well as a single IgG3 intermediate hinge repeat motif. In yet another embodiment, the polypeptide linker may include an IgG4 upper hinge, an IgG1 intermediate hinge, and an IgG2 lower hinge.

[0140] In another embodiment, the polypeptide linker includes or consists of a gly-ser linker. As used herein, the term “gly-ser linker” refers to a peptide consisting of a glycine residue and a serine residue. 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 one embodiment, a gly-ser linker may be inserted between two other polypeptide linker sequences (e.g., any of the polypeptide linker sequences described herein). In another embodiment, a gly-ser linker is attached to one or both ends of another polypeptide linker sequence (e.g., any of the polypeptide linker sequences described herein). In yet another embodiment, two or more gly-ser linkers are incorporated in series within the polypeptide linker. In one embodiment, the polypeptide linker of the present invention comprises at least a portion of an upper hinge region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule), at least a portion of an intermediate hinge region (e.g., derived from an IgG1, IgG2, IgG3, or IgG4 molecule), and a series of gly / ser amino acid residues (e.g., a gly / ser linker such as (Gly4Ser)n).

[0141] In one embodiment, the polypeptide linker of the present invention includes a non-natural immunoglobulin hinge domain, for example, a hinge domain not found naturally in a polypeptide containing a hinge domain, and / or a hinge domain modified to have a different amino acid sequence from a natural immunoglobulin hinge domain. In one embodiment, mutations can be introduced into the hinge domain to produce the polypeptide linker of the present invention. In one embodiment, the polypeptide linker of the present invention includes a hinge domain that does not contain the natural cysteine ​​count, i.e., the polypeptide linker contains either fewer or more cysteine ​​than a natural hinge molecule.

[0142] In other embodiments, the polypeptide linker of the present invention comprises a biologically relevant peptide sequence or a portion thereof. For example, the biologically relevant peptide sequence may, in no particular way, include sequences derived from rejection-inhibiting peptides or anti-inflammatory peptides. The rejection-inhibiting peptide or anti-inflammatory peptide may be selected from the group consisting of cytokine-inhibiting peptides, cell adhesion-inhibiting peptides, thrombin-inhibiting peptides, and platelet-inhibiting peptides. In one preferred embodiment, the polypeptide linker comprises 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, antipathogen 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, one of which may be used as a polypeptide linker, are disclosed in U.S. Patent No. 6,660,843, which is incorporated herein by reference.

[0143] It will be understood that variants of these exemplary polypeptide linkers can be created by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence encoding the polypeptide linker, such that one or more amino acid substitutions, additions, or deletions are introduced into the polypeptide linker. For example, mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0144] The polypeptide linker of the present invention is at least one amino acid long and can be of a variety of lengths. In one embodiment, the polypeptide linker of the present invention is about 1 to about 50 amino acid long. In this context, the term "about" refers to + / - 2 amino acid residues. Since the linker length must be a positive integer, lengths from about 1 amino acid to about 50 amino acids mean lengths from 1 amino acid to 48 to 52 amino acids. In another embodiment, the polypeptide linker of the present invention is about 10 to 20 amino acid long. In yet another embodiment, the polypeptide linker of the present invention is about 15 to about 50 amino acid long.

[0145] In another embodiment, the polypeptide linker of the present invention has an amino acid length of about 20 to about 45 amino acids. In another embodiment, the polypeptide linker of the present invention has an amino acid length of about 15 to about 25 amino acids. In another embodiment, the polypeptide linker of the present invention has an amino acid length of 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 amino acids or more.

[0146] Polypeptide linkers can be introduced into polypeptide sequences using methods known in the art. Modifications can be confirmed by DNA sequence analysis. Host cells can be transformed using plasmid DNA to ensure stable production of the polypeptide.

[0147] Nuclease domain In some cases, hybrid nuclease molecules contain nuclease domains. Therefore, the hybrid nuclease molecules of the present invention typically contain at least one nuclease domain and at least one linked Fc domain. In some cases, hybrid nuclease molecules contain multiple nuclease domains.

[0148] In some embodiments, the nuclease domain is substantially all, or at least, the enzymatically active fragment of the DNase. In some embodiments, the DNase is a type I secreted DNase, preferably a human DNase such as DNase 1. Exemplary DNase 1 domains are shown in SEQ ID NOs 48-53 and 102. Exemplary human DNase 1 is described in UniProtKB entry P24855 (SEQ ID NOs: 49 and 102). In some embodiments, the DNase is DNase 1 and / or DNase 1-like (DNase L) enzymes 1-3. Exemplary human DNase 1-like enzymes 1-3 are described in UniProtKB entry Q13609 (SEQ ID NOs: 57 and 103). In some embodiments, the DNase is TREX1 (3' repair exonuclease 1). Exemplary human TREX1 is described in UniProtKB entry Q9NSU2 (SEQ ID NO: 104). Preferably, human TREX1 is a C-terminal truncated human TREX1 lacking an intracellular nuclear targeting sequence, such as human TREX1 lacking 72 C-terminal amino acids as shown in SEQ ID NO:105.

[0149] In some embodiments, the nuclease domain is substantially all, or at least, the enzymatically active fragment of the RNase. In some embodiments, the RNase is an extracellular or secretory RNase of the RNase A superfamily, e.g., RNase A, preferably human pancreatic RNase. Exemplary human RNases are described in UniProtKB entry P07998 (SEQ ID NO: 58 and 101).

[0150] In one embodiment, the nuclease domain is functionally linked to the N-terminus of the Fc domain (e.g., chemically conjugated or genetically fused (e.g., directly or via a polypeptide linker)). In another embodiment, the nuclease domain is functionally linked to the C-terminus of the Fc domain (e.g., chemically conjugated or genetically fused (e.g., directly or via a polypeptide linker)). In yet another embodiment, the nuclease domain is functionally linked to the Fc domain via its amino acid side chain (e.g., chemically conjugated or genetically fused (e.g., directly or via a polypeptide linker)). In one exemplary embodiment, the nuclease domain is fused to the Fc domain via the hinge domain or a portion thereof of a human immunoglobulin.

[0151] In one embodiment, the hybrid nuclease molecule of the present invention comprises two or more nuclease domains and at least one Fc domain. For example, the nuclease domain may be functionally ligated to both the N-terminus and C-terminus of the Fc domain. In another exemplary embodiment, the nuclease domain may be functionally ligated to both the N-terminus and C-terminus of multiple Fc domains (e.g., two, three, four, five or more Fc domains) that are ligated together in series to form a tandem array of Fc domains.

[0152] In other embodiments, two or more nuclease domains are linked in series with each other (e.g., via a polypeptide linker), and the tandem array of nuclease domains is functionally linked (chemically conjugated or genetically fused (e.g., directly or via a polypeptide linker)) to either the C-terminus or N-terminus of either an Fc domain or a tandem array of Fc domains. In other embodiments, the tandem array of nuclease domains is functionally linked to both the C-terminus and N-terminus of either an Fc domain or a tandem array of Fc domains.

[0153] In other embodiments, one or more nuclease domains may be inserted between two Fc domains. For example, one or more nuclease domains may form all or part of the polypeptide linker of the hybrid nuclease molecule of the present invention.

[0154] A preferred hybrid nuclease molecule of the present invention comprises at least one nuclease domain (e.g., RNase or DNase), at least one linker domain, and at least one Fc domain.

[0155] In one embodiment, the hybrid nuclease molecule of the present invention comprises at least one nuclease domain specific to a target molecule that mediates a biological effect. In another embodiment, the binding of the hybrid nuclease molecule of the present invention to a target molecule (e.g., DNA or RNA) results in the reduction or elimination of the target molecule, for example, from cells, tissues, or circulating blood.

[0156] In one embodiment, the hybrid nuclease molecule of the present invention may contain two or more nuclease domains. In one embodiment, the nuclease domains are identical, for example, RNase and RNase, or TREX1 and TREX1. In another embodiment, the nuclease domains are different, for example, DNase and RNase.

[0157] In other embodiments, the hybrid nuclease molecules of the present invention may be assembled together or with other polypeptides to form a binding protein ("multimer") having two or more polypeptides, where at least one polypeptide of the multimer is the hybrid nuclease molecule of the present invention. Exemplary multimer forms include modified binding proteins that are dimers, trimers, tetramers, and hexamers. In one embodiment, the polypeptides of the multimer are identical (i.e., modified homomeric binding proteins, e.g., homodimers, homotetramers). In another embodiment, the polypeptides of the multimer are different (e.g., heteromeric).

[0158] Method for producing hybrid nuclease molecules The hybrid nuclease molecules of the present invention are primarily produced in transformed host cells using recombinant DNA techniques. To do this, recombinant DNA molecules encoding the peptides are prepared. Methods for preparing such DNA molecules are well known in the art. For example, the sequences encoding these peptides can be excised from DNA using suitable restriction enzymes. Alternatively, DNA molecules can be synthesized using chemical synthesis methods such as the phosphoramidate method. Furthermore, a combination of these methods can be used.

[0159] The present invention also includes vectors capable of expressing these peptides in a suitable host. These vectors comprise DNA molecules encoding these peptides, functionally linked to regulatory expression sequences. Methods for influencing this functional linkage, either before or after insertion of the DNA molecules into the vector, are well known. Regulatory expression sequences include 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.

[0160] The resulting vector, which contains DNA molecules, is used to transform a suitable host. This transformation can be carried out using methods well known in the art.

[0161] Any of the many available and well-known host cells can be used in carrying out the present invention. The selection of a specific host depends on several factors known in the art. These include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, transformation rate, ease of peptide recovery, expression characteristics, biological safety, and cost. These factors must be balanced, with the understanding that not all hosts are equally effective for the expression of a particular DNA sequence. In these general guidelines, useful microbial hosts include bacterial (e.g., Escherichia coli) species, yeast (e.g., Saccharomyces) and other fungi, insects, plants, mammalian (including human) cells, or other hosts known in the art.

[0162] Next, the transformed host cells are cultured and purified. The host cells may be cultured under conventional fermentation conditions to express the desired compound. Such fermentation conditions are well known in the art. Then, the peptide is purified from the culture by a method well known in the art.

[0163] Compounds can also be prepared by synthetic methods. For example, solid-phase synthesis can be used. Suitable methods are well known in the art and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifled (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 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. Solid-phase synthesis is the most cost-effective method for producing small peptides and is therefore a preferred technique for producing individual peptides. Compounds containing derivatized peptides or non-peptide groups can be synthesized by well-known organic chemical methods.

[0164] Other methods for molecular expression / synthesis are generally known to those skilled in the art.

[0165] Pharmaceutical compositions and therapeutic uses In some embodiments, the hybrid nuclease molecule is administered alone. In some embodiments, the hybrid nuclease molecule is administered before the administration of at least one other therapeutic agent. In some embodiments, the hybrid nuclease molecule is administered concurrently with the administration of at least one other therapeutic agent. In some embodiments, the hybrid nuclease molecule is administered after the administration of at least one other therapeutic agent. In other embodiments, the hybrid nuclease molecule 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 hybrid nuclease molecule is combined with other drugs / compounds. In some embodiments, the hybrid nuclease molecule and other drugs are administered simultaneously. In some embodiments, the hybrid nuclease molecule and other drugs are not administered simultaneously, but the hybrid nuclease molecule is administered before or after the administration of the drugs. In some embodiments, the subject receives both the hybrid nuclease molecule and other drugs during the same prophylactic period, at the time of injury and / or treatment period.

[0166] The pharmaceutical compositions of the present invention can be administered in the form of combination therapy, i.e., in combination with other agents. In one embodiment, the combination therapy comprises a nuclease molecule and at least one other agent. The agents include, but are not limited to, chemical compositions prepared by in vitro synthesis, antibodies, antigen-binding regions, and combinations and conjugates thereof. In one embodiment, the agents can act as agonists, antagonists, allosteric modulators, or toxins.

[0167] In one embodiment, the present invention provides a pharmaceutical composition comprising a hybrid nuclease molecule together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or auxiliary agent.

[0168] In one embodiment, the present invention provides a pharmaceutical composition comprising a hybrid nuclease molecule and a therapeutically effective amount of at least one additional therapeutic agent, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0169] In some embodiments, the acceptable formulation materials are preferably non-toxic to the recipient at the dosage and concentration used. In some embodiments, the formulation materials are for subcutaneous and / or intravenous administration. In some embodiments, the pharmaceutical composition may include formulation materials to modify, maintain, or preserve, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, aroma, sterility, stability, rate of dissolution or release, adsorption or permeability of the composition. In one embodiment, suitable formulation materials include, but are not limited to, the following: amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffering agents (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulin); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (poly Vinylpyrrolidone, etc.); low molecular weight polypeptides; salt-forming counterions (sodium, etc.); preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide, etc.); solvents (glycerin, propylene glycol, or polyethylene glycol, etc.); sugar alcohols (mannitol or sorbitol, etc.); suspending agents; surfactants or wetting agents (pluronic acid, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate 80, etc., triton, tromethamine, lecithin, cholesterol, tyroxapol, etc.); stability enhancers (sucrose or sorbitol, etc.); tonicity enhancers (alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, etc.); delivery media; 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.)

[0170] In some embodiments, hybrid nuclease molecules and / or therapeutic molecules are linked to half-life-extending media known in the Art. Such media include, but are not limited to, polyethylene glycol, glycogen (e.g., glycosylation of the hybrid nuclease molecule), and dextran. Such media are described, for example, in U.S. Patent Application No. 09 / 428,082, now U.S. Patent No. 6,660,843, and PCT Publication No. WO 99 / 25044, which are incorporated herein by reference, regardless of their purpose.

[0171] In one embodiment, the optimal pharmaceutical composition can be determined by those skilled in the art, for example, depending on the intended route of administration, delivery method, and desired dose. See, for example, Remington's Pharmaceutical Sciences, above. In one embodiment, such a composition may affect the physical state, stability, in vivo release rate, and in vivo efflux rate of the antibody of the present invention.

[0172] In some embodiments, the primary medium or carrier in the pharmaceutical composition may be either aqueous or non-aqueous. For example, in some embodiments, suitable mediums or carriers may be water for injection, physiological saline, or artificial cerebrospinal fluid, optionally with the addition of other materials common in compositions for parenteral administration. In some embodiments, physiological saline includes isotonic phosphate-buffered saline. In some embodiments, neutral buffered saline, or saline mixed with serum albumin, are further exemplary mediums. In some embodiments, the pharmaceutical composition comprises Tris buffer with a pH of about 7.0–8.5, or acetate buffer with a pH of about 4.0–5.5, which may further include sorbitol or a suitable substitute thereof. In some embodiments, a composition comprising a hybrid nuclease molecule with or without at least one additional therapeutic agent can be prepared for storage in the form of a lyophilized solid (cake) or aqueous solution by mixing a selected composition having the desired purity with an optional formulation agent (Remington's Pharmaceutical Sciences, hereafter). Furthermore, in one embodiment, a composition comprising a hybrid nuclease molecule with or without at least one additional therapeutic agent can be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0173] In one embodiment, a pharmaceutical composition may be selected for parenteral delivery. In another embodiment, a composition may be selected for inhalation or delivery through the gastrointestinal tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0174] In one embodiment, the components of the formulation are present at concentrations acceptable to the administration site. In another embodiment, a buffer is used to maintain the composition at or slightly below the physiological pH, typically in the pH range of about 5 to about 8.

[0175] In some embodiments, when parenteral administration is intended, the therapeutic composition may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired hybrid nuclease molecule, with or without additional therapeutic agents, in a pharmaceutically acceptable medium. In some embodiments, the medium for parenteral infusion is sterile distilled water, in which the hybrid nuclease molecule, with or without additional therapeutic agents, is formulated as a properly preserved sterile isotonic solution. In some embodiments, the preparation may comprise a formulation of the desired molecule with a substance such as an injectable microsphere, biodegradable particles, a high molecular weight compound (polylactic acid or polyglycolic acid), beads, or liposomes, which results in controlled or sustained release of the product, and which may then be delivered via depot injection. In some embodiments, hyaluronic acid may also be used, which may have the effect of improving the duration of action in the circulating blood. In some embodiments, other suitable means for introducing the desired molecule include implantable drug delivery devices.

[0176] In one embodiment, a pharmaceutical composition can be formulated for inhalation. In one embodiment, a hybrid nuclease molecule can be formulated as a dry powder for inhalation, with or without at least one additional therapeutic agent. In one embodiment, an inhalation solution containing a hybrid nuclease molecule, with or without at least one additional therapeutic agent, can be formulated together with a spray for aerosol delivery. In one embodiment, the solution can also be atomized. Administration to the lungs is further described in PCT application PCT / US94 / 001875, which describes the pulmonary delivery of chemically modified proteins.

[0177] In some embodiments, the formulation is intended to be administered orally. In some embodiments, the hybrid nuclease molecule administered in this manner can be formulated with or without a carrier commonly used in the formulation of solid dosage forms such as tablets and capsules, with or without at least one additional therapeutic agent. In some embodiments, the capsule can be designed to release the active portion of the formulation at a location in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. In some embodiments, at least one additional agent may be included to enhance the absorption of the hybrid nuclease molecule and / or any additional therapeutic agent. In some embodiments, diluents, flavoring agents, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0178] In one embodiment, the pharmaceutical composition may contain an effective amount of a hybrid nuclease molecule in a mixture with a non-toxic excipient suitable for the manufacture of tablets, with or without at least one additional therapeutic agent. In one embodiment, the solution can be prepared as a unit dose dosage form by dissolving the tablets in sterile water or another suitable medium. In one embodiment, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; or binders such as starch, gelatin or gum arabic; or lubricants such as magnesium stearate, stearic acid or talc.

[0179] Other pharmaceutical compositions, including formulations containing, with or without, a hybrid nuclease molecule as an additional therapeutic agent in a sustained-release or controlled-release formulation, will be apparent to those skilled in the art. In some embodiments, methods for formulating various other means of sustained-release or controlled-release, such as liposome carriers, biodegradable 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 the delivery of pharmaceutical compositions. In some embodiments, sustained-release formulations may include a semipermeable polymer matrix in the form of a molded article, such as a thin film or microcapsule. The sustained-release matrix may include polyester, hydrogel, polylactide (U.S. Patent No. 3,773,919 and EP No. 058,481), copolymer of L-glutamic acid and γ-ethyl-L-glutamic acid (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., as described above), or poly-D(-)-3-hydroxybutyric acid (EP No. 133,988). In some embodiments, the sustained-release composition may also include liposomes that can be prepared by any of several methods known in the art. For example, see Eppstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

[0180] Pharmaceutical compositions intended for in vivo administration are typically sterile. In some embodiments, this can be achieved by filtration through a sterile filtration membrane. In some embodiments, when the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. In some embodiments, compositions for parenteral administration can be stored in lyophilized form or as a solution. In some embodiments, parenteral compositions are generally placed in containers with a sterile access port, such as intravenous solution bags or vials with a stopper that can be punctured by a subcutaneous injection needle.

[0181] In some embodiments, after a pharmaceutical composition has been formulated, it may be stored in a suitable sterile vial as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In some embodiments, such formulations may be stored either in a ready-to-use form or in a form that is reconstituted before administration (e.g., lyophilized).

[0182] In one embodiment, a kit for preparing single-dose units is provided. In one embodiment, the kit may include both a first container containing a dry protein and a second container containing an aqueous formulation. In one embodiment, the kit may also include single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes).

[0183] In some embodiments, the effective dose of a pharmaceutical composition containing a hybrid nuclease molecule, used therapeutically, with or without at least one additional therapeutic agent, is considered to depend, for example, on the context and purpose of the treatment. Those skilled in the art will understand that, according to some embodiments, and therefore in part, the appropriate dose level for treatment will vary depending on the indication, route of administration, and the patient's physique (weight, body surface area, or organ size) and / or condition (age and overall health) when the hybrid nuclease molecule is used with or without at least one additional therapeutic agent. In some embodiments, a clinician may set the dose and modify the route of administration to obtain the optimal therapeutic effect. In some embodiments, a typical dose may range from about 0.1 μg / kg to a maximum of about 100 mg / kg or more, depending on the factors described above. In one embodiment, the dosage may range from 0.1 μg / kg to a maximum of approximately 100 mg / kg; or from 1 μg / kg to a maximum of approximately 100 mg / kg; or from 5 μg / kg to a maximum of approximately 100 mg / kg.

[0184] In one embodiment, the frequency of administration is considered to take into account the pharmacokinetic parameters of the hybrid nuclease molecule and / or any additional therapeutic agent in the formulation used. In one embodiment, the clinician administers the composition until the dose reaches the desired effect. In one embodiment, the composition can therefore be administered as a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implantable device or catheter. Further fine-tuning of the appropriate dose is routinely performed by those skilled in the art and is within the scope of routine practice. In one embodiment, the appropriate dose can be determined by using appropriate dose-response data.

[0185] In one embodiment, the route of administration of the pharmaceutical composition is, for example, by oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal, or intrafocal route of injection; by a continuous-release system or by an implantable device, etc. In one embodiment, the composition may be administered by bolus injection, by continuous infusion, or by an implantable device.

[0186] In one embodiment, the composition can be administered topically via implantation of a membrane, sponge, or other suitable substance on which the desired molecule is adsorbed or encapsulated. In one embodiment, if an implantation device is used, the device can be implanted in any suitable tissue or organ, and the delivery of the desired molecule may be via diffusion, sustained-release bolus, or continuous administration.

[0187] In one embodiment, it may be desirable to use an ex vivo pharmaceutically acceptable

[0188] In some embodiments, hybrid nuclease molecules and / or any additional therapeutic agents can be delivered by transplanting certain cells that have been genetically engineered using methods such as those described herein to express and secrete their polypeptides. In some embodiments, such cells may be animal cells or human cells and may be autologous, xenogeneic, or xenogeneic. In some embodiments, the cells may be immortalized. In some embodiments, the cells may be encapsulated to prevent invasion of surrounding tissue for the purpose of reducing the likelihood of an immunological response. In some embodiments, the encapsulation material is typically a biocompatible, semipermeable polymer encapsulation or membrane that allows for the release of protein products but prevents destruction of the cells by the patient's immune system or other harmful factors from surrounding tissue.

[0189] The hybrid nuclease molecules of the present invention are particularly effective in treating autoimmune disorders or abnormal immune responses. In this regard, it will be understood that the hybrid nuclease molecules of the present invention can be used to suppress, inhibit, modulate, treat, or eliminate unwanted immune responses to both external and autoantigens. In yet another embodiment, the polypeptides of the present invention are effective in treating insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes mellitus, Goodpasture syndrome, and pemphigus vulgaris. It can be used to treat immunodeficiencies, including but not limited to bullous pemphigoid, sympathetic ophthalmitis, lens-induced uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, idiopathic cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus erythematosus, systemic lupus erythematosus, or connective tissue diseases.

[0190] kit The kit may include the hybrid nuclease molecules disclosed herein and instructions for use. The kit may also include, in a suitable container, the hybrid nuclease molecules disclosed herein, one or more controls, and various buffers, reagents, enzymes, and other standard components well known in the art.

[0191] The container may include at least one vial, well, test tube, flask, bottle, syringe, or other container means that can contain and optionally divide the hybrid nuclease molecule. If additional components are provided, the kit may include additional containers that can contain these components. The kit may also include means for tightly housing containers of the hybrid nuclease molecule and any other reagents for sale. Such containers may include injection-molded or blow-molded plastic containers that hold the desired vials inside. The container and / or kit may include labels with instructions for use and / or warnings. [Examples]

[0192] The following are examples of specific embodiments for carrying out the present invention. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. While efforts have been made to be accurate with respect to the figures used (e.g., quantities, temperatures, etc.), some degree of experimental error and deviation should naturally be acceptable.

[0193] Unless otherwise specified, the implementation of this invention shall utilize conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the scope of the art. Such methods are well described in the literature. For example, T.E. Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundherg, Advanced Organic Chemistry 3. rd See Ed (Plenum Press) Vols. A and B (1992).

[0194] Example 1. General approach for producing hybrid nuclease molecules Hybrid nuclease molecules were designed as modular cassettes with compatible restriction enzyme sites for shuttling and domain exchange, allowing for the incorporation of desired structures and functional activities in mono-enzyme or multi-enzyme configurations. Schematic structures of various embodiments of hybrid nuclease molecules are shown in Figure 1. The nucleotide and amino acid sequences of representative hybrid nuclease molecules are shown in Table 1.

[0195] Human cDNA was isolated from human pancreatic RNA (Ambion) or human PBMC RNA derived from normal human peripheral blood lymphocytes (approximately 5 × 10⁶ cells) using the QIAgen RNAeasy kit (Valencia, CA) and the QIAshredder kit (Qiagen, Valencia, CA) for homogenizing cell lysates. Human PBMCs were isolated from heparinized human blood diluted 1:1 in D-PBS and layered on a Ficoll gradient in LSM Lymphocyte Separation Medium (MP Biomedicals, Irvine, CA).

[0196] Mouse spleen RNA was isolated from approximately 5 × 10⁶ splenocytes using the QIAgen RNAeasy kit (Valencia, CA). Cells were pelleted from the culture medium by centrifugation, and 5 × 10⁶ cells were used for RNA preparation. RNA was isolated from cells using the QIAGEN RNAeasy kit (Valencia, Calif.) total RNA isolation kit and QIAGEN QIAshredder according to the manufacturer's instructions provided with the kit. 1–2 micrograms (1–2 μg) of total RNA was used as a template for cDNA preparation by reverse transcription. This RNA, 300 ng of random primers, 500 ng of Oligo dT (12–18), and 1 μl of 25 mM dNTPs were mixed and denatured at 80°C for 5 minutes, after which enzymes were added. Superscript III reverse transcriptase (Invitrogen, Life Technologies) was added to a total volume of 25 μl of RNA + primer mixture in the presence of 5× second-chain buffer and 0.1 M DTT, which were provided with the enzyme. The reverse transcription reaction was carried out at 50°C for 1 hour.

[0197] 10–100 ng of cDNA was used in PCR amplification reactions using primers specific to the nuclease gene of interest (RNaseA, RNase1, DNase1, Trex1, DNase1L3, etc.). For the initial cloning reaction, primers were designed to isolate full-length cDNA or truncated products encoding the gene of interest. Full-length or truncated PCR fragments were isolated by agarose gel electrophoresis and purified using a Qiagen QIAquick column to remove nucleotides, primers, and unwanted amplification products. The purified fragments were cloned into a pCR2.1 TOPO cloning vector (Invitrogen, Carlsbad, CA) and transformed into TOP10 competent bacteria. Isolated colonies were excised and grown overnight in Luria Broth medium containing 50 ug / ml carbenicillin to isolate plasmids. TOPO clones were screened for correct-sized inserts by digestion with EcoRI (NEB, Ipswich, MA) restriction enzymes and agarose gel electrophoresis of the digested fragments. DNA sequencing of positive clones was performed using ABI Ready Reaction Mix v 3.1 and analyzed using an ABI 3730 XL DNA sequencer. Once correct clones were obtained, further sequence modifications were designed, and PCR reactions were performed to produce the desired alleles or expression cassettes. Shortened products and alleles were produced by PCR mutagenesis using duplicate primers to introduce mutations at specific locations in the gene. Linkers were synthesized by duplicate PCR using internal duplicate primers, followed by sequential PCR to attach additional sequences to each end. Hybrid nuclease molecules were assembled as chains of several interchangeable cassettes.A preferred embodiment of the molecule comprises a default leader peptide, a nuclease cassette, an optional cassette encoding one selected from several different polypeptide linkers, an Ig Fc domain cassette having either a stop codon or linker at the carboxyl terminus of the CH3 domain, and, in the case of a resolvICase-type molecule, a second linker cassette followed by a second nuclease cassette. Figure 1 illustrates the cassette structure of these hybrid nuclease molecules and examples of sequences that may be inserted at each position. Once the hybrid nuclease molecules are assembled, they are translocated to a suitable mammalian expression plasmid pDG for transient expression in COS7 or other cells, and for stable expression in CHO DG44 cells, using methotrexate-mediated selection for DHFR.

[0198] Transient expression of hybrid nuclease molecules COS-7 cells were transiently transfected with an expression vector pDG containing an insert of a hybrid nuclease molecule gene. The day before transfection, cells were seeded at a rate of 4 × 10⁵ cells per 60 mm dish in 4 ml DMEM (ThermoFisher / Mediatech cell gro) + 10% FBS tissue medium. 4.5 g / L of glucose, sodium pyruvate, 4 mM L-glutamine, and non-essential amino acids were added to the DMEM base medium. Fetal bovine serum (Hyclone, Logan, UT ThermoFisher Scientific) was added to the medium to a final volume ratio of 10%. Cells were incubated overnight at 37°C and 5% CO₂, resulting in a cell density of approximately 40–80% on the day of transfection. Plasmid DNA was prepared using the Qiagen (Valencia, CA) QIAprep miniprep kit according to the manufacturer's instructions and eluted in 50 μl of EB buffer. DNA concentration was measured using a Nanodrop 1000 (ThermoFisher Scientific, Wilmington DE) spectrophotometer. Plasmid DNA was transfected using Polyfect (Qiagen, Valencia, CA) transfection reagent according to the manufacturer's instructions, with 2.5 ug of plasmid DNA per 60 mm dish and 15 ug of Polyfect reagent in a 150 ug serum-free DMEM transfection cocktail. After complex formation, the reaction was diluted in 1 ml of cell growth medium containing serum and all additives and dropped onto a plate containing 3 ml of fresh DMEM complete medium. After incubation of transient transfections for 48–72 hours, the culture supernatant was collected for further analysis.

[0199] Preparation of a stable CHO DG44 transfectant expressing a hybrid nuclease molecule of interest. Stable production of the hybrid nuclease molecule was achieved by electroporation of a selectable and amplified plasmid pDG containing nuclease-Ig cDNA under the control of the CMV promoter into Chinese hamster ovary (CHO) cells. This pDG vector is a modified version of pcDNA3 encoding a DHFR selection marker, with a weakened promoter to increase the selective pressure on the plasmid. Plasmid DNA was prepared using the Qiagen maxiprep kit, and the purified plasmid was linearized at a single AscI site, followed by phenol extraction and ethanol precipitation. Salmon sperm DNA (Sigma-Aldrich, St. Louis, Mo.) was added as the carrier DNA, and 100 μg each of plasmid and carrier DNA were used for 10 7CHO DG44 cells were transfected by electroporation. The cells were grown until the logarithmic phase in Excell 302 medium (JRH Biosciences), containing glutamine (4 mM), pyruvate, recombinant insulin, penicillin-streptomycin, and 2× DMEM non-essential amino acids (all from Life Technologies, Gaithersburg, Md.), hereafter referred to herein as "Excell 302 Complete" medium. The medium for untransfected cells also contained HT (diluted from a 100× solution of hypoxanthine and thymidine) (Invitrogen / Life Technologies). For selective transfection, the medium contained various levels of methotrexate (Sigma-Aldrich) ranging from 50 nM to 1 μM as a selective agent. Electroporation was performed at 280 volts and 950 microfarads. After transfected cells were allowed to recover overnight in non-selective medium, selective plating was performed into 96-well flat-bottom plates (Costar) at various serial dilutions ranging from 125 cells / well to 2000 cells / well. The medium for cell cloning was Excell 302 complete medium containing 50 nM methotrexate. Once sufficient clonal growth was achieved, serial dilutions of the culture supernatant from the master well were screened for the expression of hybrid nuclease molecules using an IgG sandwich ELISA. Briefly, NUNC immulon II plates were coated overnight at 4°C with 7.5 micrograms / ml of F(ab'2) goat anti-mouse IgG (KPL Labs, Gaithersburg, MD) or 2 ug / ml of goat anti-human or anti-mouse IgG (Jackson Immunoresearch, WestGrove PA) in PBS. The plates were blocked in PBS / 2-3% BSA, and serial dilutions of the culture supernatant were incubated at room temperature for 2-3 hours.The plates were washed three times in PBS / 0.05% Tween 20 and incubated for 1-2 hours at room temperature with either a mixture of horseradish peroxidase-conjugated F(ab'2) goat anti-mouse IgG2a (Southern Biotechnologies) and goat anti-mouse IgG (KPL) in PBS / 1.0% BSA at a ratio of 1:3500, or with horseradish peroxidase-conjugated F(ab')2 goat anti-human IgG1 (Jackson Immunoresearch, WestGrove, PA) at a ratio of 1:2500. The plates were washed four times in PBS / 0.05% Tween 20, and binding was detected using SureBlue Reserve and TMB substrate (KPL Labs, Gaithersburg, MD). The reaction was stopped by adding an equal volume of 1N HCl, and the plates were read at 450 nM using a Spectramax Pro plate reader (Microdevices, Sunnyvale CA). The clones with the highest fusion protein production were grown in T25 flasks, followed by T75 flasks, to obtain a sufficient number of cells for freezing and for scaling up fusion protein production. Production levels in cultures from the top four superior clones were further increased by progressive amplification in methotrexate-containing medium. The concentration of methotrexate in Excell 302 complete medium was gradually increased with each successive cell passage to ensure that only cells with amplified DHFR plasmids could survive.

[0200] The supernatant was collected from CHO cells expressing hybrid nuclease molecules, filtered through a 0.2 μm PES express filter (Nalgene, Rochester, NY), and then passed through a protein A-agarose (IPA 300 cross-linked agarose) column (Repligen, Needham, Mass.). The column was washed with column washing buffer (90 mM Tris base, 150 mM NaCl, 0.05% sodium azide, pH 8.7), and the bound protein was eluted using 0.1 M citrate buffer, pH 3.0. The fractions were collected, and the protein concentration was determined at 280 nM using a Nanodrop (Wilmington DE) microsample spectrophotometer. A blank test was also performed using 0.1 M citrate buffer, pH 3.0. After pooling the fraction containing the hybrid nuclease molecule, buffer exchange was performed by continuous spinning in PBS using a centricon concentrator followed by filtration through a 0.2 μm filter device to reduce the risk of endotoxin contamination.

[0201] Example 2: Construction of an RNase-Ig fusion gene Mouse RNase 1 was amplified as full-length cDNA from an EST library (by Dr. C. Raine, Albert Einstein School of Medicine, Bronx, NY, who sent clones to our laboratory without exchanging MTAs). The sequence-specific 5' and 3' primers used were based on publicly available sequences. The clone sequences were verified by sequence analysis. The Genebank accession number is NCBI geneID 19752. Full-length human RNase 1 was isolated from cDNA derived from human pancreatic total RNA using the random primer method and the oligo-dT primer method (Ambion / Applied Biosystems, Austin, TX).

[0202] After isolating a full-length clone, primers were designed to create a fusion gene with the Fc domain of mouse IgG2a or human IgG1 (SEQ ID NO: 40). Two primers were designed for the 5' sequence fused at the amino terminus of the Fc tail; the first incorporated a native leader peptide derived from mouse (or human) RNase, while the second attached the AgeI site to the amino terminus of the RNase at the expected signal peptide cleavage site, with the aim of fusing the RNase with a human VKIII leader peptide that the inventors had already cloned and were using for other expression tests. For mouse RNase, the sequence of the first primer is as follows: mribNL5' 30mer (RNase 5' with native leader and HindIII+Kozak) TIFF0007897613000001.tif7159

[0203] The second primer creates a gene fusion ligation site between the existing leader sequence and the mature 5' end sequence of the RNase, at or near the expected leader peptide cleavage site. 27mer (RNase 5' mature sequence (no leader, contains Age I site)) TIFF0007897613000002.tif7157

[0204] The sequences of the 3' primers for fusion of the carboxyl terminus and the amino terminus of the Fc tail of the RNase to mouse IgG2a are as follows: mrib3NH2 28mer (RNase 3' terminus, containing an XhoI site for fusion with mIgG2a) TIFF0007897613000003.tif7158

[0205] To create the -Ig-RNase fusion gene located at the amino terminus of the -Ig tail relative to the RNase enzyme domain, two additional oligonucleotides were designed. mrib5X A 36-mer RNase 5' end having an XbaI site for fusion with the linker aa and the carboxyl terminus of the Fc domain. TIFF0007897613000004.tif14136mrib3X The 31-mer RNase 3' end has two stop codons and an XbaI site for fusion with the carboxyl terminus of the Fc domain. TIFF0007897613000005.tif7169

[0206] Example 3: Isolation of human Fc domains and mouse Fc domains, and introduction of mutations into coding sequences. For the isolation of mouse and human Fc domains (SEQ ID NO: 40), RNA was obtained from mouse or human tissue as follows: Single-cell suspensions were prepared from mouse spleen in RPMI medium. Alternatively, human PBMCs were isolated from fresh whole blood using Lymphocyte Separation Media (LSM) Organon Teknika (Durham, NC). The buffy coat was collected according to the manufacturer's instructions, and the cells were washed three times in PBS before use. Cells were pelleted from the medium by centrifugation and 2 × 10⁶ cells were obtained. 7Cells were used for RNA preparation. RNA was isolated from cells using the QIAGEN RNAeasy kit (Valencia, Calif.) total RNA isolation kit and QIAGEN QIAshredder column according to the manufacturer's instructions provided with the kit. 1 microgram (4 μg) of total RNA was used as a template for cDNA preparation by reverse transcription. This RNA, 300 ng of random primers, 500 ng of Oligo dT (12-18), and 1 μl of 25 mM dNTPs were mixed and denatured at 80°C for 5 minutes, after which the enzyme was added. Superscript III reverse transcriptase (Invitrogen, Life Technologies) was added to a total volume of 25 μl of RNA + primer mixture in the presence of the second-chain buffer and 0.1 M DTT provided with the enzyme. The reverse transcription reaction proceeded at 50°C for 1 hour. The cDNA was purified using a QIAquick (QIAGEN) PCR purification column, eluted in 40 microliters of EB buffer, and then used for PCR.

[0207] Wild-type mouse and human-Fc domains were isolated by PCR amplification using the above cDNA as a template. The following primers were used for initial amplification of the wild-type sequence, and the desired mutagenic changes were incorporated into the hinge domain. mahIgG1CH2M:47mer TIFF0007897613000006.tif7128hIgG1-5scc:49mer TIFF0007897613000007.tif7128mahIgG1S:51mer TIFF0007897613000008.tif7129muIgG2aCH2:58mer TIFF0007897613000009.tif7140mIgG2a-5scc:47mer TIFF0007897613000010.tif7128mIgG2a3S:48mer TIFF0007897613000011.tif6128

[0208] PCR reactions were performed using a C1000 thermal cycler (BioRad, Hercules CA) or an Eppendorf thermal cycler (ThermoFisher Scientific, Houston TX). The reaction included 34 cycles consisting of an initial denaturation phase at 95°C for 2 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 1 minute, followed by a final extension at 72°C for 4 minutes. After isolating the wild-type tail, the fragment was TOPO cloned into a pCR2.1 vector, DNA was prepared using the QIAGEN spin plasmid miniprep kit according to the manufacturer's instructions, and the clones were sequenced using the ABI Dye Terminator v3.1 sequencing reaction according to the manufacturer's instructions.

[0209] DNA derived from the correct clone was used as a template in duplicate extension PCR to introduce mutations at desired locations in the coding sequences of mouse IgG2a or human-IgG1. The PCR reaction was set up using a full-length wild-type clone (1 microliter) as a template, 50 pmol of 5' and 3' primers for PCR from each direction to the desired mutation site and for each portion of the -Fc domain containing it, and PCR hi fidelity Supermix (Invitrogen, Carlsbad CA) in a reaction volume of 50 microliters, with short amplification cycles. As an example of duplicate PCR mutagenesis, the primer combination used to introduce the P331S mutation into human-IgG1 was as follows:

[0210] The 5' partial fragment was amplified using a full-length wild-type clone as a template, where the 5' primer was hIgG1-5scc: TIFF0007897613000012.tif6128, while the 3' primer is P331AS: The result was TIFF0007897613000013.tif7128. The 3' partial fragment was amplified using a full-length wild-type clone as a template, where the 5' primer was P331S: TIFF0007897613000014.tif6128, while the 3' primer is mahIgG1S: The filename was TIFF0007897613000015.tif7130.

[0211] Partial fragments were amplified and isolated by agarose gel electrophoresis, then purified using a QIAquick gel purification column and eluted in 30 microliters of EB buffer according to the manufacturer's instructions. Subsequently, two PCR cycles were performed using the two partial fragments as duplicate templates in the new reaction. The cycler was paused, and 5'(hIgG1-5scc, see above) and 3'(mahIgG1S, see above) flanking primers were added to the reaction mixture (50 pmol each). PCR amplification was then performed for 34 cycles under the conditions described above for the wild-type molecule. Full-length fragments were isolated by gel electrophoresis and TOPO cloned into the pCR2.1 vector for sequence analysis. Subsequently, clone-derived fragments with the correct sequence were subcloned into expression vectors to produce the various hybrid nuclease molecules described herein.

[0212] Example 4: Determination of RSLV-124 protein and RNase enzyme activity in mouse serum In vivo stability analysis of the RSLV-124 construct (SEQ ID NO: 106) in mice. Four mice (C220, C221, C222, C223) were administered a single intravenous injection of RSLV-124 at time zero. Blood samples were collected at various time points after injection and analyzed for the presence of RSLV-124 protein (human wild-type RNase (SEQ ID NO: 106) linked to the wild-type human IgG1 Fc domain) and RNase enzyme activity. To detect the RSLV-124 compound in mouse serum, an ELISA was developed that captures human Fc from mouse serum and then detects human RNase. ELISA was performed on blood samples from the four mice, and RSLV-124 protein was detected at concentrations of 38 μg / ml to 55 μg / ml 5 minutes after a single 150 ug intravenous injection (Figure 2). One day after injection, the concentration of RSLV-124 rapidly decreased to 8 μg / ml to 12 μg / ml. The drug's blood concentration remained relatively stable over the 7-day analysis period, with blood drug levels approximately 5 μg / ml. The RNase enzyme activity of the drug was quantified using the same blood samples used to measure the RSLV-124 protein by ELISA. The enzymatic reaction rate of the RSLV-124 protein in mouse blood samples was measured using the Ambion RNaseAlert QC system (catalog number AM1966) with some modifications. The drug compound was captured from mouse serum onto an RNaseAlert assay plate using a human anti-Fc monoclonal antibody and quantified by fluorescence measurement as instructed in the Ambion kit. Analysis of the relative fluorescence units (RFU) of the RSLV-124 molecule showed 80,000–140,000 RFU 5 minutes after injection (Figure 3). RFU decreased rapidly in parallel with the protein concentration and remained relatively stable between 18,000–40,000 RFU until day 7. Using the RNaseAlert QC system, a standard curve was created using known amounts of protein, and the RSLV-124 protein concentration was extrapolated from this standard curve using the RFU of RSLV-124 in blood samples. This analysis revealed that the protein concentration present in mouse blood over a 7-day experiment, calculated using the RNase enzyme activity assay, was remarkably similar to the value measured using ELISA (Figure 4).These experiments concluded that the RSLV-124 compound was stable in vivo in mouse circulating blood for 7 days, maintaining its enzymatic activity. This suggests that the compound is not degraded in vivo in mice, as its enzymatic activity was almost 100% maintained in mouse circulating blood for 7 days. Since Fc fusion proteins are often susceptible to degradation in circulating blood, this finding further supports the potential use of RNase-Fc fusion proteins as beneficial drugs.

[0213] Example 5: Phenotype of TLR7.1×RNaseA dual transgenic mouse We created mice that overexpress RNaseA (RNase Tg). This nuclease is expressed at high levels in RNase Tg mice. We developed a one-way diffusion-radiation (SRED) method (Figure 5) and a much more quantitative ELISA (see Figure 6) for quantifying serum RNase. Both assays showed a significant increase in RNase activity in RNase Tg mice. The quantification of RNase levels in Figure 6 showed that RNase was increased approximately 10-fold in RNase Tg mice compared to wild-type B6 mice. We created double transgenic (DTg) mice by crossing RNaseA Tg with TLR7.1 Tg mice. TLR7.1 mice have 8 to 16 copies of TLR7 and develop a highly malignant, rapidly progressive lupus-like disease, beginning to die at 3 months of age, with a median survival time of 6 months. In a preliminary analysis, the inventors raised DTg mice and littermates (bled) to 3 months of age to investigate whether DTg mice showed signs of improvement. As shown in Figure 5, DTg mice had very high levels of RNase in their serum (corresponding to RNase levels above 13 U / ml based on the inventors' standard substance with a specific activity of 993 U / mg). As shown in Figure 6, RNase A concentrations in Tg and DTg mice were also measured by ELISA assay. RNase A Tg mice and TLR7.1×RNaseA Dtg mice had serum concentrations of 1–2 ng / ml.

[0214] Detailed method for RNase A ELISA 1. Coat the plate with anti-RNaseA Abcam Ab(ab6610): 2.5-10 ug / ml over 4C. Wash the plate three times with 2.0.05% Tween / 1×PBS. 3. Block with 1% BSA in PBS for at least one hour. Wash the plate three times with 4.0.05% Tween / 1×PBS. 5. Load the sample. The sample dilution ratio is 1:50. 6. Incubate at room temperature for 2 hours. Wash the plate three times with 7.0.05% Tween / 1×PBS. 8. Prepare a 1:4500 (2.2 ug / ml) dilution of biotin-labeled anti-RNase Ab. Let stand at room temperature for 1 hour (Rockland 200-4688: 10 mg / ml). 9. Wash the plate three times. 10. Dilute StrepAV HRP (Biolegend 405210) to 1:2500. Cover with foil and leave at room temperature for 25-30 minutes. 11. Wash 6 times, allowing the liquid to stand in the well for at least 30 seconds between washes. 12. Add BD OptEIA substrates A and B in a 1:1 ratio. Wait until the color reaches its maximum in 5-10 minutes. Ensure the standard in the top well does not exceed 1.0. Add 80 μl. (Catalog numbers: 51-2606KC; Reagent A, 51-2607KC; Reagent B) 13. Add 40 µl of 1M sulfuric acid to stop the reaction.

[0215] Product / reagent information: RNaseA Ab:ab6610 (90 mg / ml) ELISA buffer: 1% BSA in PBS ELISA washing buffer: 0.05% Tween / 1×PBS Anti-RNase A biotin-binding Ab: Rockland: 200-4688 (10 mg / ml) Strep AV HRP: Biolegend 405210 BD OptEIA Reagents A and B: 51-2606KC and 51-2607KC

[0216] Example 6. Survival curve of the TLR7.1 transgenic mouse strain A significant difference in survival was observed between DTg mice and TLR7.1 littermates / controls. As shown in Figure 7, 61% of TLR7.1 mice died at 10 months, compared to 31% of DTg mice. This data indicates that RNaseA overexpression exerted a strong therapeutic effect. The reason for the early death of TLR7.1 mice is not fully clear, but severe anemia, thrombocytopenia, and glomerulonephritis may have played a role. To determine whether erythrocyte and platelet counts were positively affected by RNaseA expression in DTg mice, the inventors performed complete blood counts, but no difference was found between TLR7.1 and DTg mice. In contrast, significant improvement in renal histopathology was observed in DTg mice. The inventors observed reduced IgG and C3 deposition in DTg mice. PAS staining, which reflects inflammation in the mesangium, was also reduced in DTg mice compared to TLR7.1 littermates / controls. The inventors compared macrophage infiltration in the kidney using an anti-MAC-2 (galectin 3) antibody (Lyoda et al. Nephrol Dial Transpiat 22: 3451, 2007) and found that the number of MAC-2-positive cells was significantly lower in the glomeruli of DTg mice. Using 5 mice in each group, and calculating with 20 glomeruli per mouse, the mean + / -SE for single Tg and DTg mice was 3.8 + / - 1.1 and 1.4 + / - 0.2, respectively, with p=0.05. In addition, the inventors quantified glomerular capillary size and observed a significant decrease in glomerular capillary size in DTg mice (179 + / - 41 and 128 + / - 16.8 μm² for single Tg and DTg mice, respectively, p=0.037). In summary, TLR7.1×RNaseA DTg mice survived longer than their single Tg TLR7.1 littermates, and their renal inflammation and injury were less severe. This finding suggests that removing RNA immune complexes in this mouse model significantly improved overall mortality and reduced renal damage and overall inflammation associated with this lupus-like condition.

[0217] Example 7. Analysis of IRG in the spleen of TLR Tg mice Analysis of interferon-responsive genes (IRGs) in the spleen of TLR7.1 Tg mice and TLR7.1×RNaseA DTg mice showed that the expression of the IRF7 gene (interferon regulator 7 (UniProtKB P70434)) was significantly lower in DTg mice (p=0.03). Several other IRGs, including MX1 (interferon-inducible GTP-binding protein Mx1 (UniProtKB P09922)) and VIG1 (radical S-adenosylmethionine domain-containing protein 2 (UniProtKB Q8CBB9)), were lower in DTg mice compared to Tg mice, but the difference was not statistically significant (Figure 8). Quantitative PCR was performed as follows. Total RNA was isolated from mouse spleen using the RNeasy mini kit (Qiagen, Valencia, CA, USA), treated with DNase using Turbo DNA-free (Applied Biosystems, Foster City, CA, USA), and first-strand cDNA was generated using the RNA-to-cDNA kit (Applied Biosystems) with random primers. For the isolated RNA, the 260 / 280 value measured using NanoDrop (Thermo Scientific, Waltham, MA, USA) was between 1.7 and 2.0. The cDNA was diluted to 1 ng / µl equivalent to total RNA, and 8 µl was used per reaction. Primers for the reference gene (18s) and the gene of interest (GOI) were synthesized (IDT, Coralville, Iowa, USA) and diluted to appropriate concentrations for qPCR using molecular-grade water. BLAST results of the primers showed specific sequence homology only to the reference gene or GOI. Using a 1:1 mixture of template and primer for SensiMix SYBR low-ROX master mix (Bioline, London, UK), two reaction mixtures (20 μl each) were administered using the ABI Fast7500 system. Age-matched wild-type B6 mice were used as a baseline to determine the change multiplier for each GOI.-ddCT Relative quantification was calculated using the appropriate method. Dissociation curves for the reactants showed a single melting peak for each gene. Standard curves showed similar amplification efficiencies for each gene, and the template concentrations were within the linear dynamic range for each primer set.

[0218] Example 8. Construction and expression of DNase1-Ig mono- and bienzyme hybrid nuclease molecules. Native alleles of human DNase1 molecules or DNase1-like molecules have been reported. The A114F mutation has previously been reported to be present in native variants of human DNase1-like enzymes, and that this sequence alteration leads to actin resistance in the enzyme. See Pan, CQ, Dodge TH, Baker DL, Prince WE, Sinicropi DV, and Lazarus RA. J Biol Chem 273: 18374-18381, (1998); Zhen A, Parmelee D, Hyaw H, Coleman TA, Su K, Zhang J, Gentz ​​R, Ruben S, Rosen C, and Li Y. Biochem and Biophys Res Comm 231: 499-504 (1997); and Rodriguez AM, Rodin D, Nomura H, Morton CC, Weremowicz S, and Schneider MC. Genomics 42: 507-513 (1997), all of which are incorporated herein by reference.

[0219] Similarly, the G105R mutation has recently been reported as a single nucleotide polymorphism in the gene encoding human DNase 1 that is polymorphic in some populations or the entire population and is associated with autoimmunity (see Yasuda T, Ueki M, Takeshita H, Fujihara J, Kimura-Kataoka K, Lida R, Tsubota E, Soejima M, Koda Y, Dato H, Panduro A. Int J Biochem Cell Biol 42(7): 1216-1225 (2010), which is incorporated herein by reference). The allelic variant at this position resulted in a DNase 1 isoform with higher activity compared to the wild type. Another natural polymorphic mutation (R21S) has also been reported to confer higher activity (see Yasuda, supra).

[0220] It has been reported that the level of DNase1 activity is significantly lower in SLE patients (see Martinez-Valle F, Balada E, Ordi-Ros J, Bujan-Rivas S, Sellas-Femandez A, Vilardell-Tarres M. Lupus 18(5): 418-423 (2009), which is incorporated herein by reference).

[0221] Therefore, natural enzyme variants may be less immunogenic when administered to patients because these isoforms exist in the human population. The inventors inferred that a novel allelic variant of human DNase1 with the potential to show improved clinical activity in vitro and in vivo would be generated by combining the actin resistance characteristics of alleles similar to A114F and the increased enzyme activity of alleles such as G105R. To the inventors' knowledge, their report is the first report on this new mutant form of DNase1 created by the combination of the two natural variants, G105R and A114F.

[0222] Human DNase 1 was isolated from human pancreatic RNA (Ambion) by cDNA reverse transcription using the random primer method and PCR using the following primer sets as previously described. 5'hDNase1-age: TIFF0007897613000016.tif71475'hDNase1-bx: TIFF0007897613000017.tif7156

[0223] Alternatively, the 3'DNase cassette was amplified by PCR using the following primer pair. 3'hDNase1-RV: TIFF0007897613000018.tif71473'hDNase1-Termination: TIFF0007"

[0224] The PCR reaction was performed using Platinum PCR Supermix as previously described, with 50 pmol of each primer and 2 μl of cDNA in a total volume of 50 μl. The amplification profile was 35 cycles of 94°C for 30 seconds; 55°C for 30 seconds; 68°C for 90 seconds.

[0225] The wild-type gene was amplified by PCR, and the fragments were subjected to gel electrophoresis. The 850 bp fragment was purified by QIAquick column purification. As described for other constructs, the fragments were cloned into pCR2.1 and transformed by TOPO cloning according to the manufacturer's instructions. After sequence verification, PCR primers were used to create partial fragments containing the native allele of DNase1, which has been reported to improve specific activity and resistance to actin inhibitory activity. These partial fragments contained duplicate sequences, thus enabling amplification of complete DNase1 subclones containing the desired allele mutations. COS7 cells were transiently transfected in a 60 mm dish using Polyfect (Qiagen, Valencia, CA) transfection reagent. Plasmid DNA was prepared using the Qiagen QIAprep miniprep kit according to the manufacturer's instructions. Plasmids were eluted in 50 μl of EB buffer. DNA concentration was measured using Nanodrop, and an aliquot equivalent to 2.5 ug of plasmid DNA was used for each transfection reaction. Each DNaseIg or RNase-Ig-DNase expression cassette was inserted into pDG, a mammalian expression vector derived from pcDNA3.1. Transfected cells were incubated at 37°C and 5% CO2 for 72 hours, after which the culture supernatant was collected for further analysis. After collecting the culture supernatant, the remaining cells were centrifuged and the liquid was transferred to a new tube.

[0226] COS-7 cells were transiently transfected with a plasmid containing a fusion of the wild-type or native DNase 1 mutant allele (G105R and / or A114F) of human DNase 1 with the wild-type human IgG1 Fc domain. This hinge-CH2-CH3 cassette contains a single C→S mutation in the hinge region that eliminates the first cysteine ​​in this domain, resulting in unpairing due to the absence of its pairing partner in the antibody's light chain. In addition, more complex polynuclease fusion proteins were also expressed by transient transfections of COS cells.

[0227] Example 9: Isolation of human Ig tail, introduction of mutations into coding sequence, and construction of mutant nuclease molecule To isolate mutant human-Ig Fc domains, RNA was obtained from human PBMCs isolated from fresh whole blood using Lymphocyte Separation Media (LSM) Organon Teknika (Durham, NC). The buffy coat was collected according to the manufacturer's instructions, and the cells were washed three times in PBS before use. The cells were pelleted from the medium by centrifugation, resulting in 2 × 10⁶ cells. 7Cells were used for RNA preparation. RNA was isolated from cells using the QIAGEN RNAeasy kit (Valencia, Calif.) total RNA isolation kit and QIAGEN QIAshredder column according to the manufacturer's instructions provided with the kit. 1 microgram (4 μg) of total RNA was used as a template for cDNA preparation by reverse transcription. RNA, 300 ng of random primers, 500 ng of Oligo dT (12-18), and 1 μl each of 25 mM dNTPs were denatured at 80°C for 5 minutes, after which the enzyme was added. Superscript III reverse transcriptase (Invitrogen, Life Technologies) was added to the RNA plus primer mixture to a total volume of 25 μl in the presence of the second-chain buffer and 0.1 M DTT provided with the enzyme. The reverse transcription reaction was carried out at 50°C for 1 hour. cDNA was purified using a QIAquick (QIAGEN) PCR purification column according to the manufacturer's instructions, eluted in 40 microliters of EB buffer, and then used for the PCR reaction.

[0228] The wild-type human-Ig Fc domain was isolated by PCR amplification using the above cDNA as a template. Mutant Ig fragments were isolated by directional PCR mutagenesis using appropriate PCR primers containing the desired mutation and a wild-type cassette as a template. The PCR reaction was performed using a C1000 thermal cycler (BioRad, Hercules CA). The reaction included 34 cycles of an initial denaturation step at 95°C for 2 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, followed by a final extension at 72°C for 4 minutes. After isolating the full-length mutant tail, the fragment was TOPO cloned into a pCR2.1 vector, DNA was prepared using the QIAGEN spin plasmid miniprep kit according to the manufacturer's instructions, and the clones were sequenced using the ABI Dye Terminator v3.1 sequencing reaction according to the manufacturer's instructions.

[0229] Recombinant molecules were prepared by PCR mutagenesis using duplicate extension PCR with mutant oligonucleotides.

[0230] The following oligonucleotides were used to obtain these molecules. CS-P238S 5-1: TIFF0007897613000020.tif14128SSSH-5-2: TIFF0007897613000021.tif14128P331S-S: TIFF0007897613000022.tif14128P331S-AS: TIFF0007897613000023.tif14128hIgG1-3'WTnogt: TIFF0007897613000024.tif15128

[0231] The P238S mutation and the substitution of SCC to SSS were introduced by PCR mutagenesis using two duplicated 5' oligonucleotides in sequential PCR reactions. The first PCR reaction included the following 5' primers that incorporated the P238S mutation into its sequence. CS-P238S 5-1: TIFF0007897613000025.tif14129

[0232] The second PCR reaction included the following 5' primer, which overlapped with the first primer and added a mutant hinge residue to the P238S mutant. SSSH-5-2: TIFF0007897613000026.tif14166

[0233] DNA derived from the correct clone was used as a template in duplicate extension PCR to introduce mutations at desired internal locations within the coding sequence of human-IgG1. The PCR reaction was set up using a full-length clone as a template (1 microliter), 50 pmol of 5' and 3' primers for PCR of each portion of the -Ig tail from each direction to the desired mutation site and containing it, and PCR hi fidelity Supermix (Invitrogen, Carlsbad CA) in a 50 microliter reaction volume with short amplification cycles. As an example of duplicate PCR mutagenesis, the primer combination used to introduce a P238S mutation into human-IgG1 that already had a P331S mutation introduced was as follows:

[0234] The 5' partial fragment was amplified using a full-length wild-type clone as a template, where the 5' primer was SSSH-5-2: TIFF0007897613000027.tif14128, while the 3' primer is P331S-AS: The file was TIFF0007897613000028.tif14138.

[0235] The 3' partial fragment was amplified using a full-length wild-type clone as a template, where the 5' primer was P331S-S: TIFF0007897613000029.tif6166, while the 3' primer is hIgG1-3'WTnogt: The file was TIFF0007897613000030.tif14166.

[0236] The partial fragments were amplified, isolated by agarose gel electrophoresis, purified by QIAquick Gel Extraction Column, and eluted in 30 μl of EB buffer according to the manufacturer's instructions. Subsequently, two rounds of PCR were performed using the two partial fragments as overlapping templates in a new reaction. The cycler was paused and 5' and 3' flanking primers were added to the reaction (50 pmol each). Subsequently, PCR amplification was carried out for 34 cycles under the conditions described above for the wild-type molecule. The full-length fragment was isolated by gel electrophoresis and TOPO cloned into the pCR2.1 vector for sequence analysis. Subsequently, the fragments from the clones with the correct sequence were subcloned into expression vectors to create various nuclease molecules described herein.

[0237] For the multispecific nuclease molecule, a PCR reaction was performed using alternative primers for the 3' end of the Fc domain. The stop codon was removed and an NLG linker and an EcoRV restriction site were added to facilitate the fusion of the remaining part of the cassette. The primer sequences are listed below. TIFF0007897613000031.tif7139

[0238] The RSLV mutant nuclease was constructed by fusing the mutated human-Ig tail with the wild-type RNase domain with or without a linker separating the two domains. In RSLV125 and RSLV126, the human RNase is fused with the mutant hinge and IgG1 Fc domain. RSLV125 does not contain a linker, while RSLV126 contains a (gly4ser)4 linker as an inserted (BglII-XhoI) fragment between the nuclease domain and the hinge region. RSLV-125 incorporates a wild-type RNase cassette directly fused to an SSS-type (CCC or not wild-type) human IgG1 hinge and P238S, P331S mutant human IgG1 Fc domain (SEQ ID NO: 61-62).

[0239] RSLV126 incorporates a wild-type RNase cassette fused with the (gly4ser)4 linker domain, followed by an SSS mutant hinge and a P238S-P331S double mutant Fc domain (SEQ ID NO: 63~64).

[0240] RSLV-127 is a polynuclease fusion construct (SEQ ID NO: 65~66) that incorporates an amino-terminal human DNase (G105R / A114F) fused with a (gly4ser)4 linker domain, followed by an SSS mutant hinge and a P238S-P331S double mutant Fc domain, which then fuses with an NLG linker domain, followed by a C-terminal wild-type RNase domain.

[0241] RSLV-128 is a polynuclease fusion construct (SEQ ID NO: 67~68) that incorporates an amino-terminal wild-type human RNase domain fused with a (gly4ser)4 linker domain, followed by an SSS mutant hinge and a P238S-P331S double mutant Fc domain, which then fuses with an NLG linker domain, followed by a C-terminal mutant DNase (G105R / A114F) domain.

[0242] RSLV-129 is a multispecific fusion construct (SEQ ID NO: 69~70) that incorporates an amino-terminal wild-type human RNase domain fused with an SSS mutant hinge and a P238S-P331S double mutant Fc domain, which then fuses with an NLG linker domain, followed by a C-terminal mutant DNase (G105R / A114F) domain.

[0243] RSLV-132 incorporates a wild-type RNase cassette directly fused with an SCC-type human IgG1 hinge and P238S, P331S mutant human IgG1Fc domain (SEQ ID NO: 91-92 and 95-96).

[0244] RSLV-133 is a multispecific fusion construct incorporating an amino-terminal wild-type human RNase domain fused with an SCC mutant hinge and a P238S-P331S double mutant Fc domain, which then fuses with an NLG linker domain, followed by a C-terminal mutant DNase (G105R / A114F) domain (SEQ ID NO: 93-94 and 97-98).

[0245] Other models of RSLV-125 to RSLV-129 with SCC hinges are shown in Table 1 as RSLV-125-2 (SEQ ID NO: 77~78), RSLV-126-2 (SEQ ID NO: 79~80), RSLV-127-2 (SEQ ID NO: 81~82), RSLV-128-2 (SEQ ID NO: 83~84), and RSLV-129-2 (SEQ ID NO: 85~86).

[0246] Example 10: Western blot of RSLV 125-129 fusion proteins expressed by COS7 transfection. Figure 9 shows Western blots of COS transfection supernatants from RSLV 125-129 constructs. Expression plasmids containing RSLV 125, 126, 127, 128, or 129 were transfected into COS7 cells using Polyfect transfection reagent, and the supernatant was collected after 48 hours. In addition to the single nuclease molecules contained in RSLV 125 and 126, more complex multinuclease fusion proteins encoded by RSLV 127, 128, and 129 were also expressed from transient transfected COS cells. Western blot analysis was performed on the supernatants from the transient transfectants. The molecules shown in Figure 9 include those containing human RNase1 fused with the human SSSIgG1 hinge and the Ig G1 P238S-P331S Fc domain, or those containing human RNase1 (wild type) fused with the SSS hinge-(P238S-331S)CH2-CH3 Fc domain of human IgG1, followed by a novel linker containing an N-linked glycosylation site to protect the linker domain from protease cleavage, and a mutant allele G105R-A114F type human DNase1 at the carboxyl terminus of the molecule. In addition, RSLV 127 encodes the above human DNase1 mutant at the amino terminus of the mutant-Ig tail and RNase 1 WT at the carboxyl terminus. The COS supernatant was collected after 72 hours, and 0.5 ml of the sample was immunoprecipitated overnight at 4°C with 100 μl of protein A-agarose beads. Protein A beads were centrifuged, washed twice in PBS, and then resuspended in SDS-PAGE loading buffer, or in reducing or non-reducing LDS sample buffer (Invitrogen, Carlsbad, CA) for NuPAGE gels. The sample was heated according to the manufacturer's instructions, the protein A beads were centrifuged to pelletize, and the sample buffer was loaded onto a 5-12% NuPAGE gradient gel. Electrophoresis of the sample was performed at 150 volts for 1.5-2 hours, and the gel was blotted onto a nitrocellulose membrane at 30 mAmp for 1 hour. Western blots were blocked overnight in TBS / 5% skim milk.The blots were incubated with 1:2500 HRP (horseradish peroxidase)-conjugated goat anti-human IgG (Fc-specific, Jackson Immunoresearch) at room temperature for 1.5 hours, washed five or more times with PBS / 0.5% Tween20, and developed using ECL reagent. The results demonstrate successful construction of the nuclease Fc fusion protein and its readily expressed in COS cells. Furthermore, analysis of the reductive and non-reductive profiles of these nuclease Fc fusion proteins confirms that the DNA constructs encode proteins of the appropriate molecular weight. The pattern on non-reductive SDS-PAGE confirms that the disulfide bond properties of the protein are consistent with the expected behavior of the constructs.

[0247] Example 11: SRED analysis of affinity-purified proteins derived from COS7 transfectant Figure 10 shows SRED analysis comparing aliquots of purified protein A from COS supernatant transfected with RSLV according to Example 10. A 2% agarose gel was prepared with distilled water. Poly-IC(Sigma) was dissolved in distilled water at 3 mg / ml. The gel plate was prepared as follows: 1.5 ml of reaction buffer (0.2 M Tris-HCl pH 7.0, 40 mM EDTA and 0.1 mg / ml ethidium bromide), 1 ml of Poly-IC and 0.5 ml of water were placed in a tube and maintained at 50°C for 5 minutes. 3 ml of agarose (maintained at 50°C) was added to the tube. This mixture was immediately poured onto a glass plate. Sampling wells were prepared by perforating the gel. 2 μl each of control, serum sample, or affinity-purified RSLV protein was loaded into the wells, and the gel was incubated in a humid chamber at 37°C for 4 hours. The gel was then incubated on ice for 30 minutes in buffer (20 mM sodium acetate, pH 5.2, 20 mg / ml ethidium bromide) and read under UV light. Photographs of the gel were taken on a UV transilluminator using a Kodak DC290 digital camera system equipped with an ethidium bromide filter, and analyzed using Kodak Molecular Imaging software. The results of the RNase enzyme activity assay indicate that all constructs contain catalytically active RNase molecules.

[0248] Example 12: DNase activity of RSLV nuclease molecule in gel Figure 11 shows the results of a DNase nuclease activity assay performed on purified protein A from COS7 supernatant transfected with the RSLV fusion plasmid in Example 10. Figure 11 shows five panels (11a, 1lb, 11c), each gel panel showing the digestion pattern by the indicated fusion protein in decreasing amounts and 1 microgram of plasmid DNA. Each protein was sequentially diluted in nuclease-free water in 2x increments from 500 ng to 4 ng of enzyme. 1 ug of PDG plasmid DNA was added to each sample and incubated at 37°C for 30 minutes. Half of each sample was subjected to agarose gel electrophoresis at 100 volts for 30 minutes using a 1.2% TAE agarose gel. Figure 11c shows the results of a gel-in-gel DNase enzyme activity assay using commercially available DNase 1 (Biolabs, Inc.). The rightmost lane is a negative control of DNA alone without the enzyme, and the lane to its left is another negative control, this time an RNase-Ig molecule with RNase activity but no DNase activity. As expected, in both cases the plasmid DNA remained intact and undigested. These results demonstrate that the commercially available DNase1 enzyme is highly active and digested all of the DNA at most of the concentrations tested. The results in Figures 11a and 11b show the DNase activity of four different nuclease Fc fusion constructs. The upper panel in Figure 11a shows the ability of the DNase-Ig fusion protein to digest plasmid DNA, and as can be seen from the gel, this enzyme digested all of the DNA at all concentrations tested with the same or greater activity than commercially available DNase 1. The lower panel of Figure 11a shows bispecific nuclease Fc fusion proteins (SED IDs 65-66) that have DNase at the amino terminus of Fc. These also exhibit robust DNase enzymatic activity, though somewhat weaker than that of DNase-Ig in the upper panel of Figure 11a. The upper panel of Figure 11b shows the DNase enzymatic activity of another bispecific nuclease, this Fc fusion protein having DNase linked to Fc at the C terminus via a specially constructed NLG linker (SEQ IDs 67-68).As the data shows, this enzyme also possesses robust DNase activity, and appears to be more active than the other bispecific nucleases examined here. The lower panel of Figure 11b shows the DNase activity of another bispecific nuclease molecule (SEQ ID 69-70) that lacks the (G4S)4 linker that connects the RNase module to Fc. This bispecific nuclease also exhibits good DNase activity, but appears somewhat weaker than the other two bispecific nuclease Fc fusion proteins shown in this experiment. This data suggests that all bispecific nucleases possess good DNase activity, which is unexpected given previous work by others in this area (Dwyer et al. JBC; Vol 271, No. 14; pp 9738-9743). Furthermore, the position of the DNase in the construct, as well as the length and composition of the linker connecting the DNase to Fc, are critically important for producing a highly active DNase enzyme in the context of a bispecific nuclease-Fc fusion protein.

[0249] Example 13: Enzyme reaction kinetics analysis Figures 12-13 show the results of a kinetic fluorescence enzyme activity assay comparing the RNase enzyme activities of recombinant RNase A (Ambion), RSLV 125, RSLV 126, hRNase WT-SCCH-WThIgG1, and hRNase G88D-SCCH-(P238S / K322S / P331S)hIgG1. To further clarify the functional characteristics of bivalent mRNAase-Ig fusion proteins, the inventors investigated the enzymatic reaction rates of various nuclease fusion proteins using RNase Alert Substrate (Ambion / IDT) and quantified fluorescence using a Biotek Synergy2 microplate reader. The data were analyzed using Gen5 software (Biotek Instruments, Inc., Winooski, Vermont). Relative fluorescence units as a function of time were assayed minute by minute throughout a 45-minute experimental process, incubated at 37C according to the manufacturer's instructions, with the enzyme concentration starting at 10 pg / ul and decreasing in 0.67-fold increments down to 0.1 pg / ul. Each sample contained a fixed concentration of RNase Alert substrate (200 nM) in 1 × RNase Alert reaction buffer.

[0250] Figure 12 shows the RFU (relative fluorescence units) for each protein at equimolar concentrations in the presence of a 200 nM RNase Alert substrate, with the test protein at 4.5 pg / ul or 4.5 ng / ml and the recombinant RNase A control at 1.3 pg / ul, in comparison to time.

[0251] Figure 13 shows the Lineweaver-Burk plots for various molecules. To estimate Vmax and Km, an RNase Alert kinetic fluorescence assay was prepared using 105 pM enzyme, and the substrate concentration was decreased in 4-fold increments from 200 nM to 50 pM. That is, the enzyme concentration was fixed and the substrate concentration was fine-tuned in this series of experiments. These data show the Lineweaver-Burk plots prepared for various fusion proteins under these conditions. Taken together, the data in Figures 12 and 13 demonstrate high activity of the RNase moiety in the three RNase Fc fusion protein constructs constructed and tested herein.

[0252] Example 14: Evaluation of in vitro cytotoxic effects on human THP-1 cell lines Figures 14-15 show the results of an in vitro study analyzing the effect of RNaseIg fusion proteins with wild-type or mutant (including SCC, P238S, and P331S) IgG Fc domains on the survival of the human monocyte cell line THP1. THP1 cells were maintained under logarithmic growth in RPMI / 10% FBS and then harvested for assay. Cell viability before use in the cytotoxicity assay exceeded 98%. THP1 cells were plated in 96-well plates at 1 × 10⁶ cells / ml or 100,000 cells per well. Hybrid nuclease proteins were added to sequential wells using a 2-fold serial dilution series, starting at 5 micrograms / ml per reaction and ending at 0.01 micrograms / ml of fusion protein per reaction. In this experiment, we compared the ability of RNase-Fc fusion proteins (wtRNasewtIgG) with wild-type IgG1 Fc to RNase-Fc (mtRNasemgIgG) with mutant Fc (P238S, P331S) exhibiting significantly reduced binding to the Fc receptor and internal translocation, in terms of their ability to induce cytotoxicity in cultured THP1 cells. The reaction mixture was incubated in 96-well plates at 37°C under 5% CO2 for 3 days, after which cells were collected and analyzed. After 3 days, cells were collected by centrifugation at 1000 rpm, washed with PBS / 2% FBS, and incubated with the FITC Annexin V Apoptosis Detection Kit reagent (#556547, Becton Dickinson / Pharmingen) according to the manufacturer's instructions. The cells were washed with 100 microliters of chilled binding buffer supplied with the kit, and Annexin V-FITC / propidium iodide (PI) was added to 100 µl of binding buffer at a ratio of 1:100. The samples were incubated on ice for 20 minutes, after which 400 μl of additional binding buffer was added to each sample. The stained samples were analyzed by flow cytometry using FACS Canto (Becton Dickinson), and the data was analyzed using Flowjo software (Treestar, Ashland, OR).

[0253] Figure 14 shows the effect of RNase Fc fusion proteins with wild-type or mutant Fc domains on cell death, evaluated by two methods: annexin V binding (upper panel) and propidium iodide binding (lower panel), both of which are highly sensitive measures of cell death. This experiment demonstrates that the binding affinity of RNase fusion proteins with mutant Fc (P238S, P331S) to Fc receptors on the surface of THP1 cells is reduced, as is the subsequent internal translocation of the protein. This result indicates that cell death is significantly reduced by RNase-Fc mutants compared to RNase-wild-type Fc fusion proteins (e.g., approximately one-third reduction at 1.25 μg / ml protein). Figure 15 presents the results of fluorescence-activated cell sorting (FACS) experiments to examine the cytotoxic effects of RNase Fc fusion constructs with wild-type or mutant Fc domains (RNase-wtIgG or RNase-mtIgG, respectively). These data demonstrate a significant reduction in the number of dead cells when THP1 cells are incubated with an RNase Fc construct containing a mutant Fc (the peak size is smaller for the RNase Fc mutant on the right side of the graph compared to RNase-wtIgG). These data indicate that cell death is reduced by approximately one-third to one-fifth compared to the wild type with the RNase Fc mutant. These and other experiments examining binding to the Fc receptor clearly show that, in the presence of cells possessing the Fc receptor, the RNase Fc construct with the mutant Fc region exhibits reduced binding to the Fc receptor and decreased internal translocation by cells, resulting in less cell death attributable to the construct's RNase activity. Such constructs are particularly useful in the treatment of autoimmune diseases because the use of protein therapies that have cytotoxic effects on cells possessing the Fc receptor may be undesirable.

[0254] Example 15: Addition of RSLV-132 to cultures in vitro inhibits IFN-α production by human PBMCs. The addition of RSLV132 eliminated the induction of interferon-α from human peripheral blood mononuclear cells stimulated with immune complexes formed from serum from three SLE patients combined with necrotic cell extract (NCE) (Figure 16). To measure RSLV-132's ability to bind to and degrade RNA contained in lupus patient immune complexes, an in vitro bioassay was developed. This experiment involved in vitro immune complex formation using autoantibodies from lupus patients and NCE derived from cultured human cells (U937). Combining lupus patient serum with NCE resulted in the formation of immune complexes (ICs), which are highly potent inducers of interferon, whereas normal human serum did not stimulate interferon production. The ICs were incubated with normal human peripheral blood mononuclear cells (PBMCs) as reporter cells. Interferon production by the reporter cells was measured using interferon-α ELISA. Reporter cells were obtained from normal volunteers by Ficoll density gradient centrifugation. Serum from lupus patients or healthy volunteers was obtained in accordance with University of Washington Institutional Review Board #HSD No. 3971. The serum was diluted to 1 / 1000 and added at 10% (v / v) to necrotic cell extract (NCE) obtained from cultured U937 cells as described above. The diluted lupus patient or healthy volunteer serum was incubated with NCE at room temperature for 15 minutes, and the resulting IC was incubated for 15 minutes with or without various doses of RSLV-132, RSLV-124, or wild-type RNase, followed by incubation with normal PBMCs for 20 hours in the presence of 500 U / mL Universal IFN, after which the amount of IFN secreted from the PBMC cultures was measured. Serum was obtained from three different lupus patients with varying disease activity ranging from mild to active. NCEs were incubated with either lupus patient serum or healthy volunteer serum at room temperature for 15 minutes, followed by a 20-hour incubation with PBMCs.IFN-α was quantified by ELISA, which involved capturing IFN-α with a mouse MAb (MMHA-11) against human IFNα [PBL Biomedical Laboratories, product #2112-1], detecting it with a rabbit polyclonal antibody against IFNα [PBL Biomedical Laboratories, product #31101-1], and then developing the quantification using anti-rabbit HRP [Jackson Immuno Research, product #711-035-152] and a TMB substrate. In some cases, before adding NCE to PBMCs, the test substance (RSLV-124 or RSLV-132) was added to the NCE at concentrations of 0.16, 0.5, 1.6, and 5.0 ug / mL, or RNase was added at concentrations of 0.05, 0.16, 0.5, and 1.6 ug / mL (equomolar). The ability of lupus patient serum to stimulate IFN production from PBMCs was reduced by approximately 50% with the addition of 5.0 ug / mL of RSLV-124. This inhibition reflected that of equimolar amounts of RNase. The addition of the same concentration of huRSLV-132 was equivalent to or better than RSLV-124 in inhibiting IFN, and with the addition of 5.0 ug / mL of huRSLV-132, IFN production was almost completely abolished. Combined with NCE, anti-RNA / DNA antibodies from lupus patients potently induce IFN from newly isolated PBMCs. Serum from normal volunteers does not have this same ability to induce IFN production from reporter cells. This data suggests that circulating autoantibodies in lupus patient serum may form immune complexes that likely induce TLR7 and subsequent IFN production. The exact type and subtype of IFN were not analyzed. This data indicates that RSLV-132 binds to its molecular target, RNA associated with intracytoplasmic sperm injection (IC) in lupus patients, and potently degrades it, thereby inhibiting IFN stimulation by PBMCs (Figure 16). In this assay, RSLV-132 appears to be more active than RSLV-124.

[0255] Example 16: RSLV-132 is a potent in vivo inhibitor of RNA-induced interferon activation. To evaluate the ability of RSLV-132 to bind to and degrade RNA in mouse circulating blood, a pharmacodynamic model was developed using polyinosine:polycytidic acid (PolyI:C), an RNA mimetic and potent activator of the interferon pathway. Poly(I:C) is a mismatched double-stranded RNA in which one strand is a polymer of inosinic acid and the other is a polymer of cytidic acid. It is known to interact with Toll-like receptor 3 (TLR3) expressed on the membranes of B cells, macrophages, and dendritic cells. Poly(I:C) is available from Invitrogen. The effect of PolyI:C can be quantified by measuring the expression levels of interferon-stimulating genes in the mouse spleen after administration. On day 0, ten 3-month-old B6 mice were administered either RSLV-132 (250 ug per mouse) or intravenous immunoglobulin (IVIG) (Privigen, Behring) (250 ug per mouse) as a control, both by intraperitoneal injection. Twenty hours after RSLV-132 or IVIG injection, the mice were intraperitoneally injected with poly(I:C) at a dose of 200 ug per mouse. Two hours later, the mice were sacrificed by CO2 exposure, and their spleens were collected in RNAlater (Qiagen) and stored at -80C for later testing of interferon-stimulated gene (ISG) expression. Spleen samples were subjected to qPCR to test the expression of ISGs, including Ifit1 (an interferon-inducible protein with tetratricopeptide repeat sequence 1 (UniProt Q64282)), Irf7 (interferon regulator 7 (UniProt P70434)), and the Mx1 gene. The results of these experiments demonstrate that intraperitoneal injection of RSLV-132 leads to serum concentrations of RSLV-132 that can bind to poly(I:C) in the circulating blood and effectively degrade this RNA mimetic, thereby effectively inhibiting the stimulation of the interferon pathway and the three monitored ISGs (Figure 17).

[0256] Example 17. Enzyme reaction kinetic analysis of RSLV-132 and RSLV-133 RSLV-132 and RSLV-133 were transiently expressed in CHO cells and purified using protein-A. The RNase activity of these RNase Fc fusion proteins was quantified using the Ambion RNaseAlert QC kit (catalog number AM 1966). Various amounts of RNase Fc fusion protein were used, and the results are shown in Figure 18 as relative fluorescence units (RFU) over time. These results demonstrate that RSLV-132 is a highly active RNase enzyme, exhibiting higher RNase activity compared to other RNase Fc fusion constructs such as RSLV-124 and wild-type RNase. For example, using the same amount (400 pM) of RSLV-132 and RSLV-124, RSLV-132 produced more than twice the RFU (80,000 vs. 35,000) compared to RSLV-124. In addition, two production lots were tested for their stability at 4C. RSLV-132.1 was stored at 4C for 8 weeks before this experiment, while RSLV-132.2 was stored at -80C and thawed immediately before the test. This demonstrated that the protein is stable at 4C for up to 2 months. The increased drug stability and catalytic activity may lead to improved efficacy in therapeutic settings.

[0257] Figure 19 shows the RNase enzyme activity as RFU over time, comparing the RNase activity of the bispecific RSLV-133 molecule with that of monospecific RSLV-132 and wild-type RNase. As demonstrated in Figure 19, the RSLV-133 molecule exhibited significantly higher RNase activity compared to the monospecific RSLV-124 molecule, or the earlier bispecific nuclease Fc RSLV-123, or wild-type RNase, yielding more than twice the RFU for the same amount of protein. Figure 20 shows the results of a DNase enzyme activity assay of the RSLV-133 molecule compared with the earlier bispecific nuclease construct RSLV-123 and wild-type DNase. In this experiment, DNase enzyme activity was quantified using the DNaseAlert Kit from Integrated DNA Technologies. Fluorescence was induced by DNA substrate cleavage and quantified using a Synergy2 Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT). Figure 20 shows the RFU of DNase enzyme activity over time for RSLV-133, RSLV-123, and wild-type DNase. The experimental results demonstrate that RSLV-133 exhibits higher DNase activity than wild-type DNase and the earlier bispecific nuclease molecule RSLV-123, showing more than three times the DNase activity in the linear range experiment. Figure 21 demonstrates the ability of the RSLV-133 molecule to digest DNA in a gel digestion experiment. This result indicates that RSLV-133 can digest DNA as effectively as wild-type DNase in this assay (compare lanes 5 and 7). Considering the relative molecular weights of RSLV-133 and wild-type DNase, RSLV-133 appears to be more effective in digesting DNA in this assay as well.

[0258] Example 18: RSLV-132 demonstrates reduced binding affinity to Fc receptors. To investigate the ability of RNase Fc fusion proteins to bind to Fc receptors in vitro, RSLV124 (wild-type Fc domain) and RSLV-132 (mutant Fc domain; P238S / P331S) were incubated with the Fc-containing human myeloid cell line THP1, and specific binding to cells was quantified by fluorescence-activated cell sorting (FACS) analysis. RSLV-124 and RSLV-132 were fluorescently labeled with the Alexa fluor dye AF-647 (catalog number A20006) using Invitrogen. After dialysis of the RNase Fc fusion proteins to remove unbound dyes, various amounts of the labeled proteins were incubated with THP1 cells for 1 hour. The cells were then vigorously washed to remove unbound RNase Fc fusion proteins, and the specifically bound proteins were quantified by FACS measuring the mean fluorescence intensity. The results in Figure 22 demonstrate that the RSLV-132 protein with the mutant Fc domain has significantly lower binding affinity to the Fc receptor than RSLV-124 with the wild-type Fc domain, with binding affinity reduced to less than one-quarter. This finding is consistent with our previous findings that RNase Fc fusion proteins with the mutant Fc domain (P238S / P331S) exhibit significantly reduced cytotoxicity.

[0259] Example 19: In vitro evaluation of the biological activity of hybrid nuclease molecules One or more hybrid nuclease molecules are purified, for example, by affinity chromatography or ion exchange chromatography, as previously described in the above examples. In some cases, the hybrid nuclease molecule is a polypeptide. In some cases, the hybrid nuclease molecule contains one or more sequences from Table 1. In some cases, the hybrid nuclease molecule contains a nuclease domain linked to a mutant Fc domain. In some cases, the hybrid nuclease molecule contains a mutant Fc domain. In some cases, the mutant Fc domain contains mutations in the hinge domain, CH2 domain, and / or CH3 domain. In some cases, the mutant Fc domain may contain P238S and / or P331S, and also contain mutations in one or more of the three cysteines in the hinge region. In some cases, the mutant Fc domain contains P238S and / or P331S, and / or mutations in the three cysteines in the hinge region. In some cases, the mutant Fc domain contains mutations to P238S and / or P331S, and / or SSS in the three cysteines of the hinge region. In some cases, the mutant Fc domain contains mutations to P238S and P331S, and in the three cysteines of the hinge region. In some cases, the mutant Fc domain contains P238S and P331S and SSS. In some cases, the mutant Fc domain is shown in SEQ ID NO 59, 60, 61. In some cases, the hybrid nuclease molecule is shown in SEQ ID NO. Various linker domains (e.g., those described herein) can be used to link the Fc domain to the nuclease domain. For example, linker domains can be used that have a length of 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 or more amino acids.The molecules are assayed in vitro for their specific nuclease activity using qualitative assays to confirm that they possess the desired nuclease function. Subsequently, the specific activity is generally determined by fluorescence-based kinetic assays using a substrate such as an RNase or DNase Alert Kit reagent and a fluorescence plate reader set to provide readings as a function of time. In addition, the protein solution is generally tested for endotoxin contamination using a commercially available kit, such as the Pyrotell Limulus Amebocyte Lysate (LAL) kit from Cape Cod, Inc. (E. Palmouth, MA), which has a detection limit of 0.06 EU / ml. Subsequently, the molecules are assayed using various in vitro assays for biological activity.

[0260] A series of in vitro assays will measure the effect of molecules on cytokine production by human PBMCs in response to various stimuli, in or out of the presence of molecules in the culture. Normal or patient-human PBMCs (approximately 1 × 10⁶ cells) will be cultured for 24, 48, or 96 hours, depending on the assay. PBMCs will be cultured in the presence of stimuli such as TLR ligands, costimulatory antibodies, immune complexes, and normal or autoimmune serum. The effect of molecules on cytokine production will be measured using commercially available reagents, such as the Biolegend (San Diego, CA) antibody pairing kit for IL-6, IL-8, IL-10, IL-4, IFN-γ, and TNF-α. To determine the effect of molecules on cytokine production, culture supernatants from the in vitro cultures will be collected at 24, 48 hours, or later. IFN-α production is measured using anti-human IFN-α antibodies and standard curve reagents, which can be obtained, for example, from the PBL interferon supplier (Piscataway, NJ). A similar group of assays are performed using a subpopulation of human lymphocytes (isolated monocytes, B cells, pDCs, T cells, etc.); and purification is performed using commercially available isolation kits with magnetic beads, for example, from Miltenyi Biotech (Auburn, CA).

[0261] In addition, the effects of molecules on the expression of lymphocyte-activating receptors such as CD5, CD23, CD69, CD80, CD86, and CD25 will be evaluated at various time points after stimulation. To clarify how these molecules affect the expression of various receptors associated with immune cell activation, PBMCs or isolated cell subpopulations will be subjected to multicolor flow cytometry.

[0262] Another group of assays are thought to measure the effects of these molecules on the proliferation of various lymphocyte subpopulations in vitro. These assays, for example, utilize CFDA-SE staining of human PBMCs (Invitrogen, Carlsbad, CA) before stimulation. 5 mM CFSE is diluted 1:3000 in PBS / 0.5% BSA with 10e7–10e8 PBMCs or a purified cell subset, and the labeled reaction is incubated at 37C for 3–4 minutes, followed by several washes in RPMI / 10% FBS to remove any remaining CFSE. Subsequently, CFSE-labeled cells are incubated in the co-culture reaction with various stimuli (TLR ligands, co-stimulatory antibodies, etc.) and molecules for 4 days, and then cell proliferation is analyzed by flow cytometry using antibodies specific to the cell subpopulation, conjugated to the dye.

[0263] Another assay measures the cytotoxic effect of one or more molecules. This assay measures toxicity using Annexin 5 staining (e.g., Annexin 5-FITC). Cells of interest (e.g., monocytes or monocyte cell lines) are brought into contact with a hybrid nuclease molecule of interest (e.g., a hybrid nuclease molecule with a mutant Fc domain) or one or more controls. At various time points after contact, the cells are isolated from the culture and stained with Annexin 5. Subsequently, the number of apoptotic or dead cells is counted, for example, using flow cytometry or fluorescence microscopy. The number of cells that stain positively for Annexin 5 in cells that have been brought into contact with the hybrid nuclease molecule of interest is lower compared to the positive controls.

[0264] The effects of these molecules on the in vitro maturation of monocytes to DCs and macrophages will also be evaluated using both normal and patient PBMC samples.

[0265] The efficacy of hybrid nuclease molecules is demonstrated by comparing the results of assays using cells treated with the hybrid nuclease molecules disclosed herein with the results of assays using cells treated with a control formulation. After treatment, the levels of the various markers described above (e.g., cytokines, cell surface receptors, proliferation) generally improve in the group treated with the effective molecule compared to the marker levels present before treatment or compared to the levels measured in the control group.

[0266] Example 20: Administration of a hybrid nuclease molecule to mammals that require it. This study uses mammals (e.g., mice, rats, rodents, humans, guinea pigs). One or more hybrid nuclease molecules containing one or more sequences from Table 1, or a control, are administered to the mammals (e.g., intravenously). In some cases, the hybrid nuclease molecule is a polypeptide. In some cases, the hybrid nuclease molecule contains one or more sequences from Table 1. In some cases, the hybrid nuclease molecule contains a nuclease domain linked to a mutant Fc domain. In some cases, the hybrid nuclease molecule contains a mutant Fc domain. In some cases, the mutant Fc domain contains mutations within the hinge domain, CH2 domain, and / or CH3 domain. In some cases, the mutant Fc domain may contain P238S and / or P331S, and may contain mutations in one or more of the three cysteine ​​molecules in the hinge region. In some cases, the mutant Fc domain contains mutations in P238S and / or P331S, and / or three cysteines in the hinge region. In some cases, the mutant Fc domain contains mutations to SSS in P238S and / or P331S, and / or three cysteines in the hinge region. In some cases, the mutant Fc domain contains mutations in P238S and P331S, and three cysteines in the hinge region. In some cases, the mutant Fc domain contains P238S and P331S and SSS. In some cases, the mutant Fc domain is shown in SEQ ID NO 59, 60, 61. In some cases, the hybrid nuclease molecule is shown in SEQ ID NO. Various linker domains (e.g., those described herein) can be used to link the Fc domain to the nuclease domain. For example, linker domains can be used that have a length of 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 or more amino acids.In some cases, the hybrid nuclease molecule is formulated with a pharmaceutically acceptable carrier. In some cases, the molecule is formulated as a pharmaceutically acceptable composition as described in the section on pharmaceutically acceptable compositions above. The hybrid nuclease molecule targets RNase and / or DNase.

[0267] If deemed beneficial, multiple doses will be used. Effects on IFN-α levels, IFN-α-responsive gene levels, autoantibody titers, renal function and pathophysiology, and / or immune complex levels in circulating blood will be monitored in mammals. Similar studies will be conducted using different treatment protocols and routes of administration (e.g., intramuscular administration). The efficacy of the hybrid nuclease molecule will be demonstrated by comparing IFN-α levels, IFN-α-responsive gene levels, autoantibody titers, renal function and pathophysiology, and / or immune complex levels in circulating blood in mammals treated with the hybrid nuclease molecule disclosed herein with those in mammals treated with a control formulation.

[0268] In one example, human subjects requiring treatment may be selected or identified. These subjects may, for example, require a reduction in the cause or symptoms of SLE. The identification of subjects may be performed in a clinical setting or elsewhere, for example, at the subject's home, by the subject using a self-testing kit.

[0269] At time zero, an appropriate initial dose of the hybrid nuclease molecule is administered to the target. The hybrid nuclease molecule is formulated as described herein. After a certain period following the initial administration, for example, 7, 14, and 21 days, the target's condition is assessed by measuring, for example, IFN-α levels, IFN-α-responsive gene levels, autoantibody titers, renal function and pathology, and / or immune complex levels in circulating blood. Other relevant criteria may also be measured. The frequency and intensity of dosing are adjusted according to the patient's needs.

[0270] Following treatment, the subject's IFN-α levels, IFN-α-responsive gene levels, autoantibody titers, renal function and pathogenesis, and / or circulating immune complex levels are reduced and / or improved compared to levels present before treatment or compared to levels in similarly affected but untreated / control subjects.

[0271] In another example, a rodent subject requiring treatment is selected or identified. The identification of the subject may take place in a laboratory setting or elsewhere.

[0272] At time zero, an appropriate initial dose of the hybrid nuclease molecule is administered to the subject. The hybrid nuclease molecule is formulated as described herein. After a certain period following the initial administration, for example, 7, 14, and 21 days, the subject's condition is assessed by measuring, for example, IFN-α levels, IFN-α-responsive gene levels, autoantibody titers, renal function and pathology, and / or immune complex levels in circulating blood. Other relevant criteria may also be measured. The frequency and intensity of dosing are adjusted according to the subject's needs.

[0273] Following treatment, the subject's IFN-α levels, IFN-α-responsive gene levels, autoantibody titers, renal function and pathogenesis, and / or circulating immune complex levels are reduced and / or improved compared to levels present before treatment or compared to levels in similarly affected but untreated / control subjects.

[0274] While the present invention has been described and illustrated in detail with reference to preferred embodiments and various alternative embodiments, those skilled in the art will understand that various modifications of the form and details can be made without departing from the spirit and scope of the invention.

[0275] All references, granted patents, and patent applications cited in the text of this specification, regardless of their purpose, are incorporated herein by reference in their entirety.

[0276] [Table 1] TIFF0007897613000033.tif241168TIFF0007897613000034.tif239168TIFF0007897613000035.tif239168TIFF0007897613000036.tif241168TIFF0007897613000037.tif240168TIFF0007897613000038.tif240168TIFF0007897613000039.tif240168TIFF0007897613000040.tif241168TIFF0007897613000041.tif241168TIFF0007897613000042.tif240168TIFF0007897613000043.tif240168TIFF0007897613000044.tif241168TIFF0007897613000045.tif240168TIFF0007897613000046.tif241168TIFF0007897613000047.tif243168TIFF0007897613000048.tif72168

[0277] Array information SEQUENCE LISTING <110> UNIVERSITY OF WASHINGTON <120> THERAPEUTIC NUCLEASE COMPOSITIONS AND METHODS <150> US 61 / 617,241 <151> 2012-03-29 <150> US 61 / 480,961 <151> 2011-04-29 <160> 126 <170> PatentIn version 3.5 <210> 1 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 1 gttaagcttg ccaccatggg tctggagaag tccctcattc tg 42 <210> 2 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 2 gataccaccg gtagggaatc tgcagcacag aagtttcag 39 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 3 ggctcgagca cagtagcatc aaagtggact ggtacgtagg 40 <210> 4 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 4 aaatctagac ctcaaccagg tagggaatct gcagcacaga agtttcag 48 <210> 5 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 5 tctagactat cacacagtag catcaaagtg gactggtacg tag 43 <210> 6 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 6 tgtccaccgt gtccagcacc tgaactcctg ggtggatcgt cagtcttcc 49 <210> 7 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 7 agatctcgag cccaaatctt ctgacaaaac tcacacatgt ccaccgtgt 49 <210> 8 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 8 tctagattat catttacccg gagacagaga gaggctcttc tgcgtgtagt g 51 <210> 9 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 9 cctccatgca aatgcccagc acctaacctc ttgggtggat catccgtctt catcttcc 58 <210> 10 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 10 gaagatctcg agcccagagg tcccacaatc aagccctctc ctcca 45 <210> 11 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 11 gtttctagat tatcatttac ccggagtccg agagaagctc ttagtcgt 48 <210> 12 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 12 agatctcgag cccaaatctt ctgacaaaac tcacacatgt ccaccgtgt 49 <210> 13 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 13 gttttctcga tggaggctgg gagggctttg ttggagacc 39 <210> 14 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 14 aaggtctcca acaaagccct cccagcctcc atcgagaaaa caatctcc 48 <210> 15 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 15 tctagattat catttacccg gagacagaga gaggctcttc tgcgtgtagt g 51 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 16 gttaccggtc tgaagatcgc agccttcaac atccag 36 <210> 17 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 17 gttctcgaga tctttcagca tcacctccac tggatagtg 39 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 18 gttgatatcc tgaagatcgc agccttcaac atccag 36 <210> 19 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 19 gtttctagat tatcacttca gcatcacctc cactggatag tg 42 <210> 20 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 20 tctccaccga gcccagcacc tgaactcctg ggaggatcgt cagtcttcct cttccccc 58 <210> 21 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 21 agatctcgag cccaaatctt ctgacaaaac tcacacatct ccaccgagcc cagcacct 58 <210> 22 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 22 gtctccaaca aagccctccc agcctccatc gagaaaacca tctcca 46 <210> 23 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 23 tggagatggt tttctcgatg ggggctggga gggctttgtt ggagacc 47 <210> 24 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 24 tctagattat cattttcccg gagagagaga gaggctcttc tgcgtgtagt g 51 <210> 25 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 25 tctccaccga gcccagcacc tgaactcctg ggaggatcgt cagtcttcct cttccccc 58 <210> 26 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 26 agatctcgag cccaaatctt ctgacaaaac tcacacatct ccaccgagcc cagcacct 58 <210> 27 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 27 tggagatggt tttctcgatg ggggctggga gggctttgtt ggagacc 47 <210> 28 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 28 tctagattat cattttcccg gagagagaga gaggctcttc tgcgtgtagt g 51 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 29 gatatcctgc acgctagggc tgctcacatt 30 <210> 30 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 30 agatctctcc ggaggaggtg gctcaggtgg tggaggatct ggaggaggtg ggagtggtgg 60 aggtggttct accggtctcg ag 82 <210> 31 <211> 97 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 31 agatctctcc ggaggaggtg gctcaggtgg tggaggatct ggaggaggtg gctcaggtgg 60 tggaggatct ggaggaggtg ggagtaccgg tctcgag 97 <210> 32 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 32 agatctctcc ggaggaggtg gctcaggtgg tggaggatct ggaggaggtg gctcaggtgg 60 tggaggatct ggaggaggtg ggagtctcga g 91 <210> 33 <211> 462 <212> DNA <213> Homo sapiens <400> 33 gtcgacggag ctagcagccc cgtgaacgtg agcagcccca gcgtgcagga tatcccttcc 60 ctgggcaagg aatcccgggc caagaaattc cagcggcagc atatggactc agacagttcc 120 cccagcagca gctccaccta ctgtaaccaa atgatgaggc gccggaatat gacacagggg 180 cggtgcaaac cagtgaacac ctttgtgcac gagcccctgg tagatgtcca gaatgtctgt 240 ttccaggaaa aggtcacctg caagaacggg cagggcaact gctacaagag caactccagc 300 atgcacatca cagactgccg cctgacaaac gactccaggt accccaactg tgcataccgg 360 accagcccga aggagagaca catcattgtg gcctgtgaag ggagcccata tgtgccagtc 420 cactttgatg cttctgtgga ggactctacc tataatcta ga 462 <210> 34 <211> 798 <212> DNA <213> Homo sapiens <400> 34 gatatcctga agatcgcagc cttcaacatc cagacatttg gggagaccaa gatgtccaat 60 gccaccctcg tcagctacat tgtgcagatc ctgagccgct atgacatcgc cctggtccag 120 gaggtcagag acagccacct gactgccgtg gggaagctgc tggacaacct caatcaggat 180 gcaccagaca cctatcacta cgtggtcagt gagccactgg gacggagacag ctataaggag 240 cgctacctgt tcgtgtacag gcctgaccag gtgtctgcgg tggacagcta ctactacgat 300 gatggctgcg agccctgcag gaacgacacc ttcaaccgag agccattcat tgtcaggttc 360 ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 420 gacgcagtag ccgagatcga cgctctcat gacgtctacc tggatgtcca agagaaatgg 480 ggcttggagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 540 tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 600 agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 660 atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 720 tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 780 ctgaagtgat aatctaga 798 <210> 35 <211> 798 <212> DNA <213> Homo sapiens <400> 35 gatatcctga agatcgcagc cttcaacatc cagacatttg gggagaccaa gatgtccaat 60 gccaccctcg tcagctacat tgtgcagatc ctgagccgct atgacatcgc cctggtccag 120 gaggtcagag acagccacct gactgccgtg gggaagctgc tggacaacct caatcaggat 180 gcaccagaca cctatcacta cgtggtcagt gagccactgg gacggagacag ctataaggag 240 cgctacctgt tcgtgtacag gcctgaccag gtgtctgcgg tggacagcta ctactacgat 300 gatgggctcg agccctgcgg gaacgacacc ttcaaccgag agccagccat tgtcaggttc 360 ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 420 gacgcagtag ccgagatcga cgctctcat gacgtctacc tggatgtcca agagaaatgg 480 ggcttggagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 540 tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 600 agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 660 atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 720 tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 780 ctgaaatgat aatctaga 798 <210> 36 <211> 798 <212> DNA <213> Homo sapiens <400> 36 gatatcctga agatcgcagc cttcaacatc cagacatttg gggagaccaa gatgtccaat 60 gccaccctcg tcagctacat tgtgcagatc ctgagccgct atgacatcgc cctggtccag 120 gaggtcagag acagccacct gactgccgtg gggaagctgc tggacaacct caatcaggat 180 gcaccagaca cctatcacta cgtggtcagt gagccactgg gacggaacag ctataaggag 240 cgctacctgt tcgtgtacag gcctgaccag gtgtctgcgg tggacagcta ctactacgat 300 gatggctgcg agccctgcgg gaacgacacc ttcaaccgag agccattcat tgtcaggttc 360 ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 420 gacgcagtag ccgagatcga cgctctctat gacgtctacc tggatgtcca agagaaatgg 480 ggcttagagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 540 tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 600 agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 660 atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 720 tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 780 ctgaagtgat aatctaga 798 <210> 37 <211> 795 <212> DNA <213> Homo sapiens <400> 37 accggtctga agatcgcagc cttcaacatc cagacatttg gggagaccaa gatgtccaat 60 gccaccctcg tcagctacat tgtgcagatc ctgagccgct atgacatcgc cctggtccag 120 gaggtcagag acagccacct gactgccgtg gggaagctgc tggacaacct caatcaggat 180 gcaccagaca cctatcacta cgtggtcagt gagccactgg gacggaacag ctataaggag 240 cgctacctgt tcgtgtacag gcctgaccag gtgtctgcgg tggacagcta ctactacgat 300 gatggctgcg agccctgcag gaacgacacc ttcaaccgag agccattcat tgtcaggttc 360 ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 420 gacgcagtag ccgagatcga cgctctctat gacgtctacc tggatgtcca agagaaatgg 480 ggcttggagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 540 tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 600 agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 660 atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 720 tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 780 ctgaaagatc tcgag 795 <210> 38 <211> 795 <212> DNA <213> Homo sapiens <400> 38 accggtctga agatcgcagc cttcaacatc cagacatttg gggagaccaa gatgtccaat 60 gccaccctcg tcagctacat tgtgcagatc ctgagccgct atgacatcgc cctggtccag 120 gaggtcagag acagccacct gactgccgtg gggaagctgc tggacaacct caatcaggat 180 gcaccagaca cctatcacta cgtggtcagt gagccactgg gacggagacag ctataaggag 240 cgctacctgt tcgtgtacag gcctgaccag gtgtctgcgg tggacagcta ctactacgat 300 gatgggctcg agccctgcgg gaacgacacc ttcaaccgag agccagccat tgtcaggttc 360 ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 420 gacgcagtag ccgagatcga cgctctcat gacgtctacc tggatgtcca agagaaatgg 480 ggcttggagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 540 tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 600 agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 660 atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 720 tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 780 ctgaaagatc tcgag 795 <210> 39 <211> 795 <212> DNA <213> Homo sapiens <400> 39 accggtctga agatcgcagc cttcaacatc cagacatttg gggagaccaa gatgtccaat 60 gccaccctcg tcagctacat tgtgcagatc ctgagccgct atgacatcgc cctggtccag 120 gaggtcagag acagccacct gactgccgtg gggaagctgc tggacaacct caatcaggat 180 gcaccagaca cctatcacta cgtggtcagt gagccactgg gacggagacag ctataaggag 240 cgctacctgt tcgtgtacag gcctgaccag gtgtctgcgg tggacagcta ctactacgat 300 gatggctgcg agccctgcgg gaacgacacc ttcaaccgag agccattcat tgtcaggttc 360 ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 420 gacgcagtag ccgagatcga cgctctcat gacgtctacc tggatgtcca agagaaatgg 480 ggcttagagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 540 tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 600 agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 660 atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 720 tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 780 ctgaaagatc tcgag 795 <210> 40 <211> 715 <212> DNA <213> Homo sapiens <400> 40 agatctcgag cccaaatctt ctgacaaaac tcacacatgt ccaccgtgcc cagcacctga 60 actcctgggg ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat 120 ctcccggacc cctgaggtca catgcgtggt ggtggacgtg agccacgaag acctgaggt 180 caagttcaac tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga 240 ggagcagtac aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg 300 gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga 360 gaaaaccatc tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc 420 atcccgggat gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta 480 tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac 540 cacgctccc gtgctggact ccgacggctc cttcttctc tacagcaagc tcaccgtgga 600 caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca 660 caaccactac acgcagaaga gcctctctct gtctccgggt aaatgataat ctaga 715 <210> 41 <211> 858 <212> DNA <213> Homo sapiens <400> 41 gttaagcttg ccaccatgga aaccccagcg cagcttctct tcctcctgct actctggctc 60 ccagatacca ccggtctgaa gatcgcagcc ttcaacatcc agacatttgg ggagaccaag 120 atgtccaatg ccaccctcgt cagctacatt gtgcagatcc tgagccgcta tgacatcgcc 180 ctggtccagg aggtcagaga cagccacctg actgccgtgg ggaagctgct ggacaacctc 240 aatcaggatg caccagacac ctatcactac gtggtcagtg agccactggg acggaacagc 300 tataaggagc gctacctgtt cgtgtacagg cctgaccagg tgtctgcggt ggacagctac 360 tactacgatg atggctgcga gccctgcggg aacgacacct tcaaccgaga gccagccatt 420 gtcaggttct tctcccggtt cacagaggtc agggagttttg ccattgttcc cctgcatgcg 480 gccccggggg acgcagtagc cgagatcgac gctctctatg acgtctacct ggatgtccaa 540 gagaaatggg gcttggagga cgtcatgttg atgggcgact tcaatgcggg ctgcagctat gtgagaccct cccagtggtc atccatccgc ctgtggacaa gccccacctt ccagtggctg 660 atccccgaca gcgctgacac cacagctaca cccacgcact gtgcctatga caggatcgtg gttgcaggga tgctgctccg aggcgccgtt gttcccgact cggctcttcc ctttaacttc 780 caggctgcct atggcctgag tgaccaactg gcccaagcca tcagtgacca ctatccagtg 840 gaggtgatgc tgaagtga <210> 42 <211> 918 <212> DNA <213> Homo sapiens <400> 42 atgtcacggg agctggcccc actgctgctt ctcctcctct ccatccacag cgccctggcc atgaggatct gctccttcaa cgtcaggtcc tttggggaaa gcaagcagga agacaagaat 120 gccatggatg tcattgtgaa ggtcatcaaa cgctgtgaca tcatactcgt gatggaaatc 180 aaggacagca acaacaggat ctgccccata ctgatggaga agctgaacag aaattcaagg 240 agaggcataa catacaacta tgtgattagc tctcggcttg gaagaaacac atataaagaa 300 caatatgcct ttctctacaa ggaaaagctg gtgtctgtga agaggagtta tcactaccat 360 gactatcagg atggagacgc agatgtgttt tccagggagc cctttgtggt ctggttccaa 420 tctccccaca ctgctgtcaa agacttcgtg attatccccc tgcacaccac cccagagaca 480 tccgttaagg agatcgatga gttggttgag gtctacacgg acgtgaaaca ccgctggaag 540 gcggagaatt tcattttcat gggtgacttc aatgccggct gcagctacgt ccccaagaag 600 gcctggaaga acatccgctt gaggactgac cccaggtttg tttggctgat cggggaccaa 660 gaggacacca cggtgaagaa gagcaccaac tgtgcatatg acaggattgt gcttagagga 720 caagaaatcg tcagttctgt tgttcccaag tcaaacagtg ttttgactt ccagaaagct 780 tacaagctga ctgaagagga ggccctggat gtcagcgacc actttccagt tgaatttaaa 840 ctacagtctt caagggcctt caccaacagc aaaaaatctg tcactctaag gaagaaaaca 900 aagagcaaac gctcctag 918 <210> 43 <211> 459 <212> DNA <213> Homo sapiens <400> 43 atgggtctgg agaagtctct tgtccggctc cttctgcttg tcctgatact gctggtgctg 60 ggctgggtcc agccttccct gggcaaggaa tcccgggcca agaaattcca gcggcagcat 120 atggactcag acagttcccc cagcagcagc tccacctact gtaaccaaat gatgaggcgc 180 cggaatatga cacaggggcg gtgcaaacca gtgaacacct ttgtgcacga gcccctggta 240 gatgtccaga atgtctgttt ccaggaaaag gtcacctgca agaacgggca gggcaactgc 300 tacaagagca actccagcat gcacatcaca gactgccgcc tgacaaacgg ctccaggtac 360 cccaactgtg cataccggac cagcccgaag gagacaca tcattgtggc ctgtgaaggg 420 agcccatatg tgccagtcca ctttgatgct actgtgtag 459 <210> 44 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 44 gtcgacggcg cggccgccag ccccgtgaac gtgagcagcc ccagcgtgca ggatatc 57 <210> 45 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 46 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 46 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <210> 47 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 47 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 48 <211> 260 <212> PRT <213> Homo sapiens <400> 48 Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys Met 1 5 10 15 Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser Arg Tyr 20 25 30 Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr Ala Val 35 40 45 Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp Thr Tyr His 50 55 60 Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu Arg Tyr 65 70 75 80 Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp Ser Tyr Tyr 85 90 95 Tyr Asp Asp Gly Cys Glu Pro Cys Arg Asn Asp Thr Phe Asn Arg Glu 100 105 110 Pro Phe Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg Glu Phe 115 120 125 Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala Glu Ile 130 135 140 Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp Gly Leu 145 150 155 160 Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val 165 170 175 Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr Phe 180 185 190 Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr His 195 200 205 Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala 210 215 220 Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala Tyr Gly 225 230 235 240 Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His Tyr Pro Val Glu 245 250 255 Val Met Leu Lys 260 <210> 49 <211> 260 <212> PRT <213> Homo sapiens <400> 49 Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys Met 1 5 10 15 Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser Arg Tyr 20 25 30 Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr Ala Val 35 40 45 Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp Thr Tyr His 50 55 60 Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu Arg Tyr 65 70 75 80 Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp Ser Tyr Tyr 85 90 95 Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Asn Arg Glu 100 105 110 Pro Ala Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg Glu Phe 115 120 125 Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala Glu Ile 130 135 140 Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp Gly Leu 145 150 155 160 Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val 165 170 175 Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr Phe 180 185 190 Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr His 195 200 205 Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala 210 215 220 Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala Tyr Gly 225 230 235 240 Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His Tyr Pro Val Glu 245 250 255 Val Met Leo Lys 260 <210> 50 <211> 260 <212> PRT <213> Homo sapiens <400> 50 Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys Met 1 5 10 15 Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser Arg Tyr 20 25 30 Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr Ala Val 35 40 45 Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp Thr Tyr His 50 55 60 Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu Arg Tyr 65 70 75 80 Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp Ser Tyr Tyr 85 90 95 Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Asn Arg Glu 100 105 110 Pro Phe Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg Glu Phe 115 120 125 Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala Glu Ile 130 135 140 Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp Gly Leu 145 150 155 160 Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val 165 170 175 Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr Phe 180 185 190 Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr His 195 200 205 Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala 210 215 220 Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala Tyr Gly 225 230 235 240 Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His Tyr Pro Val Glu 245 250 255 Val Met Leu Lys 260 <210> 51 <211> 260 <212> PRT <213> Homo sapiens <400> 51 Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys Met 1 5 10 15 Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser Arg Tyr 20 25 30 Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr Ala Val 35 40 45 Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp Thr Tyr His 50 55 60 Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu Arg Tyr 65 70 75 80 Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp Ser Tyr Tyr 85 90 95 Tyr Asp Asp Gly Cys Glu Pro Cys Arg Asn Asp Thr Phe Asn Arg Glu 100 105 110 Pro Ala Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg Glu Phe 115 120 125 Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala Glu Ile 130 135 140 Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp Gly Leu 145 150 155 160 Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val 165 170 175 Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr Phe 180 185 190 Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr His 195 200 205 Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala 210 215 220 Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala Tyr Gly 225 230 235 240 Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His Tyr Pro Val Glu 245 250 255 Val Met Leo Lys 260 <210> 52 <211> 260 <212> PRT <213> Homo sapiens <400> 52 Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys Met 1 5 10 15 Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser Arg Tyr 20 25 30 Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr Ala Val 35 40 45 Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp Thr Tyr His 50 55 60 Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu Arg Tyr 65 70 75 80 Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp Ser Tyr Tyr 85 90 95 Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Asn Arg Glu 100 105 110 Pro Ala Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg Glu Phe 115 120 125 Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala Glu Ile 130 135 140 Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp Gly Leu 145 150 155 160 Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val 165 170 175 Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr Phe 180 185 190 Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr His 195 200 205 Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala 210 215 220 Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala Tyr Gly 225 230 235 240 Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His Tyr Pro Val Glu 245 250 255 Val Met Leu Light 260 <210> 53 <211> 260 <212> PRT <213> Homo sapiens <400> 53 Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys Met 1 5 10 15 Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser Arg Tyr 20 25 30 Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr Ala Val 35 40 45 Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp Thr Tyr His 50 55 60 Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu Arg Tyr 65 70 75 80 Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp Ser Tyr Tyr 85 90 95 Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Asn Arg Glu 100 105 110 Pro Phe Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg Glu Phe 115 120 125 Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala Glu Ile 130 135 140 Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp Gly Leu 145 150 155 160 Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val 165 170 175 Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr Phe 180 185 190 Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr His 195 200 205 Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala 210 215 220 Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala Tyr Gly 225 230 235 240 Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His Tyr Pro Val Glu 245 250 255 Val Met Leu Lys 260 <210> 54 <211> 233 <212> PRT <213> Homo sapiens <400> 54 Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 20 25 30 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 35 40 45 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 50 55 60 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 65 70 75 80 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 85 90 95 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 100 105 110 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 115 120 125 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 130 135 140 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 145 150 155 160 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 165 170 175 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 180 185 190 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 195 200 205 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 210 215 220 Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 55 <211> 128 <212> PRT <213> Homo sapiens <400> 55 Lys Glu Ser Arg Ala Lys Lys Phe Gln Arg Gln His Met Asp Ser Asp 1 5 10 15 Ser Ser Pro Ser Ser Ser Ser Thr Tyr Cys Asn Gln Met Met Arg Arg 20 25 30 Arg Asn Met Thr Gln Gly Arg Cys Lys Pro Val Asn Thr Phe Val His 35 40 45 Glu Pro Leu Val Asp Val Gln Asn Val Cys Phe Gln Glu Lys Val Thr 50 55 60 Cys Lys Asn Gly Gln Gly Asn Cys Tyr Lys Ser Asn Ser Ser Met His 65 70 75 80 Ile Thr Asp Cys Arg Leu Thr Asn Asp Ser Arg Tyr Pro Asn Cys Ala 85 90 95 Tyr Arg Thr Ser Pro Lys Glu Arg His Ile Ile Val Ala Cys Glu Gly 100 105 110 Ser Pro Tyr Val Pro Val His Phe Asp Ala Ser Val Glu Asp Ser Thr 115 120 125 <210> 56 <211> 280 <212> PRT <213> Homo sapiens <400> 56 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly 20 25 30 Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile 35 40 45 Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His 50 55 60 Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro 65 70 75 80 Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr 85 90 95 Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val 100 105 110 Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr 115 120 125 Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe Thr Glu 130 135 140 Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala 145 150 155 160 Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu 165 170 175 Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly 180 185 190 Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr 195 200 205 Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala 210 215 220 Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu 225 230 235 240 Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln 245 250 255 Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His 260 265 270 Tyr Pro Val Glu Val Met Leu Lys 275 280 <210> 57 <211> 305 <212> PRT <213> Homo sapiens <400> 57 Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile His 1 5 10 15 Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly 20 25 30 Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val 35 40 45 Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn 50 55 60 Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg 65 70 75 80 Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg Asn 85 90 95 Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu Val Ser 100 105 110 Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly Asp Ala Asp 115 120 125 Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln Ser Pro His Thr 130 135 140 Ala Val Lys Asp Phe Val Ile Ile Pro Leu His Thr Thr Pro Glu Thr 145 150 155 160 Ser Val Lys Glu Ile Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys 165 170 175 His Arg Trp Lys Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala 180 185 190 Gly Cys Ser Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg 195 200 205 Thr Asp Pro Arg Phe Val Trp Leu Ile Gly Asp Gln Glu Asp Thr Thr 210 215 220 Val Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly 225 230 235 240 Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp 245 250 255 Phe Gln Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser 260 265 270 Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr 275 280 285 Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg 290 295 300 Looking 305 <210> 58 <211> 156 <212> PRT <213> Homo sapiens <400> 58 Met Ala Leu Glu Lys Ser Leu Val Arg Leu Leu Leu Leu Val Leu Ile 1 5 10 15 Leu Leu Val Leu Gly Trp Val Gln Pro Ser Leu Gly Lys Glu Ser Arg 20 25 30 Ala Lys Lys Phe Gln Arg Gln His Met Asp Ser Asp Ser Ser Pro Ser 35 40 45 Ser Ser Ser Thr Tyr Cys Asn Gln Met Met Arg Arg Arg Asn Met Thr 50 55 60 Gln Gly Arg Cys Lys Pro Val Asn Thr Phe Val His Glu Pro Leu Val 65 70 75 80 Asp Val Gln Asn Val Cys Phe Gln Glu Lys Val Thr Cys Lys Asn Gly 85 90 95 Gln Gly Asn Cys Tyr Lys Ser Asn Ser Ser Met His Ile Thr Asp Cys 100 105 110 Arg Leu Thr Asn Gly Ser Arg Tyr Pro Asn Cys Ala Tyr Arg Thr Ser 115 120 125 Pro Lys Glu Arg His Ile Ile Val Ala Cys Glu Gly Ser Pro Tyr Val 130 135 140 Pro Val His Phe Asp Ala Ser Val Glu Asp Ser Thr 145 150 155 <210> 59 <211> 693 <212> DNA <213> Homo sapiens <400> 59 cccaaatctt ctgacaaaac tcacacatct ccaccgtctc cagcacctga actcctgggg 60 ggaccgtcag tcttctctctt ccccccaaaaa cccaaggaca ccctcatgat ctcccggacc 120 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 180 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 240 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 300 aaggagtca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaccatc 360 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 420 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 480 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 540 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 600 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 660 Your account gcctctctct gtctccgggt aaa 693 <210> 60 <211> 233 <212> PRT <213> Homo sapiens <400> 60 Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro 1 5 10 15 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 20 25 30 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 35 40 45 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 50 55 60 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 65 70 75 80 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 85 90 95 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 100 105 110 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 115 120 125 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 130 135 140 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 145 150 155 160 Ser Asp Has More Glu Val Than Glu Ser Asn Gly Gln Pro Glu Asn Asn 165 170 175 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 180 185 190 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 195 200 205 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 210 215 220 Lys Ser Leu Ser Leu Ser Pro Gly Lys 225 230 <210> 61 <211> 1146 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 61 atggaaaccc ctgcccagct gctgttcctg ctgctgctgt ggctgccga caccaccggt 60 aaggaatccc gggccaagaa attccagcgg cagcatatgg actcagacag ttccccagc 120 agcagctcca cctactgtaa ccaatgatg aggcgccgga attgacaca ggggcggtgc 180 aaaccagtga acacctttgt gcacgagccc ctggtagatg tccagaatgt ctgtttccag 240 gaaaaggtca cctgcaagaa cgggcagggc aactgctaca agagcaactc cagcatgcac 300 atcacagact gccgcctgac aaacggctcc aggtacccca actgtgcata ccggaccagc 360 ccgaaggaga gacacatcat tgtggcctgt gaagggagcc catatgtgcc agtccacttt 420 gatgcttctg tggaggactc taccctcgag cccaaatctt ctgacaaaac tcacacatct 480 ccaccgagcc cagcacctga actcctggga ggatcgtcag tcttctctctt ccccccaaaaa 540 cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 600 660 gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 720 accgtcctgc accaggactg gctgaatggc aaggagtca agtgcaaggt ctccaacaaa 780 gccctcccag cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 840 caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 900 tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 960 ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1020 tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1080 gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctctct ctctccggga 1140 August 1146 <210> 62 <211> 381 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 62 Put Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Lys Glu Ser Arg Ala Lys Lys Phe Gln Arg Gln His 20 25 30 Met Asp Ser Asp Ser Pro Pro Ser Ser Ser Thr Tyr Cys Asn Gln 35 40 45 Met Met Arg Arg Arg Asn Met Thr Gln Gly Arg Cys Lys Pro Val Asn 50 55 60 Thr Phe Val His Glu Pro Leu Val Asp Val Gln Asn Val Cys Phe Gln 65 70 75 80 Glu Lys Val Thr Cys Lys Asn Gly Gln Gly Asn Cys Tyr Lys Ser Asn 85 90 95 Ser Ser Met His Ile Thr Asp Cys Arg Leu Thr Asn Gly Ser Arg Tyr 100 105 110 Pro Asn Cys Ala Tyr Arg Thr Ser Pro Lys Glu Arg His Ile Ile Val 115 120 125 Ala Cys Glu Gly Ser Pro Tyr Val Pro Val His Phe Asp Ala Ser Val 130 135 140 Glu Asp Ser Thr Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser 145 150 155 160 Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu 165 170 175 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 180 185 190 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 195 200 205 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 210 215 220 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 225 230 235 240 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 245 250 255 Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys 260 265 270 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 275 280 285 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 290 295 300 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 305 310 315 320 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 325 330 335 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 340 345 350 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 355 360 365 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <210> 63 <211> 1221 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 63 atggaaaccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60 aaggaatccc gggccaagaa attccagcgg cagcatatgg actcagacag ttcccccagc 120 agcagctcca cctactgtaa ccaaatgatg aggcgccgga atatgacaca ggggcggtgc 180 aaaccagtga acacctttgt gcacgagccc ctggtagatg tccagaatgt ctgtttccag 240 gaaaaggtca cctgcaagaa cgggcagggc aactgctaca agagcaactc cagcatgcac 300 atcacagact gccgcctgac aaacggctcc aggtacccca actgtgcata ccggaccagc 360 ccgaaggaga gacacatcat tgtggcctgt gaagggagcc catatgtgcc agtccacttt 420 gatgcttctg tggaggactc tacagatctc tccggaggag gtggctcagg tggtggagga 480 tctggaggag gtgggagtgg tggaggtggt tctaccggtc tcgagcccaa atcttctgac 540 aaaactcaca catctccacc gagcccagca cctgaactcc tgggaggatc gtcagtcttc 600 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 660 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 720 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 780 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 840 aaggtctcca acaaagccct cccagcctcc atcgagaaaa ccatctccaa agccaaaggg 900 cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 960 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1020 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1080 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1140 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1200 tctctctctc cgggaaaatg a 1221 <210> 64 <211> 401 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 64 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Lys Glu Ser Arg Ala Lys Lys Phe Gln Arg Gln His 20 25 30 Met Asp Ser Asp Ser Ser Pro Ser Ser Ser Ser Thr Tyr Cys Asn Gln 35 40 45 Met Met Arg Arg Arg Asn Met Thr Gln Gly Arg Cys Lys Pro Val Asn 50 55 60 Thr Phe Val His Glu Pro Leu Val Asp Val Gln Asn Val Cys Phe Gln 65 70 75 80 Glu Lys Val Thr Cys Lys Asn Gly Gln Gly Asn Cys Tyr Lys Ser Asn 85 90 95 Ser Ser Met His Ile Thr Asp Cys Arg Leu Thr Asn Gly Ser Arg Tyr 100 105 110 Pro Asn Cys Ala Tyr Arg Thr Ser Pro Lys Glu Arg His Ile Ile Val 115 120 125 Ala Cys Glu Gly Ser Pro Tyr Val Pro Val His Phe Asp Ala Ser Val 130 135 140 Glu Asp Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Pro Lys Ser Ser Asp Lys 165 170 175 Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser 180 185 190 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 195 200 205 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 210 215 220 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 225 230 235 240 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 245 250 255 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 260 265 270 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys 275 280 285 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 290 295 300 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 305 310 315 320 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 325 330 335 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 340 345 350 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 355 360 365 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 370 375 380 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 385 390 395 400 Lys <210> 65 <211> 2064 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 65 atggaaaccc cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccggt 60 ctgaagatcg cagccttcaa catccagaca tttggggaga ccaagatgtc caatgccacc 120 ctcgtcagct acattgtgca gatcctgagc cgctatgaca tcgccctggt ccaggaggtc 180 agagacagcc acctgactgc cgtggggaag ctgctggaca acctcaatca ggatgcacca 240 gacacctatc actacgtggt cagtgagcca ctgggacgga acagctataa ggagcgctac 300 ctgttcgtgt acaggcctga ccaggtgtct gcggtggaca gctactacta cgatgatggc 360 tgcgagccct gcaggaacga caccttcaac cgagagccat tcattgtcag gttcttctcc 420 cggttcacag aggtcaggga gtttgccatt gttcccctgc atgcggcccc gggggacgca 480 gtagccgaga tcgacgctct ctatgacgtc tacctggatg tccaagagaa atggggcttg 540 gaggacgtca tgttgatggg cgacttcaat gcgggctgca gctatgtgag accctcccag 600 tggtcatcca tccgcctgtg gacaagcccc accttccagt ggctgatccc cgacagcgct 660 gacaccacag ctacacccac gcactgtgcc tatgacagga tcgtggttgc agggatgctg 720 ctccgaggcg ccgttgttcc cgactcggct cttcccttta acttccaggc tgcctatggc 780 ctgagtgacc aactggccca agccatcagt gaccactatc cagtggaggt gatgctgaaa 840 gatctctccg gaggaggtgg ctcaggtggt ggaggatctg gaggaggtgg gagtggtgga 900 ggttctaccg gtctcgagcc caaatcttct gacaaaactc acacatctcc accgagccca 960 gcacctgaac tcctgggagg atcgtcagtc ttcctcttcc ccccaaaacc caaggacacc 1020 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 1080 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 1140 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 1200 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 1260 tccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1320 ctgcccccat cccgggatga gctgaccaag aaccaggtca gcctgacctg cctggtcaaa 1380 ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1440 tacaagacca cgctcccgt gctggactcc gacggctcct tcttctctca cagcaagctc 1500 accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1560 gctctgcaca accactacac gcaagaagc ctctctctct ctccgggaaa agtcgacgga 1620 gctagcagcc ccgtgaacgt gagcagcccc agaatgcagg atatcccttc cctgggcaag 1680 gaatcccggg ccaagaaatt ccagcggcag catatggact cagacagttc ccccagcagc 1740 agctccacct actgtaacca aatgatgagg cgccggata tgacacaggg gcggtgcaaa 1800 ccagtgaaca cctttgtgca cgagcccctg gtagatgtcc agaatgtctg tttccaggaa 1860 aaggtcacct gcaagaacgg gcagggcaag tggtacaaga gcaactccag catgcacatc 1920 acagactgcc gcctgacaaa cggctccagg taccccaact gtgcataccg aaccagcccg 1980 aaggagac acatcattgt ggcctgtgaa ggagcccata tgtgccagtc cactttgatg 2040 cttgctgtgg aggactctac ctaa 2064 <210> 66 <211> 679 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 66 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly 20 25 30 Glu Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile 35 40 45 Leu Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His 50 55 60 Leu Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro 65 70 75 80 Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr 85 90 95 Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val 100 105 110 Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Arg Asn Asp Thr 115 120 125 Phe Asn Arg Glu Pro Phe Ile Val Arg Phe Phe Ser Arg Phe Thr Glu 130 135 140 Val Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala 145 150 155 160 Val Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu 165 170 175 Lys Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly 180 185 190 Cys Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr 195 200 205 Ser Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala 210 215 220 Thr Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu 225 230 235 240 Leu Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln 245 250 255 Ala Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His 260 265 270 Tyr Pro Val Glu Val Met Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly 275 280 285 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Pro Lys 290 295 300 Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu 305 310 315 320 Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 325 330 335 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 340 345 350 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 355 360 365 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 370 375 380 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 385 390 395 400 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 405 410 415 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 420 425 430 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 435 440 445 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 450 455 460 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 465 470 475 480 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 485 490 495 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 500 505 510 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 515 520 525 Leu Ser Pro Gly Lys Val Asp Gly Ala Ser Ser Pro Val Asn Val Ser 530 535 540 Ser Pro Ser Val Gln Asp Ile Lys Glu Ser Arg Ala Lys Lys Phe Gln 545 550 555 560 Arg Gln His Met Asp Ser Asp Ser Ser Pro Ser Ser Ser Ser Thr Tyr 565 570 575 Cys Asn Gln Met Met Arg Arg Arg Asn Met Thr Gln Gly Arg Cys Lys 580 585 590 Pro Val Asn Thr Phe Val His Glu Pro Leu Val Asp Val Gln Asn Val 595 600 605 How To Get Gl Glu Lys Val Thr From Cys Lys And Gly Gln Gly And Cys Tyr 610 615 620 Lys Ser Asn Ser Met His Ile Thr Asp Cys Arg Leu Thr Asn Gly 625 630 635 640 Ser Arg Tyr Pro Asn Cys Ala Tyr Arg Thr Ser Pro Lys Glu Arg His 645,650,655 Ile Ile Val Ala Cys Glu Gly Ser Pro Tyr Val Pro Val His Phe Asp 660,665,670 Only Ser Val Glu Asp Ser Thr 675 <210> 67 <211> 2055 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 67 atggaaaccc cagcgcagct tctctcctc ctgctactct ggctcccaga tctctcct 60 aaggaatccc gggccaagaa attccagcgg cagcatatgg actcagacag ttccccagc 120 agcagctcca cctactgtaa ccaatgatg aggcgccgga attgacaca ggggcggtgc 180 aaaccagtga acacctttgt gcacgagccc ctggtagatg tccagaatgt ctgtttccag 240 gaaaaggtca cctgcaagaa cgggcagggc aactgctaca agagcaactc cagcatgcac 300 atcacagact gccgcctgac aaacggctcc aggtacccca actgtgcata ccggaccagc 360 ccgaaggaga gacacatcat tgtggcctgt gaagggagcc catatgtgcc agtccacttt 420 gatgcttctg tggagctc tacagatctc tccggaggag gtggctcagg tggtggagga 480 tctggaggag gtgggagtgg tggaggtggt tctaccggtc tcgagcccaa atcttctgac 540 aaaactcaca catctccacc gagcccagca cctgaactcc tgggaggatc gtcagtcttc 600 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 660 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 720 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 780 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 840 aaggtctcca acaaagccct cccagcctcc atcgagaaaa ccatctccaa agccaaaggg 900 cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 960 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1020 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1080 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1140 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1200 tctctctctc cgggaaaagt cgacggagct agcagccccg tgaacgtgag cagccccaga 1260 atgcaggata tcctgaagat cgcagccttc aacatccaga catttgggga gaccaagatg 1320 tccaatgcca ccctcgtcag ctacattgtg cagatcctga gccgctatga catcgccctg 1380 gtccaggagg tcagagacag ccacctgact gccgtgggga agctgctgga caacctcaat 1440 caggatgcac cagacaccta tcactacgtg gtcagtgagc cactgggacg gaacagctat 1500 aaggagcgct acctgttcgt gtacaggcct gaccaggtgt ctgcggtgga cagctactac 1560 tacgatgatg gctgcgagcc ctgcaggaac gacaccttca accgagagcc attcattgtc 1620 aggttcttct cccggttcac agaggtcagg gagtttgcca ttgttcccct gcatgcggcc 1680 ccgggggacg cagtagccga gatcgacgct ctctatgacg tctacctgga tgtccaagag 1740 aaatggggct tggaggacgt catgttgatg ggcgacttca atgcgggctg cagctatgtg 1800 agaccctccc agtggtcatc catccgcctg tggacaagcc ccaccttcca gtggctgatc 1860 cccgacagcg ctgacaccac agctacaccc acgcactgtg cctatgacag gatcgtggtt 1920 gcagggatgc tgctccgagg cgccgttgtt cccgactcgg ctcttccctt taacttccag 1980 gctgcctatg gcctgagtga ccaactggcc caagccatca gtgaccacta tccagtggag 2040 gtgatgctga aatga 2055 <210> 68 <211> 679 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 68 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Lys Glu Ser Arg Ala Lys Lys Phe Gln Arg Gln His 20 25 30 Met Asp Ser Asp Ser Ser Pro Ser Ser Ser Ser Thr Tyr Cys Asn Gln 35 40 45 Met Met Arg Arg Arg Asn Met Thr Gln Gly Arg Cys Lys Pro Val Asn 50 55 60 Thr Phe Val His Glu Pro Leu Val Asp Val Gln Asn Val Cys Phe Gln 65 70 75 80 Glu Lys Val Thr Cys Lys Asn Gly Gln Gly Asn Cys Tyr Lys Ser Asn 85 90 95 Ser Ser Met His Ile Thr Asp Cys Arg Leu Thr Asn Gly Ser Arg Tyr 100 105 110 Pro Asn Cys Ala Tyr Arg Thr Ser Pro Lys Glu Arg His Ile Ile Val 115 120 125 Ala Cys Glu Gly Ser Pro Tyr Val Pro Val His Phe Asp Ala Ser Val 130 135 140 Glu Asp Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu Pro Lys Ser Ser Asp Lys 165 170 175 Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser 180 185 190 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 195 200 205 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 210 215 220 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 225 230 235 240 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 245 250 255 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 260 265 270 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys 275 280 285 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 290 295 300 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 305 310 315 320 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 325 330 335 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 340 345 350 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 355 360 365 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 370 375 380 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 385 390 395 400 Lys Val Asp Gly Ala Ser Ser Pro Val Asn Val Ser Ser Pro Ser Val 405 410 415 Gln Asp Ile Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu 420 425 430 Thr Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu 435 440 445 Ser Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu 450 455 460 Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp 465 470 475 480 Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys 485 490 495 Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp 500 505 510 Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Arg Asn Asp Thr Phe 515 520 525 Asn Arg Glu Pro Phe Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val 530 535 540 Arg Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val 545 550 555 560 Ala Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys 565 570 575 Trp Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys 580 585 590 Ser Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser 595 600 605 Pro Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr 610 615 620 Pro Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu 625 630 635 640 Arg Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala 645,650,655 Ala Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His Tyr 660 665 670 Pro Val Glu Val Met Leu Lys 675 <210> 69 <211> 1980 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 69 atggaaaccc ctgcccagct gctgttcctg ctgctgctgt ggctgcccga caccaccggt 60 aaggaatccc gggccaagaa attccagcgg cagcatatg actcagacag ttcccccagc 120 agcagctcca cctactgtaa ccaaatgatg aggcgccgga atatgacaca ggggcggtgc 180 aaaccagtga acaccttttgt gcacgagccc ctggtagatg tccagaatgt ctgtttccag 240 gaaaaggtca cctgcaagaa cgggcagggc aactgctaca agagcaactc cagcatgcac 300 atcacagact gccgcctgac aaacggctcc aggtacccca actgtgcata ccggaccagc 360 ccgaaggaga gacacatcat tgtggcctgt gaagggagcc catatgtgcc agtccacttt 420 gatgcttctg tggaggactc taccctcgag cccaaatctt ctgacaaaac tcacacatct 480 ccaccgagcc cagcacctga actcctggga ggatcgtcag tcttctctctt ccccccaaaaa 540 cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 600 660 gccaagacaa agccgcggga ggagcagtac aacagcacgt accgtgtggt cagcgtcctc 720 accgtcctgc accaggactg gctgaatggc aaggagtca agtgcaaggt ctccaacaaa 780 gccctcccag cctccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 840 caggtgtaca ccctgccccc atcccgggat gagctgacca agaaccaggt cagcctgacc 900 tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 960 ccggagaaca actacaagac cacgctccc gtgctggact ccgacggctc cttcttctc 1020 tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1080 gtgatgcatg aggctctgca caaccactac acgcaagaga gcctctctct ctctccggga 1140 aaagtcgacg gagctagcag ccccgtgaac gtgagcagcc ccagaatgca ggatatcctg 1200 aagatcgcag ccttcaacat ccagacattt ggggagacca agatgtccaa tgccaccctc 1260 gtcagctaca ttgtgcagat cctgagccgc tatgacatcg ccctggtcca ggaggtcaga 1320 gacagccacc tgactgccgt ggggaagctg ctggacaacc tcaatcagga tgcaccagac 1380 acctatcact acgtggtcag tgagccactg ggacggagaca gctataagg gcgctacctg 1440 ttcgtgtaca ggcctgacca ggtgtctgcg gtggacagct actactacga tgatggctgc 1500 gagccctgca ggaacgacac cttcaaccga gagccattca ttgtcaggtt cttctcccgg 1560 ttcacagagg tcagggagtt tgccattgtt cccctgcatg cggccccggg ggacgcagta 1620 gccgagatcg acgctctcta tgacgtctac ctggatgtcc aagagaaatg gggcttggag 1680 gacgtcatgt tgatgggcga cttcaatgcg ggctgcagct atgtgagacc ctcccagtgg 1740 tcatccatcc gcctgtggac aagccccacc ttccagtggc tgatccccga cagcgctgac 1800 accacagcta caccacgca ctgtgcctat gacaggatcg tggttgcagg gatgctgctc 1860 cgaggcgccg ttgttcccga ctcggctctt ccctttaact tccaggctgc ctatggcctg 1920 agtgaccaac tggcccaagc catcagtgac cactatccag tggaggtgat gctgaaatga 1980 <210> 70 <211> 659 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 70 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Lys Glu Ser Arg Ala Lys Lys Phe Gln Arg Gln His 20 25 30 Met Asp Ser Asp Ser Ser Pro Ser Ser Ser Ser Thr Tyr Cys Asn Gln 35 40 45 Met Met Arg Arg Arg Asn Met Thr Gln Gly Arg Cys Lys Pro Val Asn 50 55 60 Thr Phe Val His Glu Pro Leu Val Asp Val Gln Asn Val Cys Phe Gln 65 70 75 80 Glu Lys Val Thr Cys Lys Asn Gly Gln Gly Asn Cys Tyr Lys Ser Asn 85 90 95 Ser Ser Met His Ile Thr Asp Cys Arg Leu Thr Asn Gly Ser Arg Tyr 100 105 110 Pro Asn Cys Ala Tyr Arg Thr Ser Pro Lys Glu Arg His Ile Ile Val 115 120 125 Ala Cys Glu Gly Ser Pro Tyr Val Pro Val His Phe Asp Ala Ser Val 130 135 140 Glu Asp Ser Thr Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser 145 150 155 160 Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu 165 170 175 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 180 185 190 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 195 200 205 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 210 215 220 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 225 230 235 240 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 245 250 255 Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys 260 265 270 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 275 280 285 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 290 295 300 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 305 310 315 320 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 325 330 335 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 340 345 350 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 355 360 365 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Val Asp Gly 370 375 380 Ala Ser Ser Pro Val Asn Val Ser Ser Pro Ser Val Gln Asp Ile Leu 385 390 395 400 Lys Ile Ala ...

Claims

1. A composition containing a nuclease effective for degrading RNA, DNA, or both RNA and DNA-containing immune complexes, comprising a pharmaceutically acceptable carrier and a polypeptide comprising an RNase domain and a modified Fc domain, wherein the polypeptide is (i) an amino acid sequence that is at least 90% to less than 100% identical to the amino acid sequence described in SEQ ID NO: 96, 92, 62, or 78, and that retains the activity of a polypeptide containing the amino acid sequence described in SEQ ID NO: 96, 92, 62, or 78, or (ii) An amino acid sequence that is at least 90% to less than 100% identical to the amino acid sequence described in SEQ ID NO: 98 or 94, and that retains the activity of the polypeptide containing the amino acid sequence described in SEQ ID NO: 98 or 94. A composition comprising, wherein the composition is formulated for intravenous administration, and the pharmaceutically acceptable carrier comprises a citrate buffer.

2. A composition containing the nuclease according to claim 1, wherein the polypeptide is at least 90% to less than 100% identical to the amino acid sequence described in SEQ ID NO: 96, and retains the activity of the polypeptide containing the amino acid sequence described in SEQ ID NO:

96.

3. A composition containing the nuclease according to claim 1, wherein the polypeptide is at least 90% to less than 100% identical to the amino acid sequence described in SEQ ID NO: 98, and retains the activity of the polypeptide containing the amino acid sequence described in SEQ ID NO:

98.

4. A composition containing a nuclease according to any one of claims 1 to 3, comprising a dimeric polypeptide.

5. A composition containing the nuclease according to claim 4, wherein the dimeric polypeptide is a homodimer.

6. A composition containing the nuclease according to any one of claims 1 to 5, wherein the modified Fc domain is modified to have reduced binding affinity to the Fcγ receptor, complement protein, or both.

7. A composition comprising the nuclease according to any one of claims 1 to 5, wherein the polypeptide has a cytotoxic effect that is reduced by at least half, one-third, one-quarter, or one-fifth compared to a polypeptide having a wild-type Fc domain.

8. A composition containing the nuclease according to any one of claims 1 to 5, wherein the modified Fc domain comprises an amino acid sequence having one or more mutations selected from the group consisting of P238S, P331S, SCC (residues 220, 226, and 229), SSS (residues 220, 226, and 229), G236R, L328R, L234A, and L235A.

9. A composition containing the nuclease according to any one of claims 1 to 5, wherein the modified Fc domain comprises an amino acid sequence having mutant SCC or SSS, P238S, and P331S.

10. A composition containing the nuclease according to any one of claims 1 to 5, which has a longer serum half-life compared to the RNase domain alone.

11. A composition containing the nuclease according to any one of claims 1 to 5, which inhibits interferon-α production.

12. A composition containing the nuclease according to any one of claims 1 to 5, wherein the activity of the RNase is one-ninth or more of the activity of the control RNase molecule.

13. A composition containing the nuclease according to any one of claims 1 to 5, wherein the activity of the RNase is equal to the activity of a control RNase molecule.

14. A composition containing the nuclease according to any one of claims 1 to 5, wherein the polypeptide comprises a DNase, and the activity of the DNase is one-ninth or more of the activity of a control DNase molecule.

15. A composition containing the nuclease according to any one of claims 1 to 5, wherein the polypeptide comprises a DNase, and the activity of the DNase is equal to the activity of a control DNase molecule.

16. A composition containing the nuclease according to any one of claims 1 to 5, wherein the polypeptide comprises a DNase and has a longer serum half-life compared to the DNase domain alone.

17. A pharmaceutical composition for treating or preventing a medical condition associated with an abnormal immune response, comprising a composition containing the nuclease described in any one of claims 1 to 5.

18. The pharmaceutical composition according to claim 17, wherein the medical condition associated with an abnormal immune response is an autoimmune disease.

19. The aforementioned autoimmune diseases include insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyroidopathy, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes mellitus, Goodpasture syndrome, pemphigus vulgaris, bullous pemphigoid, sympathetic ophthalmitis, and water A pharmaceutical composition according to claim 18, selected from the group consisting of crystallogenic uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, idiopathic cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus erythematosus, systemic lupus erythematosus (SLE), lupus nephritis, and connective tissue disease.

20. A pharmaceutical composition for treating SLE, comprising a composition containing the nuclease described in any one of claims 1 to 5.

21. A pharmaceutical composition for treating lupus nephritis, comprising a composition containing the nuclease described in any one of claims 1 to 5.

22. A pharmaceutical composition for treating discoid lupus erythematosus, comprising a composition containing the nuclease described in any one of claims 1 to 5.

23. A pharmaceutical composition for treating Sjögren's syndrome, comprising a composition containing the nuclease described in any one of claims 1 to 5.

24. A pharmaceutical composition for treating autoimmune diseases by degrading RNA, DNA, or immune complexes containing both RNA and DNA, comprising a composition containing a nuclease, wherein the nuclease-containing composition comprises a polypeptide containing an amino acid sequence that is at least 90% to less than 100% identical to the amino acid sequence described in SEQ ID NO: 96 or 98 and retains the activity of the polypeptide containing the amino acid sequence described in SEQ ID NO: 96 or 98, and a pharmaceutically acceptable carrier, wherein the nuclease-containing composition is formulated for intravenous administration, and the pharmaceutically acceptable carrier comprises a citrate buffer.

25. The pharmaceutical composition according to claim 24, wherein the composition containing the nuclease comprises a dimeric polypeptide.

26. The pharmaceutical composition according to claim 25, wherein the dimeric polypeptide is a homodimer.

27. The aforementioned autoimmune diseases include insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyroidopathy, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes mellitus, Goodpasture syndrome, pemphigus vulgaris, bullous pemphigoid, sympathetic ophthalmitis, and lens-related diseases. A pharmaceutical composition according to any one of claims 24 to 26, selected from the group consisting of uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, idiopathic cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus erythematosus, systemic lupus erythematosus (SLE), lupus nephritis, and connective tissue disease.

28. The pharmaceutical composition according to any one of claims 24 to 27, wherein the autoimmune disease is SLE.

29. The pharmaceutical composition according to any one of claims 24 to 27, wherein the autoimmune disease is lupus nephritis.

30. The pharmaceutical composition according to any one of claims 24 to 27, wherein the autoimmune disease is discoid lupus erythematosus.

31. The pharmaceutical composition according to any one of claims 24 to 27, wherein the autoimmune disease is Sjögren's syndrome.

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