Therapeutic nuclease compositions and methods

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 April 29, 2011, and U.S. Provisional Application No. 61 / 617,241, filed March 29, 2012. This application is also related to International Patent Application No. PCT / US2010 / 055131, filed November 2, 2010; U.S. Provisional Application No. 61 / 257,458, filed November 2, 2009; and U.S. Provisional Application No. 61 / 370,752, filed August 4, 2010. The disclosures of all of the foregoing applications are incorporated herein by reference in their entirety for all purposes.

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

[0003] background Excessive release of (ribonucleoprotein) particles from dead and dying cells can cause lupus pathology through two mechanisms: (i) deposition or in situ formation of chromatin / antichromatin complexes causes nephritis and leads to impaired renal function; and (ii) nuclear proteins activate innate immunity via toll-like receptors (TLRs) 7, 8, and 9 and TLR-independent pathways. Release of nuclear proteins may serve as potent antigens for autoantibodies in SLE, leading to the amplification of B cell and DC activation through co-engagement of antigen receptors and TLRs. Therefore, there is a need for means to remove triggering antigens and / or attenuate immune stimulation, immune amplification, and immune complex-mediated disease in subjects in need thereof. Summary of the Invention

[0004] overview Disclosed herein are hybrid nuclease molecules comprising a first nuclease domain and a modified Fc domain, wherein the first nuclease domain is operably connected to the Fc domain. The Fc domain is modified to reduce the cytotoxicity of the molecule compared to a hybrid nuclease molecule having an unmodified Fc domain. In some embodiments, the hybrid nuclease molecule has an Fc domain modified to reduce binding to Fcγ receptors, complement proteins, or both. In some embodiments, the cytotoxicity of the hybrid nuclease molecule is at least one-fold, two-fold, three-fold, four-fold, or five-fold reduced compared to a control molecule, for example, a hybrid nuclease molecule without a modified Fc domain.

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

[0006] In some aspects, the hybrid nuclease molecule comprises a modified Fc domain that is a mutant IgG1 Fc domain. In some aspects, the mutant Fc domain comprises one or more mutations in the hinge domain, CH2 domain, and / or CH3 domain. In some aspects, the Fc domain comprises an amino acid sequence having one or more of the following mutations: P238S, P331S, SCC, SSS (residues 220, 226, and 229), G236R, L328R, L234A, and L235A. In some aspects, the mutant Fc domain comprises a P238S mutation. In some aspects, the mutant Fc domain comprises a P331S mutation. In some aspects, the mutant Fc domain comprises a P238S mutation and a P331S mutation. In some aspects, the mutant Fc domain comprises P238S and / or P331S and optionally a mutation in one or more of the three cysteines in the hinge region. In some aspects, the mutant Fc domain comprises 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 comprises P238S and / or P331S and / or a mutation in one of the three cysteines in the hinge region (located at residue 220 according to the EU numbering system) to SCC (where CCC refers to the three cysteines present in the wild-type hinge domain). In some aspects, the mutant Fc domain comprises P238S and / or P331S and / or a mutation in one of the three cysteines in the hinge region (located at residues 220, 226, and 229 according to the EU numbering system) to SSS. In some aspects, the mutant Fc domain comprises P238S and P331S and mutations in the three cysteines in the hinge region. In some aspects, the mutant Fc domain comprises P238S and P331S and SCC. In some aspects, the mutant Fc domain comprises P238S and P331S and SSS.In some aspects, the mutant Fc domain comprises P238S and SCC. In some aspects, the mutant Fc domain comprises P238S and SSS. In some aspects, the mutant Fc domain comprises P331S and SCC. In some aspects, the mutant Fc domain comprises P331S and SSS. In some aspects, the mutant Fc domain comprises a mutation in one or more of the three cysteines in the hinge region. In some aspects, the mutant Fc domain comprises a mutation in the three cysteines in the hinge region. In some aspects, the mutant Fc domain comprises one mutation in the three cysteines in the hinge region to SCC. In some aspects, the mutant Fc domain comprises a mutation in the three cysteines in the hinge region to SSS. In some aspects, the mutant Fc domain comprises SCC. In some aspects, the mutant Fc domain comprises SSS.

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

[0008] In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked to a mutant human IgG1 Fc domain comprising SCC, P238S, and P331S, or to a mutant human IgG1 Fc domain comprising SSS, P238S, and P331S. In some aspects, the nucleic acid encoding the hybrid nuclease molecule is as set forth in SEQ ID NO:61, 77, or 91. In some aspects, the hybrid nuclease molecule is as set forth in SEQ ID NO:209, 62, 78, 92, or 94.

[0009] In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked via a (Gly4Ser)4 linker domain to a mutant human IgG1 Fc domain comprising SCC, P238S, and P331S, or to a mutant human IgG1 Fc domain comprising SSS, P238S, and P331S. In some aspects, the nucleic acid encoding the hybrid nuclease molecule is as set forth in SEQ ID NO:63 or 79. In some aspects, the hybrid nuclease molecule is as set forth in SEQ ID NO:64 or 79.

[0010] In some aspects, the hybrid nuclease molecule comprises a human DNase 1 G105R A114F domain linked to a mutant human IgG1 Fc domain comprising SCC, P238S, and P331S via a (Gly4Ser)4 linker domain, with the Fc domain linked to a wild-type human RNase 1 domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule comprises a human DNase 1 G105R A114F domain linked to a mutant human IgG1 Fc domain comprising SSS, P238S, and P331S via a (Gly4Ser)4 linker domain, with the Fc domain linked to a wild-type human RNase 1 domain via an NLG linker domain. In some aspects, the nucleic acid encoding the hybrid nuclease molecule is as set forth in SEQ ID NO:65 or 81. In some aspects, the hybrid nuclease molecule is as set forth in SEQ ID NO:66 or 82.

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

[0012] In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked to a mutant human IgG1 Fc domain comprising SCC, P238S, and P331S via a (Gly4Ser)4 linker domain, with the Fc domain linked to a human DNase 1 G105R A114F domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked to a mutant human IgG1 Fc domain comprising SSS, P238S, and P331S via a (Gly4Ser)4 linker domain, with the Fc domain linked to a human DNase 1 G105R A114F domain via an NLG linker domain. In some aspects, the nucleic acid encoding the hybrid nuclease molecule is as set forth in SEQ ID NO:67 or 83. In some aspects, the hybrid nuclease molecule is as set forth in SEQ ID NO:68 or 84.

[0013] In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked to a mutant human IgG1 Fc domain comprising SCC, P238S, and P331S, with the Fc domain linked to a human DNase 1 G105R A114F domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked to a mutant human IgG1 Fc domain comprising SSS, P238S, and P331S, with the Fc domain linked to a human DNase 1 G105R A114F domain via an NLG linker domain. In some aspects, the nucleic acid encoding the hybrid nuclease molecule is set forth in SEQ ID NO:69, 85, or 93. In some aspects, the hybrid nuclease molecule is set forth in SEQ ID NO:70, 86, 94, or 98.

[0014] In some aspects, the cytotoxicity induced by the hybrid nuclease molecule is reduced compared to a control molecule. In some aspects, the cytotoxicity induced by the hybrid nuclease molecule is reduced compared to a control molecule by 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, 95, 96, 97, 98, 99, or 100% reduced. In some aspects, the cytotoxicity of the hybrid nuclease molecule is reduced by about 3-5 fold, or at least about 3 fold, compared to a hybrid nuclease molecule having an unmodified Fc domain (e.g., a wild-type Fc domain).

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

[0016] In some embodiments, the hybrid nuclease molecule is a polypeptide, wherein the amino acid sequence of the first nuclease domain comprises the amino acid sequence of a 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 a human mutant IgG1 Fc domain, and the first linker domain connects 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 a sequence set forth in Table 1.

[0017] In some embodiments, the hybrid nuclease molecule comprises a wild-type human DNase 1 linked to a mutated human IgG1 Fc domain. In some embodiments, the hybrid nuclease molecule comprises a (Gly4Ser) nThe hybrid nuclease molecule comprises human DNase 1 G105R A114F linked by a linker domain to a mutant human IgG1 Fc domain, where n=0, 1, 2, 3, 4, or 5. In some embodiments, the hybrid nuclease molecule comprises wild-type human RNase 1 linked to a mutant human IgG1 Fc domain linked to wild-type human DNase 1. In some embodiments, the hybrid nuclease molecule comprises wild-type human RNase 1 linked to a mutant human IgG1 Fc domain linked to human DNase 1 G105R A114F. In some embodiments, the hybrid nuclease molecule is a polypeptide, wherein the amino acid sequence of the first nuclease domain comprises the amino acid sequence of an RNase, the first linker domain is 5-32 amino acids in length, the amino acid sequence of the Fc domain comprises the amino acid sequence of a human 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 linker domain comprises (Gly4Ser)5 and the restriction sites BglII, AgeI, and XhoI. In some embodiments, the hybrid nuclease molecule is a polypeptide, wherein 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 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 Fc receptors on human cells. In some embodiments, the Fc domain is modified to reduce binding to Fcγ receptors, complement proteins, or both. In some aspects, the binding to Fc receptors is reduced by 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, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 1 , 95, 96, 97, 98, 99, or 100% reduced. In some aspects, the hybrid nuclease molecule exhibits a 3- to 5-fold, or at least about 3-fold, reduction in Fc receptor binding compared to a hybrid nuclease molecule having an unmodified Fc domain (e.g., a wild-type Fc domain).

[0019] In some embodiments, the serum half-life of the molecule is significantly longer than that of the first nuclease domain alone. In some embodiments, the nuclease activity of the first nuclease domain of the molecule is the same as or higher than that of the nuclease domain alone. In some embodiments, administration of the molecule to mice increases the survival rate of the mice as measured by assays in mouse lupus models. In some aspects, the hybrid nuclease molecule degrades RNA, DNA, or both in the circulating blood. In other aspects, the hybrid nuclease molecule degrades RNA, DNA, or both in immune complexes. In some embodiments, the hybrid nuclease molecule inhibits interferon-α production. In some aspects, interferon-α production is increased by 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, 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 2 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% decrease.

[0020] In some embodiments, the hybrid nuclease molecule comprises a leader sequence. In some embodiments, the leader sequence is a human VK3LP peptide from the human kappa light chain family, and the leader sequence is connected to the N-terminus of the first nuclease domain. In some embodiments, the VK3LP has the sequence set forth 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 at least 90% identical to the amino acid sequence of an RNase listed 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 RNase 1.

[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 at least 90% identical to the amino acid sequence of a DNase listed 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 at least 90% identical to the amino acid sequence of an Fc domain listed in Table 1.

[0025] In some embodiments, the first linker domain has a length of about 1 to about 50 amino acids. In some embodiments, the first linker domain has a length of about 5 to about 31 amino acids. In some embodiments, the first linker domain has a length of about 15 to about 25 amino acids. In some embodiments, the first linker domain has a length of about 20 to about 32 amino acids. In some embodiments, the first linker domain has a length of about 20 amino acids. In some embodiments, the first linker domain has a length of about 25 amino acids. In some embodiments, the first linker domain has a length of about 18 amino acids. In some embodiments, the first linker domain comprises a gly / ser peptide. In some embodiments, the gly / ser peptide has the formula (Gly4Ser) n wherein 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 comprises (Gly4Ser)3. In some embodiments, the gly / ser peptide comprises (Gly4Ser)4. In some embodiments, the gly / ser peptide comprises (Gly4Ser)5. In some embodiments, the first linker domain comprises at least one restriction site. In some embodiments, the first linker domain comprises about 12 or more nucleotides comprising at least one restriction site. In some embodiments, the first linker domain comprises two or more restriction sites. In some embodiments, the first linker domain comprises multiple restriction sites. In some embodiments, the first linker domain comprises an NLG peptide. The NLG peptide comprises an N-linked glycosylation consensus sequence. In some embodiments, the NLG peptide has the sequence set forth in SEQ ID NO:99. In some embodiments, the first linker domain comprises an N-linked glycosylation site.

[0026] In some embodiments, the first nuclease domain is linked to the N-terminus of the Fc domain. In some embodiments, the first nuclease domain is linked 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 operably linked 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 operably linked 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 comprising the nucleic acid molecules disclosed herein. Also disclosed herein are host cells transformed with the recombinant expression vectors disclosed herein.

[0031] Also disclosed herein is a method for producing the hybrid nucleases disclosed herein, comprising the steps of: providing a host cell containing a nucleic acid sequence encoding the 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 condition associated with an abnormal immune response, comprising administering to a patient in need thereof an effective amount of an isolated hybrid nuclease molecule disclosed herein. In some embodiments, the 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, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, early-onset diabetes mellitus, Goodpasture's syndrome, or rheumatoid arthritis. The autoimmune disease is selected from the group consisting of pemphigus folliculorum, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, idiopathic liver cirrhosis, ulcerative colitis, Sjogren'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 of treating SLE, comprising administering to a subject a composition containing a nuclease in an amount effective to degrade immune complexes containing RNA, DNA, or 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 comprising the amino acid sequence set forth in SEQ ID NO: 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, or 98. [The present invention 1001] 1. A hybrid nuclease molecule comprising a first nuclease domain and a modified Fc domain, the first nuclease domain is operably linked to the Fc domain, and the Fc domain is modified such that the molecule has reduced cytotoxicity compared to a hybrid nuclease molecule having an unmodified Fc domain; The hybrid nuclease molecule. [The present invention 1002] A hybrid nuclease molecule comprising the amino acid sequence set forth in SEQ ID NO:62, 64, 78, 80, 92, or 96, or a hybrid nuclease molecule comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:62, 64, 78, 80, 92, or 96. [The present invention 1003] A hybrid nuclease molecule comprising the amino acid sequence set forth in SEQ ID NO:96. [The present invention 1004] A hybrid nuclease molecule comprising the amino acid sequence set forth in SEQ ID NO:66, 68, 70, 82, 84, 86, 94, or 98, or a hybrid nuclease molecule comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:66, 68, 70, 82, 84, 86, 94, or 98. [The present invention 1005] A hybrid nuclease molecule comprising the amino acid sequence set forth in SEQ ID NO:98. [The present invention 1006] 1001. The hybrid nuclease molecule of the present invention, wherein the Fc domain has been modified to reduce binding to Fcγ receptors, complement proteins, or both. [The present invention 1007] 1001. A hybrid nuclease molecule of the present invention having at least one-, two-, three-, four-, or five-fold reduced cytotoxicity. [The present invention 1008] 1001. The hybrid nuclease molecule of the present invention, further comprising a second nuclease domain operably connected to the Fc domain. [The present invention 1009] 1001. The hybrid nuclease molecule of the present invention, wherein the Fc domain comprises a human immunoglobulin Fc domain, such as a human IgG1 Fc domain. [The present invention 1010] 1009. The hybrid nuclease molecule of the present invention, wherein the Fc domain comprises a hinge domain, a CH2 domain, and a CH3 domain. [The present invention 1011] 1009. The hybrid nuclease molecule of the present invention, wherein the Fc domain comprises an amino acid sequence having one or more of the following mutations: P238S, P331S, SCC, SSS (residues 220, 226, and 229), G236R, L328R, L234A, and L235A. [The present invention 1012] 1009. The hybrid nuclease molecule of the present invention, wherein the modified Fc domain comprises an amino acid sequence having the mutations SCC or SSS, P238S, and P331S. [The present invention 1013] 1001. A hybrid nuclease molecule of the present invention having an increased serum half-life compared to the first nuclease domain alone. [The present invention 1014] 1001. The hybrid nuclease molecule of the present invention, wherein the first nuclease domain is operably connected to an Fc domain via a first linker domain. [The present invention 1015] The hybrid nuclease molecule of the invention 1014, wherein the first linker domain is a polypeptide linker, such as a gly-ser linker. [The present invention 1016] 1008. The hybrid nuclease molecule of the present invention, wherein the second nuclease domain is operably connected to the Fc domain via a second linker domain. [The present invention 1017] The hybrid nuclease molecule of the invention 1016, wherein the second linker domain is a polypeptide linker, such as an NLG peptide. [The present invention 1018] Any of the hybrid nuclease molecules of the present invention, wherein the first nuclease domain comprises an RNase or a DNase. [The present invention 1019] The hybrid nuclease molecule of the invention 1018, wherein the RNase is a human RNase, such as human pancreatic RNase A. [The present invention 1020] The hybrid nuclease molecules of the present invention degrade circulating RNA and RNA in immune complexes, or inhibit interferon-α production, or both. [The present invention 1021] 1019. A hybrid nuclease molecule of the present invention, having RNase activity that is at least about 9-fold lower than the activity of a control RNase molecule. [The present invention 1022] A hybrid nuclease molecule of the present invention, wherein the RNase activity is approximately equal to the activity of a control RNase molecule. [The present invention 1023] The hybrid nuclease molecule of the invention 1008, wherein the second nuclease domain comprises a DNase or an RNase. [The present invention 1024] The hybrid nuclease molecule of the present invention 1023, wherein the DNase is selected from the group consisting of human DNase type I, human DNase 1L3, or human TREX1. [The present invention 1025] 1024. A hybrid nuclease molecule of the present invention, having DNase activity that is at least about 9-fold lower than the activity of a control DNase molecule. [The present invention 1026] 1024 hybrid nuclease molecules of the present invention, which have DNase activity approximately equal to that of a control DNase molecule. [The present invention 1027] A composition comprising any of the hybrid nuclease molecules of the present invention and a pharmaceutically acceptable carrier. [The present invention 1028] A nucleic acid molecule encoding a hybrid nuclease molecule of the present invention. [The present invention 1029] A recombinant expression vector comprising a nucleic acid molecule of the present invention. [The present invention 1030] A host cell transformed with a recombinant expression vector of the present invention. [The present invention 1031] A method of making a hybrid nuclease molecule of the present invention, comprising the steps of: providing 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. [The present invention 1032] A method for treating or preventing a condition associated with an abnormal immune response, comprising administering to a subject an effective amount of a hybrid nuclease molecule of the present invention. [The present invention 1033] The method of claim 1032, wherein the condition is an autoimmune disease. [The present 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, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, and sympathetic nervous system disorders. ophthalmitis, phacogenic uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, idiopathic liver cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus erythematosus, systemic lupus erythematosus (SLE), and connective tissue disease. [This invention 1035] The method of claim 1034, wherein the autoimmune disease is SLE. [The present invention 1036] 1. A method of treating SLE, comprising administering to a subject a composition containing a nuclease in an amount effective to degrade immune complexes containing RNA, DNA, or both RNA and DNA, The composition comprises a pharmaceutically acceptable carrier and a hybrid nuclease molecule comprising the amino acid sequence set forth in SEQ ID NO: 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, or 98; The method. [Brief explanation of the drawings]

[0034] These and other features, aspects and advantages of the present invention will become better understood in connection with the following description and accompanying drawings. [Figure 1] 1 shows prototype structures for creating various embodiments of hybrid nuclease molecules. [Figure 2] Shown are concentrations of RSLV-124 recovered from mouse serum after a single intravenous injection. [Figure 3]1 shows the results of an RNase enzyme activity assay for RLSV-124 recovered from mouse serum, measured as relative fluorescence units (RFU) over time. [Figure 4] The concentration of RSLV-124 in mouse serum is shown as extrapolated from the RNase enzymatic activity of the molecule. [Figure 5] Single radial enzyme diffusion (SRED) analysis of serum from two RNase transgenic (Tg) mice compared with normal B6 mice is shown. [Figure 6] RNase A concentrations in Tg and double Tg (DTg) mice measured by ELISA are shown. Each dot represents the concentration measured in an individual mouse. [Figure 7] Survival of TLR7.1 Tg mice and TLR7.1×RNaseA DTg mice is compared. [Figure 8] Quantitative PCR of IRGs in the spleens of Tg and DTg mice is shown. [Figure 9] A Western blot of COS transfection supernatant from the RSLV 125-129 construct (SEQ ID NOs. 208-217) is shown. [Figure 10] 1 shows an SRED analysis comparing aliquots of Protein A purified protein from RSLV-transfected COS supernatants. [Figure 11a] Results are shown from a DNase nuclease activity assay performed on Protein A purified proteins from COS7 supernatants transfected with RSLV fusion plasmids. [Figure 11b] Results are shown from a DNase nuclease activity assay performed on Protein A purified proteins from COS7 supernatants transfected with RSLV fusion plasmids. [Figure 11c] Results are shown from a DNase nuclease activity assay performed on Protein A purified proteins from COS7 supernatants transfected with RSLV fusion plasmids. [Figure 12] RFU (relative fluorescence units) as a function of time for each protein are shown. [Figure 13-1] Lineweaver-Burk plots of the various molecules tested are shown. [Figure 13-2] Lineweaver-Burk plots of the various molecules tested are shown. [Figure 14] Cytotoxicity data are shown for RNaseIg molecules with wild-type or mutant Fc domains, plotting the percentage of dead cells as a function of fusion protein concentration. [Figure 15] Shown is a histogram overlay of THP-1 stained cells after 72 hours. [Figure 16] demonstrates the ability of RSLV-132 to inhibit immune complex-induced interferon-α production in SLE patients. [Figure 17] 1 shows the in vivo ability of RSLV-132 to inhibit RNA-induced interferon-α production. [Figure 18] RNase enzyme activity assays of two production lots of RSLV-132 stored at 4C for up to 8 weeks compared to wild-type RNase and RSLV-124 are shown. [Figure 19] 1 shows an RNase enzyme activity assay of RSLV-133, RSLV-123, and RSLV-124 relative to RNase A, measured as RFU over time. [Figure 20] DNase enzyme activity assay of RSLV-133 and RSLV-123 compared to DNase 1, measured as RFU over time. [Figure 21] 1 shows the results of an in-gel digestion experiment comparing the ability of RSLV-133 to RSLV-123 and wild-type DNase 1 to digest DNA. [Figure 22]Binding of RSLV-124 and RSLV-132 to Fc receptor-bearing THP1 cells by FACS analysis measuring mean fluorescence intensity is shown. DETAILED DESCRIPTION OF THE INVENTION

[0035] Detailed Description Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by the presence of high titers of autoantibodies directed against autologous nuclear proteins. There is strong evidence that defective clearance or disposal of dead and dying cells in SLE leads 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 proinflammatory cytokines; ii) serving as antigens that generate circulating immune complexes; and iii) serving as antigens that generate in situ complex formation at localized sites, such as the kidney. The present invention is based, at least in part, on the discovery that digestion of extracellular nucleic acids has an in vivo therapeutic effect.

[0036] Thus, the present invention provides a method for treating diseases characterized by defective clearance or disposal of apoptotic cells and necrotic debris, such as SLE, by administering an effective amount of nuclease activity to degrade complexes containing extracellular RNA and DNA. Such treatment can inhibit the production of type I interferon (IFN), a cytokine prominent in SLE and strongly correlated with disease activity and nephritis.

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

[0038] In another aspect, a method for treating SLE is provided, comprising administering to a subject an effective amount of a composition containing a nuclease. In one aspect, the treatment results in the degradation of immune complexes containing RNA, DNA, or both RNA and DNA. In another aspect, the treatment results in the inhibition of type I interferon, such as interferon-α, in the subject. In one aspect, the method for treating a subject comprises administering an effective amount of a composition of a hybrid nuclease molecule comprising the amino acid sequence set forth 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 comprising the amino acid sequence set forth in SEQ ID NO: 96 or 98.

[0039] Terms used in the claims and this specification are defined as set forth below, unless otherwise specified. In the event of a conflict with a term used in the parent provisional patent application, the term used in this specification shall control.

[0040] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and those modified post-translationally, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that differs from the general chemical structure of an amino acid but function in a manner similar to a naturally occurring amino acid.

[0041] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0042] An "amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different, "replacement" amino acid residue. An "amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. An insertion will typically consist of the insertion of one or two amino acid residues, although larger "peptide insertions," e.g., insertions of about 3 to about 5, or even up to about 10, 15, or 20 amino acid residues, can also be made herein. The inserted residues can be natural or non-natural, as disclosed above. An "amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0043] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to naturally occurring and unnatural amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are chemical mimetics of a corresponding naturally occurring amino acid.

[0044] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited otherwise, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise specified, a particular nucleic acid sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly specified sequence. Specifically, degenerate codon substitutions can be obtained by creating sequences in which the third position of one or more (or all) selected codons is replaced with mixed-base 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 can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

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

[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 can be a polypeptide comprising at least one Fc domain linked to a nuclease domain, such as a DNase and / or RNase. As another example, a hybrid nuclease molecule can comprise an RNase nuclease domain, a linker domain, and an Fc domain. Examples of hybrid nuclease molecules include SEQ ID NOs: 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 molecules of the present invention can comprise further modifications. In another embodiment, the hybrid nuclease molecule may be modified to attach a functional moiety (eg, PEG, a drug, or a label).

[0047] As used herein, a "hybrid bispecific nucleic acid molecule" or "binuclease molecule" refers to a hybrid nuclease molecule having two or more nuclease domains, e.g., a DNase domain and an RNase domain.

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

[0049] As used herein, the term "gly-ser polypeptide linker" refers to a peptide consisting of glycine and serine residues. An exemplary gly / ser polypeptide linker comprises the amino acid sequence 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 "linked," "fused," or "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components or domains into one by any means, including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinkers) are known in the art.

[0051] As used herein, the term "Fc region" is intended to be 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 can also be referred to as "Ig" or "IgG." In some embodiments, the Fc domain begins at the hinge region immediately upstream of the papain cleavage site of the antibody and ends at the C-terminus. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc domain comprises at least one of the following: a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In other embodiments, the Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In one embodiment, the Fc domain comprises a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In another embodiment, the Fc domain comprises a CH2 domain (or a portion thereof) fused to 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 comprises at least a portion of an Fc molecule known in the art to be necessary for FcRn binding. In another 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 FcγR binding. In one embodiment, an Fc domain of the present invention comprises at least the portion of an Fc molecule known in the art to be necessary for Protein A binding.In one embodiment, the Fc domain of the present invention comprises at least the portion of an Fc molecule known in the art to be necessary for Protein G binding. As used herein, the term "Fc domain" generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides, for example, comprising only the hinge, CH2, and CH3 domains. The Fc domain may be derived 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 Fcs, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from whole antibodies or produced by other means. The assignment of amino acid residue numbers for the Fc domain follows the definition of Kabat (see, e.g., Sequences of Proteins of Immunological Interest (Table of Contents, Introduction and Constant Region Sequences sections), 5th edition, Bethesda, MD: NIH vol. 1:647-723 (1991); Kabat et al., "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), each of which is incorporated herein by reference for any purpose.

[0053] As described herein, one of skill in the art will understand that any Fc domain may be engineered such that it differs in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule, hi 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 comprise a CH2 and / or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the Fc domain may comprise a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the Fc domain may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0055] A polypeptide or amino acid sequence "derived from" a specified polypeptide or protein refers to the origin of the polypeptide. Preferably, a polypeptide or amino acid sequence derived from a particular sequence has an amino acid sequence essentially identical to the particular sequence or a portion thereof consisting of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or has an amino acid sequence that is otherwise identifiable by one of skill in the art as having that origin.

[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 replaced by another amino acid residue, or one or more amino acid residues inserted or deleted.

[0057] The polypeptide may comprise a non-naturally occurring amino acid sequence. Such variants necessarily have less than 100% sequence identity or similarity to the starting hybrid nuclease molecule. In one preferred embodiment, the variant will have 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% amino acid sequence identity or similarity to the amino acid sequence of the starting polypeptide, e.g., over the entire length of the variant molecule.

[0058] In one embodiment, there is a single amino acid difference between the starting polypeptide sequence and the sequence derived therefrom. Identity or similarity to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., the same residues) with the starting amino acid residues, after aligning the sequences to achieve maximum percent sequence identity and introducing gaps, if necessary.

[0059] In one embodiment, a polypeptide of the invention consists of, consists essentially of, or comprises an amino acid sequence selected from Table 1, and functionally active variants thereof. In one embodiment, the polypeptide comprises an amino acid sequence 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 an amino acid sequence set forth in Table 1. In one embodiment, the polypeptide comprises a contiguous amino acid sequence 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 a contiguous amino acid sequence set forth 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 these numbers) consecutive amino acids of an amino acid sequence set forth in Table 1.

[0060] In one embodiment, the peptides of the present invention are encoded by a nucleotide sequence. The nucleotide sequences of the present invention are useful for numerous applications, including cloning, gene therapy, protein expression and purification, mutagenesis, DNA vaccination of a host in need thereof, antibody generation for, e.g., passive immunization, PCR, primer and probe generation, siRNA design and generation (see, e.g., the Dharmacon siDesign website), and the like. In one embodiment, the nucleotide sequences of the present invention comprise, consist of, or consist essentially of a nucleotide sequence selected from Table 1. In one embodiment, the nucleotide sequence comprises a nucleotide sequence 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 a nucleotide sequence set forth in Table 1. In one embodiment, the nucleotide sequence comprises a contiguous nucleotide 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 a contiguous nucleotide sequence set forth in Table 1. In one embodiment, the nucleotide sequence comprises a nucleotide 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 these numbers) contiguous nucleotides of the nucleotide sequence set forth in Table 1.

[0061] Preferred hybrid nuclease molecules of the present invention contain sequences derived from human immunoglobulin sequences (e.g., at least one Fc domain). However, the sequences may also contain one or more sequences 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 sequences of the present invention are non-immunogenic and / or have low immunogenicity.

[0062] Those skilled in the art will also understand that hybrid nuclease molecules of the present invention can be modified so that their sequence differs from the naturally occurring or native sequence from which they are derived while retaining the desired 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-naturally occurring variants of hybrid nuclease molecules derived from immunoglobulins (e.g., Fc domains) can be created by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of the immunoglobulin, such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0063] The peptidic hybrid nuclease molecules of the present invention may contain conservative amino acid substitutions at one or more amino acid residues, for example, at essential or non-essential amino acid residues. A "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with 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), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For this reason, non-essential amino acid residues in a binding polypeptide are preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a consecutive stretch of amino acids can be replaced with a structurally similar consecutive stretch that differs in the order and / or composition of the side chain family members. Alternatively, in another embodiment, mutations can be randomly introduced throughout all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be incorporated into a binding polypeptide of the invention and screened for their ability to bind to a desired target.

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

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

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

[0067] As used herein, the term "mammal" or "subject" or "patient" includes both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0068] The term "percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of the same nucleotides or amino acid residues when compared and aligned for maximum correspondence using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN, or other algorithms available to those of skill in the art) or as determined by visual inspection. Depending on the application, the "identity" can exist over a region of the compared sequences, e.g., a functional domain, or alternatively, over the entire length of the two sequences being compared.

[0069] For sequence comparison, typically, one sequence is used as a reference sequence to be compared with test sequence.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and if necessary, coordinates of subsequence are designated, and the parameter of sequence algorithm program is designated.Then, sequence comparison algorithm calculates the sequence identity of test sequence with reference sequence according to the parameter of designated program.

[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), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity 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 (for an overview, see Ausubel et al., infra).

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

[0072] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example, an amount sufficient to alter protein aggregation in a cell.

[0073] The term "therapeutically effective amount" refers to an amount effective to ameliorate symptoms of a disease. Since prevention can be considered a treatment, a therapeutically effective amount can also be a "prophylactically effective amount."

[0074] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0075] composition Hybrid nuclease molecules In some embodiments, the compositions of the present invention comprise a hybrid nuclease molecule. In some embodiments, the hybrid nuclease molecule comprises a nuclease domain operably 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 comprises 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 molecules of the present invention comprise a leader peptide at the N-terminus of the molecule, which is subsequently cleaved from the hybrid nuclease molecule. Methods for generating nucleic acid sequences encoding leader peptides fused to recombinant proteins 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 one of skill in the art. Examples of hybrid nuclease molecules of the invention further comprising a VK3 leader peptide (VK3LP), where the leader peptide is fused to the N-terminus of the hybrid nuclease molecule, are shown in SEQ ID NOs:92 (RSLV-132) and 94 (RSLV-133). The corresponding nucleotide sequences are shown in SEQ ID NOs:91 and 93, respectively. In embodiments, after cleavage of the VK3 leader, these hybrid nuclease molecules have the sequences set forth in SEQ ID NOs:96 (RSLV-132) and 98 (RSLV-133), respectively. The corresponding nucleotide sequences are shown in SEQ ID NOs:95 and 97, respectively. In some embodiments, hybrid nuclease molecules of the invention are expressed without a leader peptide fused to its N-terminus, and the resulting hybrid nuclease molecule has an N-terminal methionine.

[0077] In some embodiments, the hybrid nuclease molecule will comprise a stop codon, hi some embodiments, the stop codon will be 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 may be C-terminal to the Fc domain. Figure 1 shows at least one embodiment of a hybrid nuclease molecule. In some embodiments, the hybrid nuclease molecule comprises a sequence set forth in Table 1.

[0079] In some embodiments, the hybrid nuclease molecule is an RNase or DNase molecule or a multienzyme molecule (e.g., both RNase and DNase, or two RNA or DNA nucleases with different substrate specificities) attached to an Fc domain that specifically binds extracellular immune complexes. In some embodiments, the Fc domain does not effectively bind to Fcγ receptors. In one aspect, the hybrid nuclease molecule does not effectively bind C1q. In another aspect, the hybrid nuclease molecule comprises an in-frame Fc domain derived from IgG1. In another aspect, the hybrid nuclease molecule further comprises a mutation in the hinge domain, CH2 domain, and / or CH3 domain. In other aspects, the mutation is P238S, P331S, or N297S, and may include a mutation in one or more of the three cysteines in the hinge region. In some such aspects, the mutation in one or more of the three cysteines in the hinge region may be SCC or SSS. In another aspect, the molecule contains an SCC hinge but is otherwise wild-type with respect to the human IgG1 Fc CH2 and CH3 domains, and effectively binds to an Fc receptor to promote uptake of the hybrid nuclease molecule into the endocytic compartment of the bound cell. In another aspect, the molecule has activity against single-stranded and / or double-stranded RNA substrates.

[0080] In some aspects, the hybrid nuclease molecule comprises a mutant Fc domain. In some aspects, the hybrid nuclease molecule comprises a mutant IgG1 Fc domain. In some aspects, the mutant Fc domain comprises one or more mutations in the hinge domain, CH2 domain, and / or CH3 domain. In some aspects, the mutant Fc domain comprises a P238S mutation. In some aspects, the mutant Fc domain comprises a P331S mutation. In some aspects, the mutant Fc domain comprises a P238S mutation and a P331S mutation. In some aspects, the mutant Fc domain comprises P238S and / or P331S and may also comprise mutations in one or more of the three cysteines in the hinge region. In some aspects, the mutant Fc domain comprises 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 comprises P238S and / or P331S, and / or mutations of three cysteines in the hinge region to SSS, or one cysteine ​​in the hinge region to SCC. In some aspects, the mutant Fc domain comprises P238S and P331S, and mutations in three cysteines in the hinge region. In some aspects, the mutant Fc domain comprises P238S and P331S, and either SCC or SSS. In some aspects, the mutant Fc domain comprises P238S and P331S, and SCC. In some aspects, the mutant Fc domain comprises P238S SSS. In some aspects, the mutant Fc domain comprises P331S, and either SCC or SSS. In some aspects, the mutant Fc domain comprises mutations in one or more of the three cysteines in the hinge region. In some aspects, the mutant Fc domain comprises mutations in the three cysteines in the hinge region, hi some aspects, the mutant Fc domain comprises mutations in the three cysteines in the hinge region to SSS.In some aspects, the mutant Fc domain comprises a mutation of one of the three cysteines in the hinge region to SCC. In some aspects, the mutant Fc domain comprises SCC or SSS. In some aspects, the mutant Fc domain is as set forth in any of SEQ ID NOs: 59, 60, 71-76, or 87-90. In some aspects, the hybrid nuclease molecule is as set forth in any of SEQ ID NOs: 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, or 98. In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked to a mutant human IgG1 Fc domain comprising SCC, P238S, and P331S, or to a mutant human IgG1 Fc domain comprising SSS, P238S, and P331S. In some aspects, the nucleic acid sequence encoding the hybrid nuclease molecule is as set forth in SEQ ID NO:61, 77, or 91. In some aspects, the hybrid nuclease molecule is as set forth in SEQ ID NO:62, 78, 92, or 96.

[0081] In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked via a (Gly4Ser)4 linker domain to a mutant human IgG1 Fc domain comprising SCC, P238S, and P331S, or to a mutant human IgG1 Fc domain comprising SSS, P238S, and P331S. In some aspects, the nucleic acid sequence encoding the hybrid nuclease molecule is set forth in SEQ ID NO:63 or 79. In some aspects, the hybrid nuclease molecule is set forth in SEQ ID NO:64 or 80.

[0082] In some aspects, the hybrid nuclease molecule comprises a human DNase 1 G105R A114F domain linked to a mutant human IgG1 Fc domain comprising SCC, P238S, and P331S via a (Gly4Ser)4 linker domain, with the Fc domain linked to a wild-type human RNase 1 domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule comprises a human DNase 1 G105R A114F domain linked to a mutant human IgG1 Fc domain comprising SSS, P238S, and P331S via a (Gly4Ser)4 linker domain, with the Fc domain linked to a wild-type human RNase 1 domain via an NLG linker domain. In some aspects, the nucleic acid sequence encoding the hybrid nuclease molecule is set forth in SEQ ID NO:65 or 81. In some aspects, the hybrid nuclease molecule is set forth in SEQ ID NO:66 or 82.

[0083] In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked to a mutant human IgG1 Fc domain comprising SCC, P238S, and P331S via a (Gly4Ser)4 linker domain, with the Fc domain linked to a human DNase 1 G105R A114F domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked to a mutant human IgG1 Fc domain comprising SSS, P238S, and P331S via a (Gly4Ser)4 linker domain, with the Fc domain linked to a human DNase 1 G105R A114F domain via an NLG linker domain. In some aspects, the nucleic acid sequence encoding the hybrid nuclease molecule is set forth in SEQ ID NO:67 or 83. In some aspects, the hybrid nuclease molecule is set forth in SEQ ID NO:68 or 84.

[0084] In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked to a mutant human IgG1 Fc domain comprising SCC, P238S, and P331S, with the Fc domain linked to a human DNase 1 G105R A114F domain via an NLG linker domain. In some aspects, the hybrid nuclease molecule comprises a wild-type human RNase 1 domain linked to a mutant human IgG1 Fc domain comprising SSS, P238S, and P331S, with the Fc domain linked to a human DNase 1 G105R A114F domain via an NLG linker domain. In some aspects, the nucleic acid sequence encoding the hybrid nuclease molecule is set forth in SEQ ID NO:69, 85, or 93. In some aspects, the hybrid nuclease molecule is set forth in SEQ ID NO:70, 86, 94, or 98.

[0085] In some aspects, the activity of the hybrid nuclease molecule is detectable in vitro and / or in vivo. In some aspects, the hybrid nuclease molecule binds to a cell, a malignant cell, or a cancer cell, and interferes with its biological activity.

[0086] In another aspect, multifunctional RNase molecules are provided that are attached to another enzyme or antibody with binding specificity, such as an scFv that targets RNA, or a second nuclease domain with the same or different specificity as the first domain.

[0087] In another aspect, multifunctional DNase molecules are provided that are attached to another enzyme or antibody with binding specificity, such as a DNA-targeting scFv, or a second nuclease domain with the same or different specificity as the first domain.

[0088] In another aspect, the hybrid nuclease molecule is adapted for preventing or treating a disease or disorder in a mammal by administering to a mammal in need thereof the hybrid nuclease molecule attached to an Fc region in a therapeutically effective amount such that the disease is prevented or treated. In another aspect, the disease or disorder is an autoimmune disease or cancer. In some such aspects, the autoimmune disease is insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, early-onset diabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, idiopathic liver cirrhosis, ulcerative colitis, Sjogren'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 the RNase hybrid nuclease molecule are primarily extracellular, consisting of, for example, RNA contained in immune complexes with anti-RNP autoantibodies and RNA expressed on the surface of cells undergoing apoptosis. In some embodiments, the RNase hybrid nuclease molecule is active in the acidic environment of endocytic vesicles. In some embodiments, the RNase hybrid nuclease molecule contains a wild-type (wt) Fc domain, allowing the molecule to bind to FcR and enter endocytic compartments through the entry pathway used by immune complexes. In some embodiments, the RNase hybrid nuclease molecule containing the wt Fc domain is adapted to be active both extracellularly and in the endocytic environment (where TLR7 may be expressed). In some aspects, this allows the RNase hybrid nuclease molecule containing the wt Fc domain to stop TLR7 signaling via previously phagocytosed immune complexes or by RNA that activates TLR7 after viral infection. In some embodiments, the wt RNase of the RNase hybrid nuclease molecule is not resistant to inhibition by cytoplasmic inhibitors of RNases. In some embodiments, the wt RNase of the RNase hybrid nuclease molecule is not active in the cytoplasm of a cell.

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

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

[0092] Alternative strategies for constructing hybrid nuclease molecules with attached Fc domains are also contemplated, and in some embodiments, the orientation of the domains can be altered to construct Ig-RNase or Ig-DNase or RNase-Ig or RNase-Ig molecules that retain FcR binding and have an active nuclease domain.

[0093] In some embodiments, the DNase hybrid nuclease molecule comprises a wt Fc domain, e.g., which allows the molecule to undergo endocytosis after binding to an FcR. In some embodiments, the DNase hybrid nuclease molecule can have activity against extracellular immune complexes containing DNA, e.g., either in soluble form or deposited as an insoluble complex.

[0094] In some embodiments, the 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 wt Fc domain, they can have activity both extracellularly and within the endocytic compartment, where TLR7 and TLR9 can be localized.

[0095] In some embodiments, the linker domain comprises a 3, 4, or 5 mutation (gly4ser), which changes the linker length by 5 amino acids. In another embodiment, the linker domain is approximately 18 amino acids in length and contains an N-linked glycosylation site, which may be susceptible to protease cleavage in vivo. In some embodiments, the N-linked glycosylation site can prevent cleavage of the hybrid nuclease molecule at the linker domain. In some embodiments, the N-linked glycosylation site can help compartmentalize the folding of independent functional domains separated by the linker domain.

[0096] In some embodiments, the hybrid nuclease molecule can comprise both mutant and / or wild-type human IgG1 Fc domains. In some embodiments, the hybrid nuclease molecule can 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 comprises a DNase 1L3-Ig-linker-RNase construct. In some embodiments, the hybrid nuclease molecule comprises a DNase 1-Ig-linker-RNase construct or an RNase-Ig-linker-DNase construct. In some embodiments, the fusion junction between the enzymatic domain and other domains of the hybrid nuclease molecule is optimized.

[0097] In some embodiments, the hybrid nuclease molecule includes a DNase-Ig hybrid nuclease molecule and / or a hybrid DNase-RNase hybrid nuclease molecule.

[0098] In some embodiments, the hybrid nuclease molecule comprises TREX1. In some embodiments, the TREX1 hybrid nuclease molecule is capable of digesting chromatin. In some embodiments, the TREX1 hybrid nuclease molecule is expressed by a cell. In some embodiments, the expressed hybrid nuclease molecule comprises mouse TREX-1 and a mouse (wt or mutant) Fc domain. In some embodiments, a 20-25 amino acid (aa) linker domain between TREX1 and the IgG hinge may be required to enable DNase activity. In some embodiments, hybrid nuclease molecules with a 15-aa linker domain have no activity. In some embodiments, the use of 20- and 25-amino acid linker domains (plus two or more amino acids to incorporate a restriction site) results in functional activity as measured by chromatin digestion. In some embodiments, an approximately 72-aa hydrophobic region can be removed from the COOH end of TREX-1 before fusing it to 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 the control and / or other hybrid nuclease molecules. In some embodiments, a kinetic enzyme assay is used to quantitatively compare the enzymatic activity of the hybrid nuclease molecule and the control.

[0099] In some embodiments, further optimization of the fusion junctions selected for truncation of the TREX1 enzyme can be used to improve expression of the hybrid nuclease molecule.

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

[0101] In some embodiments, the hybrid nuclease molecule is a TREX1-tandem hybrid nuclease molecule. In some embodiments, an alternative method for promoting head-to-tail folding of TREX1 is to create a TREX1-TREX1-Ig hybrid hybrid nuclease molecule, incorporating two TREX1 domains in tandem, followed by a linker domain and an Ig Fc domain. In some embodiments, the head-to-tail arrangement of the TREX1 cassette can be modified for head-to-tail folding on either arm of the immunoenzyme, introducing a single TREX1 functional domain into each arm of the molecule. In some embodiments, each immunoenzyme hybrid nuclease molecule has two functional TREX1 enzymes attached to a single IgG Fc domain.

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

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

[0104] In some embodiments, the hybrid nuclease molecule comprises 1L3-Ig. In some embodiments, 1L3 DNase is constructed and expressed from mouse sequences. 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 comprises human 1L3-Ig, human 1L3-Ig-RNase, and / or human RNase-Ig-1L3.

[0105] In some embodiments, the hybrid nuclease molecule comprises DNase 1-Ig. In some embodiments, the DNase 1-Ig hybrid nuclease molecule contains A114F, a naturally occurring mutant allele that exhibits reduced sensitivity to actin. In some embodiments, this mutation is introduced into the hybrid nuclease molecule to generate a more stable derivative of human DNase 1. In some embodiments, DNase 1-linker-Ig is generated, which contains a 20-aa or 25-aa linker domain. In some embodiments, the hybrid nuclease molecule comprises RNase-Ig-linker-DNase 1, where the DNase 1 domain is located on the COOH side of the Ig Fc domain. In some embodiments, hybrid nuclease molecules incorporating DNase 1 are generated, including: DNase 1-linker-Ig-linker2-RNase, and / or RNase-Ig-linker-DNase 1.

[0106] Another aspect of the present invention is the use of gene therapy methods to treat or prevent disorders, diseases, and conditions with one or more hybrid nuclease molecules. Gene therapy methods involve the introduction of nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences of the hybrid nuclease molecules into animals to achieve expression of one or more polypeptides of the present invention. This method may include the introduction of one or more polynucleotides encoding the polypeptides of the hybrid nuclease molecules of the present invention operably linked to a promoter and any other genetic elements necessary for expression of the polypeptide by the target tissue.

[0107] In gene therapy applications, the gene for the hybrid nuclease molecule is introduced into cells to achieve the in vivo synthesis of therapeutically effective gene products. "Gene therapy" includes both traditional gene therapy, which achieves a permanent effect through one treatment, and the administration of gene therapy drugs, which involve single or repeated administration of therapeutically effective DNA or mRNA. Oligonucleotides can be modified to enhance their uptake, for example, by replacing their negatively charged phosphodiester groups with uncharged groups.

[0108] Fc domain In some embodiments, the hybrid nuclease molecule comprises an Fc domain. The Fc domain does not comprise a variable region that binds to an antigen. In various embodiments, the Fc domain does not comprise a variable region. Fc domains useful for generating the hybrid nuclease molecules of the present invention can be obtained from several different sources. In preferred embodiments, the Fc domain of the hybrid nuclease molecule is derived from a human immunoglobulin. However, it will be understood that the Fc domain may also be derived from the 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 of the hybrid nuclease molecule, or a portion thereof, 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 comprises a mutant Fc domain. In some aspects, the hybrid nuclease molecule comprises a mutant IgG1 Fc domain. In some aspects, the mutant Fc domain comprises one or more mutations in the hinge domain, CH2 domain, and / or CH3 domain. In some aspects, the mutant Fc domain comprises a P238S mutation. In some aspects, the mutant Fc domain comprises a P331S mutation. In some aspects, the mutant Fc domain comprises a P238S mutation and a P331S mutation. In some aspects, the mutant Fc domain comprises P238S and / or P331S and may also comprise mutations in one or more of the three cysteines in the hinge region. In some aspects, the mutant Fc domain comprises 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 comprises a P238S and / or P331S mutation, and / or a mutation in one cysteine ​​in the hinge region to SCC or a mutation in three cysteines in the hinge region to SSS. In some aspects, the mutant Fc domain comprises P238S and P331S and a mutation in at least one of the three cysteines in the hinge region. In some aspects, the mutant Fc domain comprises P238S, P331S, and SCC. In some aspects, the mutant Fc domain comprises P238S, P331S, and SSS. In some aspects, the mutant Fc domain comprises P238S, and SCC or SSS. In some aspects, the mutant Fc domain comprises P331S, and SCC or SSS. In some aspects, the mutant Fc domain comprises a mutation in one or more of the three cysteines in the hinge region. In some aspects, the mutant Fc domain comprises mutations in the three cysteines in the hinge region.In some aspects, the mutant Fc domain comprises a mutation of one of the three cysteines in the hinge region to SCC. In some aspects, the mutant Fc domain comprises SCC. In some aspects, the mutant Fc domain comprises a mutation of three cysteines in the hinge region to SSS. In some aspects, the mutant Fc domain comprises SSS. In some aspects, the nucleic acid sequence encoding the mutant Fc domain is set forth in SEQ ID NO:59, 71, 73, 75, 87, or 89. In some aspects, the mutant Fc domain is as set forth 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 set forth 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 molecule is as set forth 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 accessible deposits. Constant region domains, including Fc domain sequences, can be selected to have specific effector functions (or lack specific effector functions) or specific modifications to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and suitable Fc domain sequences (e.g., hinge, CH2, and / or CH3 sequences, or portions thereof) can be obtained from these sequences using art-recognized techniques. The genetic material obtained using any of the above methods can then be modified or synthesized to obtain the polypeptides of the present invention. It will further be understood that the scope of the present invention encompasses alleles, variants, and mutations of constant region DNA sequences.

[0111] The Fc domain sequence can be cloned, for example, using polymerase chain reaction and primers selected to amplify the domain of interest. To clone the Fc domain sequence from an antibody, mRNA can be isolated from hybridoma cells, splenocytes, or lymphocytes, reverse transcribed into DNA, and the antibody gene can be amplified by PCR. PCR amplification methods are described in detail in U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; and, for example, "PCR Protocols: A Guide to Methods and Applications" (eds., Innis et al., 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 by consensus constant region primers or by more specific primers based on the published DNA and amino acid sequences of the heavy and light chains. As discussed above, PCR can also be used to isolate DNA clones encoding 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). Cloning of antibody sequences is further described in U.S. Pat. No. 5,658,570, issued Jan. 25, 1995, to Newman et al., which is incorporated herein by reference).

[0112] Hybrid nuclease molecules of the invention can contain one or more Fc domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Fc domains). In one embodiment, the Fc domains can be of different types. In one embodiment, at least one Fc domain present in a hybrid nuclease molecule comprises a hinge domain or a portion thereof. In another embodiment, hybrid nuclease molecules of the invention comprise at least one Fc domain comprising at least one CH2 domain or a portion thereof. In another embodiment, hybrid nuclease molecules of the invention comprise at least one Fc domain comprising at least one CH3 domain or a portion thereof. In another embodiment, hybrid nuclease molecules of the invention comprise at least one Fc domain comprising at least one CH4 domain or a portion thereof. In another embodiment, a hybrid nuclease molecule of the invention comprises at least one Fc domain comprising at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g., in a hinge-CH2 orientation). In another embodiment, a hybrid nuclease molecule of the invention comprises at least one Fc domain comprising at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (e.g., in a CH2-CH3 orientation). In another embodiment, a hybrid nuclease molecule of the invention comprises at least one Fc domain comprising at least one hinge domain or portion thereof, at least one CH2 domain or portion thereof, and at least one CH3 domain or portion thereof, e.g., in a hinge-CH2-CH3, hinge-CH3-CH2, or CH2-CH3-hinge orientation.

[0113] In certain embodiments, the hybrid nuclease molecule comprises at least one complete Fc region (e.g., an Fc domain comprising a hinge domain, a CH2 domain, and a CH3 domain, although these need not be from the same antibody) derived from one or more immunoglobulin heavy chains. In other embodiments, 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 a human IgG immunoglobulin heavy chain (e.g., human IgG1).

[0114] In another embodiment, the hybrid nuclease molecules of the present invention comprise at least one Fc domain comprising a complete CH3 domain. In another embodiment, the hybrid nuclease molecules of the present invention comprise at least one Fc domain comprising a complete CH2 domain. In another embodiment, the hybrid nuclease molecules of the present invention comprise at least one Fc domain comprising at least a CH3 domain and at least one of a hinge region and a CH2 domain. In one embodiment, the hybrid nuclease molecules of the present invention comprise at least one Fc domain comprising a hinge domain and a CH3 domain. In another embodiment, the hybrid nuclease molecules of the present invention comprise at least one Fc domain comprising a hinge domain, 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 that make up the Fc domain of the hybrid nuclease molecules of the present invention may be derived from different immunoglobulin molecules. For example, a polypeptide of the present invention may comprise 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, a hybrid nuclease molecule may comprise an Fc domain that comprises a hinge domain derived in part from an IgG1 molecule and in part from an IgG3 molecule. As described herein, those skilled in the art will understand that the Fc domain may be engineered to have a different amino acid sequence than that of a native antibody molecule.

[0116] In another embodiment, the hybrid nuclease molecules of the present invention comprise one or more truncated Fc domains that are still sufficient to confer Fc receptor (FcR) binding properties to the Fc region. Thus, the Fc domain of the hybrid nuclease molecules of the present invention can comprise or consist of an FcRn-binding portion. The FcRn-binding portion can be derived from the heavy chain of any isotype, including IgG1, IgG2, IgG3, and IgG4. In one embodiment, an FcRn-binding portion derived from an antibody of human isotype IgG1 is used. In another embodiment, an FcRn-binding portion derived from an antibody of human isotype IgG4 is used.

[0117] In one embodiment, the hybrid nuclease molecules of the present invention lack one or more constant region domains of the complete Fc region, i.e., they are partially or completely deleted. In some embodiments, the hybrid nuclease molecules of the present invention lack the entire CH2 domain (ΔCH2 construct). Those skilled in the art will appreciate that such constructs may be preferred due to the regulatory properties of the CH2 domain on the catabolic rate of antibodies. In some embodiments, the hybrid nuclease molecules of the present invention comprise a CH2 domain-deleted Fc region derived from a vector (e.g., from IDEC Pharmaceuticals, San Diego) encoding an IgG1 human constant region domain (see, e.g., WO 02 / 060955 A2 and WO 02 / 096948 A2). This exemplary vector has been engineered to delete the CH2 domain, resulting in a synthetic vector expressing the domain-deleted IgG1 constant region. It should be noted that these exemplary constructs are preferably engineered so that the binding CH3 domain is directly fused to the hinge region of each Fc domain.

[0118] In other constructs, it may be desirable to provide a peptide spacer between one or more component Fc domains. For example, a peptide spacer may be placed between the hinge region and the CH2 domain and / or between the CH2 and CH3 domains. 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 joined to the hinge region using a peptide spacer 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 free and accessible or to ensure that the hinge region remains flexible. Preferably, any linker peptide compatible with the present invention will be relatively non-immunogenic and will not interfere with proper folding of the Fc.

[0119] Changes to Fc amino acids In some embodiments, the Fc domain used in the hybrid nuclease molecules 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., an amino acid substitution, compared to the wild-type Fc from which the Fc domain is derived. For example, if the Fc domain is derived from a human IgG1 antibody, the variant will contain at least one amino acid mutation (e.g., substitution) compared to the wild-type amino acid at the corresponding position in the human IgG1 Fc region.

[0120] The amino acid substitutions of the Fc variants may be at positions within the Fc domain designated corresponding to the part number assigning that residue to the Fc region in an antibody.

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

[0122] In certain embodiments, hybrid nuclease molecules of the invention comprise Fc variants containing multiple amino acid substitutions. Hybrid nuclease molecules of the invention can contain, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions. Preferably, the amino acid substitutions are spaced apart from one another by at least one amino acid position or more, e.g., at least every 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions or more. More preferably, the engineered amino acids are spaced apart from one another by at least every 5, 10, 15, 20, or 25 amino acid positions or more.

[0123] In certain embodiments, the Fc variants provide an improvement in at least one effector function conferred by an Fc domain comprising said wild-type Fc domain (e.g., an improved ability of the Fc domain to bind an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII) or a complement protein (e.g., C1q), or to induce antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In other embodiments, the Fc variants provide an engineered cysteine ​​residue.

[0124] In some aspects, the Fc domain contains an alteration within the region of amino acids 234-238, which includes the sequence LLGGP at the beginning of the CH2 domain. In some aspects, the Fc variants alter Fc-mediated effector function, particularly ADCC, and / or reduce binding avidity for Fc receptors. In some aspects, sequence changes at positions closer to the CH2-CH3 junction, such as K322 or P331, abolish complement-mediated cytotoxicity and / or alter avidity for FcR binding. In some aspects, the Fc domain incorporates alterations at residues P238 and P331, e.g., changing the wild-type proline at these positions to serine. In some aspects, altering one or more of the three hinge cysteines in the hinge region so that these residues encode CCC, SCC, SSC, SCS, or SSS may also affect FcR binding and molecular homogeneity, for example, by eliminating unpaired cysteines that may destabilize the folded protein.

[0125] The hybrid nuclease molecules of the present invention can utilize art-recognized Fc variants known to provide improved effector function and / or FcR binding. Specifically, the hybrid nuclease molecules of the present invention can utilize Fc variants described in, for example, International PCT Publication Nos. WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, and WO02 / 060919, each of which is incorporated herein by reference. 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, Nos. WO06 / 047350A2 and WO06 / 085967A2; U.S. Patent Application Publication Nos. US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 0243188, US20070248603, US20070286859, US20080057056; or U.S. Patent Nos. 5,648,260; 5,739,277 ; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784; and 7,317,091.In one embodiment, a particular change (e.g., a particular substitution of one or more amino acids disclosed in the art) can be made at 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 at one or more of the disclosed amino acid positions.

[0126] Other amino acid mutations in the Fc domain are expected to reduce binding to Fcγ receptors and Fcγ receptor subtypes, for example, mutations at 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, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 372, 379, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305 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, as described in U.S. Patent No. 6,737,056, issued May 18, 2004, which is incorporated herein by reference in its entirety. This patent reports that changing Pro331 in IgG3 to Ser resulted in a six-fold decrease in affinity compared to IgG3 without the mutation, indicating the involvement of Pro331 in FcγRI binding. Additionally, U.S. Patent No. 5,624,821, issued April 29, 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 that may alter receptor binding affinity.

[0127] Additional mutations contemplated for use include, for example, those described in U.S. Patent Application Publication No. 2006 / 0235208, published October 19, 2006, which is incorporated herein by reference in its entirety. This publication lists the following mutations: 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 The present invention describes Fc variants that exhibit reduced binding to Fcγ receptors, reduced antibody-dependent cellular cytotoxicity, or reduced complement-dependent cytotoxicity, comprising at least one amino acid modification within the Fc region, including 236R / 237K, 236R / 325L, 236R / 328R, 237K / 325L, 237K / 328R, 325L / 328R, 235G / 236R, 267R / 269R, 234G / 235G, 236R / 237K / 325L, 236R / 325L / 328R, 235G / 236R / 237K, and 237K / 325L / 328R. Other mutations contemplated 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, 329H, 330I, 331D, 331G, 331H, 332I, 333I, 334I, 335I, 335I, 336I, 336I, 337I, 337I, 338I, 338I, 339I, 340I, 341I, 341I, 342I, 342I, 343I, 344I, 345I, 345I, 346I, 346I, 347I, 347I, 348I, 348I, 349I, 350I, 351I, 352I, 353I, 354I, 355I, 355I, 356I, 357I, 358I, 359I, 360I, 361I, 362I 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, insertion of G between positions 299 and 299 These include the insertion of A between positions 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 (numbering scheme follows EU index). Additionally, 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, 236S / 332E, 236A / 332E, 236B / 332E, 236C / 332E, 236D / 332E, 236E / 332E, 236D / 332E, 236E / 332E, 236D / 332E, 236E / 332E, 236F ... 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, 297-298 insertion of G / 332E, 297-298 insertion of A / 332E, 297-298 insertion of S / 332E, 297-298 insertion of D / 332E, 326-327 insertion of G / 332E, 326-327 insertion of A / 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, 235~236 Contemplated for use are insertion of L / 332E, insertion of G at 235-236 / 332D, insertion of A at 235-236 / 332D, insertion of S at 235-236 / 332D, insertion of T at 235-236 / 332D, insertion of N at 235-236 / 332D, insertion of D at 235-236 / 332D, insertion of V at 235-236 / 332D, and insertion of L at 235-236 / 332D (numbering system according to the EU index). Mutation L234A / L235A is described, for example, in U.S. Patent Application Publication No. 2003 / 0108548, published June 12, 2003, which is incorporated herein by reference in its entirety. In various embodiments, the described modifications are included either individually or in combination.

[0128] In certain embodiments, the hybrid nuclease molecules of the present invention contain amino acid substitutions in 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 to FcRn, and therefore, longer or shorter serum half-lives, respectively, compared to hybrid nuclease molecules lacking these substitutions. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules have useful applications in mammalian therapeutic methods where a longer half-life of the administered polypeptide is desired, for example, for treating chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful for administration to mammals where a shortened circulation time would be beneficial, such as for in vivo diagnostic imaging, or in situations where the starting polypeptide has adverse side effects if present in the circulation for a long period of time. Fc variants with reduced FcRn-binding affinity are also less likely to cross the placenta and are therefore useful for treating diseases or disorders in pregnant women. Additionally, other applications in which reduced FcRn-binding affinity may be desirable include applications in which localization to the brain, kidney, and / or liver is desired. In one exemplary embodiment, the hybrid nuclease molecules of the present invention exhibit reduced transport from the vasculature across the epithelium of the renal glomerulus. In another embodiment, the hybrid nuclease molecules of the present invention exhibit reduced transport from the brain across the blood-brain barrier (BBB) ​​to the vascular lumen. In one embodiment, a hybrid nuclease molecule with altered FcRn-binding ability comprises at least one Fc domain (e.g., one or two Fc domains) with 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 WO 05 / 047327, which is incorporated herein by reference.

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

[0130] In one embodiment, the hybrid nuclease molecule exhibits altered binding to an activating FcγR (e.g., FcγI, FcγIIa, or FcγRIIIa). In another embodiment, the hybrid nuclease molecule exhibits altered binding affinity to an inhibitory FcγR (e.g., FcγRIIb). Exemplary amino acid substitutions that alter FcR binding activity or complement fixation activity are disclosed in International PCT Publication No. WO 05 / 063815, which is incorporated herein by reference.

[0131] The hybrid nuclease molecules of the present invention may also contain amino acid substitutions that alter the glycosylation of the hybrid nuclease molecule. For example, the Fc domain of the hybrid nuclease molecule may contain an Fc domain with a mutation that leads to reduced glycosylation (e.g., N-linked or O-linked glycosylation), or may contain an altered 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 an amino acid substitution 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 No. WO 05 / 018572 and U.S. Patent Application Publication No. 2007 / 0111281, which are incorporated herein by reference.

[0132] In other embodiments, the hybrid nuclease molecules of the present invention comprise at least one Fc domain with an engineered cysteine ​​residue or analog thereof located on a solvent-exposed surface. Preferably, the engineered cysteine ​​residue or analog does not interfere with the effector function conferred by the Fc. More preferably, the modification does not interfere with the ability of the Fc to bind to an Fc receptor (e.g., FcγRI, FcγRII, or FcγRIII) or a complement protein (e.g., C1q), or to trigger immune effector function (e.g., antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)). In preferred embodiments, the hybrid nuclease molecules of the present invention comprise an Fc domain containing at least one engineered free cysteine ​​residue or analog thereof that is substantially free of disulfide bonds with a second cysteine ​​residue. Any of the above engineered cysteine ​​residues or their analogs can then be conjugated to a functional domain (e.g., conjugated with a thiol-reactive heterobifunctional linker) using art-recognized techniques.

[0133] In one embodiment, a hybrid nuclease molecule of the invention may comprise a genetically fused Fc domain having two or more of its component Fc domains independently selected from the Fc domains described herein. In one embodiment, the Fc domains are the same. In another embodiment, at least two of the Fc domains are different. For example, the Fc domains of a hybrid nuclease molecule of the invention may comprise 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 a hybrid nuclease molecule of the invention may differ at one or more amino acid positions. For example, at least two of the Fc domains may differ in sequence at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions, or about 50 positions).

[0134] Linker Domain In some embodiments, the hybrid nuclease molecule comprises a linker domain. In some embodiments, the hybrid nuclease molecule comprises 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 an amino acid sequence (which may or may not be naturally occurring) linked to a sequence (which may or may not be naturally occurring) that is not naturally linked in nature (e.g., an Fc domain sequence) in a linear sequence of amino acids. For example, the polypeptide linker may comprise a non-naturally occurring polypeptide that is a modified form of a natural polypeptide (e.g., containing mutations such as additions, substitutions, or deletions) or a non-naturally occurring polypeptide that comprises a first amino acid sequence (which may or may not be naturally occurring). The polypeptide linkers of the present invention can be used, for example, to ensure juxtaposition of Fc domains to ensure proper folding and formation of a functional Fc domain. Preferably, polypeptide linkers compatible with the present invention will be relatively non-immunogenic and will not interfere with the non-covalent association between monomeric subunits of the binding protein.

[0136] In certain embodiments, the hybrid nuclease molecules of the present invention use a polypeptide linker to join any two or more domains in-frame into a single polypeptide chain. In one embodiment, the two or more domains can be independently selected from any of the Fc domains or nuclease domains discussed herein. For example, in certain embodiments, a polypeptide linker can be used to fuse identical Fc domains, thereby forming a homomeric Fc region. In other embodiments, a polypeptide linker can be used to fuse different Fc domains (e.g., a wild-type Fc domain and an Fc domain variant), thereby forming a heteromeric Fc region. In other embodiments, the polypeptide linkers of the present invention can be used to genetically fuse the C-terminus of a first Fc domain (e.g., a hinge domain or portion thereof, a CH2 domain or portion thereof, a complete CH3 domain or portion thereof, an FcRn-binding portion, an FcγR-binding portion, a complement-binding portion, or portion thereof) to the N-terminus of a second Fc domain (e.g., a complete Fc domain).

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

[0138] In exemplary embodiments, the polypeptide linker can comprise at least a portion of an immunoglobulin hinge region. In one embodiment, the polypeptide linker comprises an upper hinge domain (e.g., the upper hinge domain of IgG1, IgG2, IgG3, or IgG4). In another embodiment, the polypeptide linker comprises a middle hinge domain (e.g., the middle hinge domain of IgG1, IgG2, IgG3, or IgG4). In another embodiment, the polypeptide linker comprises a lower hinge domain (e.g., the lower hinge domain of IgG1, IgG2, IgG3, or IgG4).

[0139] In other embodiments, the polypeptide linker can 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 an IgG1 hinge region and at least a portion of an IgG2 hinge region. In one embodiment, the polypeptide linker comprises a chimeric hinge comprising at least a portion of an IgG1 hinge region and at least a portion of an IgG3 hinge region. In another embodiment, the polypeptide linker comprises a chimeric hinge comprising at least a portion of an IgG1 hinge region and at least a portion of an IgG4 hinge region. In one embodiment, the polypeptide linker comprises a chimeric hinge comprising at least a portion of an IgG2 hinge region and at least a portion of an IgG3 hinge region. In one embodiment, the polypeptide linker comprises a chimeric hinge comprising at least a portion of an IgG2 hinge region and at least a portion of an IgG4 hinge region. In one embodiment, the polypeptide linker comprises 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 comprise an IgG1 upper and middle hinge and a single IgG3 middle hinge repeat motif. In another embodiment, the polypeptide linker may comprise an IgG4 upper hinge, an IgG1 middle hinge, and an IgG2 lower hinge.

[0140] In another embodiment, the polypeptide linker comprises or consists of a gly-ser linker. As used herein, the term "gly-ser linker" refers to a peptide consisting of glycine and serine residues. An exemplary gly / ser linker comprises an amino acid sequence of the 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 some embodiments, a gly-ser linker may be inserted between two other polypeptide linker sequences (e.g., any of the polypeptide linker sequences described herein). In other embodiments, a gly-ser linker is attached to one or both ends of another 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 tandem into the polypeptide linker. In one embodiment, a polypeptide linker of the invention comprises at least a portion of an upper hinge region (e.g., from an IgG1, IgG2, IgG3, or IgG4 molecule), at least a portion of a middle hinge region (e.g., 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, a polypeptide linker of the invention comprises a non-naturally occurring immunoglobulin hinge region domain, e.g., a hinge region domain that is not naturally found in a polypeptide that comprises a hinge region domain, and / or a hinge region domain that has been modified to differ in amino acid sequence from a naturally occurring immunoglobulin hinge region domain. In one embodiment, a hinge region domain can be mutated to generate a polypeptide linker of the invention. In one embodiment, a polypeptide linker of the invention comprises a hinge domain that does not contain the naturally occurring number of cysteines, i.e., the polypeptide linker contains either fewer or more cysteines than a naturally occurring hinge molecule.

[0142] In another embodiment, the polypeptide linker of the present invention comprises a biologically relevant peptide sequence or a portion of that sequence. For example, the biologically relevant peptide sequence may include, but is not limited to, a sequence derived from an anti-rejection peptide or an anti-inflammatory peptide. The anti-rejection peptide or anti-inflammatory peptide may be selected from the group consisting of a cytokine inhibitory peptide, a cell adhesion inhibitory peptide, a thrombin inhibitory peptide, and a platelet inhibitory peptide. In a preferred embodiment, the polypeptide linker comprises a peptide sequence selected from the group consisting of an IL-1 inhibitory or antagonist peptide sequence, an erythropoietin (EPO) mimetic peptide sequence, a thrombopoietin (TPO) mimetic peptide sequence, a G-CSF mimetic peptide sequence, a TNF antagonist peptide sequence, an integrin-binding peptide sequence, a selectin antagonist peptide sequence, an anti-pathogen peptide sequence, a vasoactive intestinal peptide (VIP) mimetic peptide sequence, a calmodulin antagonist peptide sequence, a mast cell antagonist, an SH3 antagonist peptide sequence, a urokinase receptor (UKR) antagonist peptide sequence, a somatostatin or cortistatin mimetic peptide sequence, and a macrophage and / or T cell inhibitory peptide sequence. 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] Polypeptide linkers of the invention are at least one amino acid long and can vary in length. In one embodiment, polypeptide linkers of the invention are about 1 to about 50 amino acids long. As used in this context, the term "about" refers to + / - 2 amino acid residues. Linker lengths must be positive integers, so a length of about 1 to about 50 amino acids means a length of 1 to 48-52 amino acids. In another embodiment, polypeptide linkers of the invention are about 10-20 amino acids long. In another embodiment, polypeptide linkers of the invention are about 15 to about 50 amino acids long.

[0145] In another embodiment, a polypeptide linker of the present invention is about 20 to about 45 amino acids in length. In another embodiment, a polypeptide linker of the present invention is about 15 to about 25 amino acids in length. In another embodiment, a polypeptide linker of the present invention is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids in length.

[0146] The polypeptide linker can be introduced into the polypeptide sequence using techniques known in the art. The modification can be confirmed by DNA sequence analysis. The plasmid DNA can be used to transform host cells for stable production of the polypeptide to be produced.

[0147] Nuclease domain In some aspects, the hybrid nuclease molecule comprises a nuclease domain. Thus, the hybrid nuclease molecule of the present invention typically comprises at least one nuclease domain and at least one linked Fc domain. In some aspects, the hybrid nuclease molecule comprises multiple nuclease domains.

[0148] In some embodiments, the nuclease domain is substantially all, or at least an enzymatically active fragment, of a DNase. In some embodiments, the DNase is a type I secretory DNase, preferably a human DNase, such as DNase 1. Exemplary DNase 1 domains are set forth in SEQ ID NOS: 48-53 and 102. An 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 (DNaseL) 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). An exemplary human TREX1 is described in UniProtKB entry Q9NSU2 (SEQ ID NOS: 104). Preferably, the human TREX1 is a C-terminally truncated human TREX1 lacking the intracellular nuclear targeting sequence, for example, human TREX1 lacking the 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 an enzymatically active fragment, of an RNase. In some embodiments, the RNase is an extracellular or secreted RNase of the RNase A superfamily, such as RNase A, preferably human pancreatic RNase. Exemplary human RNases are described in UniProtKB entry P07998 (SEQ ID NOs: 58 and 101).

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

[0151] In some embodiments, the hybrid nuclease molecules of the present invention comprise two or more nuclease domains and at least one Fc domain. For example, a nuclease domain may be operably linked to both the N-terminus and C-terminus of an Fc domain. In other exemplary embodiments, a nuclease domain may be operably linked to both the N-terminus and C-terminus of multiple Fc domains (e.g., two, three, four, five, or more Fc domains) that are linked together in tandem to form a tandem array of Fc domains.

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

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

[0154] Preferred hybrid nuclease molecules of the present invention comprise at least one nuclease domain (eg, RNase or DNase), at least one linker domain, and at least one Fc domain.

[0155] In some embodiments, the hybrid nuclease molecules of the invention comprise at least one nuclease domain specific for a target molecule that mediates a biological effect. In other embodiments, binding of the hybrid nuclease molecules of the invention to a target molecule (e.g., DNA or RNA) results in the reduction or elimination of the target molecule, e.g., from a cell, tissue, or from the circulation.

[0156] In some embodiments, a hybrid nuclease molecule of the invention may comprise two or more nuclease domains. In one embodiment, the nuclease domains are the same, e.g., RNase and RNase, or TREX1 and TREX1. In another embodiment, the nuclease domains are different, e.g., DNase and RNase.

[0157] In other embodiments, the hybrid nuclease molecules of the invention may be assembled together or with other polypeptides to form binding proteins having two or more polypeptides ("multimers"), where at least one polypeptide of the multimer is a hybrid nuclease molecule of the invention. Exemplary multimeric forms include modified binding proteins that are dimers, trimers, tetramers, and hexamers, and the like. 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., heteromers).

[0158] Methods 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 so, 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 the peptides can be excised from DNA using appropriate restriction enzymes. Alternatively, the DNA molecules can be synthesized using chemical synthesis techniques, such as the phosphoramidate method. A combination of these techniques can also be used.

[0159] The present invention also includes vectors capable of expressing these peptides in a suitable host. The vectors contain DNA molecules encoding these peptides operably linked to expression control sequences. Methods for affecting this functional linkage either before or after inserting the DNA molecule into the vector are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal nuclease domains, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in regulating transcription or translation.

[0160] The resulting vector carrying the DNA molecule thereon is used to transform an appropriate host, which can be accomplished using methods well known in the art.

[0161] Any of a large number of available and well-known host cells can be used in the practice of the present invention. The selection of a particular host depends on several factors recognized 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, knowing that not all hosts are equally effective for expressing a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (e.g., E. coli species), yeast (e.g., Saccharomyces species) and other fungi, insect, plant, mammalian (including human) cells in culture, or other hosts known in the art.

[0162] The transformed host cells are then 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. The peptide is then purified from the culture by methods well known in the art.

[0163] Compounds can also be produced by synthetic methods. For example, solid phase synthesis techniques can be used. Suitable techniques are well known in the art, including those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (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 preferred technique for making individual peptides because it is the most cost-effective method for making small peptides. Compounds containing derivatized peptides or containing non-peptide groups can be synthesized by well-known organic chemistry techniques.

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

[0165] Pharmaceutical Compositions and Therapeutic Methods of Use 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 simultaneously 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 agents / compounds. In some embodiments, the hybrid nuclease molecule and the other agent are administered simultaneously. In some embodiments, the hybrid nuclease molecule and the other agent are not administered simultaneously, but the hybrid nuclease molecule is administered before or after the agent is administered. In some embodiments, a subject receives both the hybrid nuclease molecule and the other agent during the same prophylactic, disorder-onset, and / or treatment period.

[0166] The pharmaceutical composition of the present invention can be administered in the form of combination therapy, i.e., in combination with other drugs.In some embodiments, the combination therapy includes a combination of a nuclease molecule and at least one other drug.The drug includes, but is not limited to, a chemical composition prepared by in vitro synthesis, an antibody, an antigen-binding region, and combinations and conjugates thereof.In some embodiments, the drug can act as an agonist, antagonist, allosteric modulator, or toxin.

[0167] In certain embodiments, the present invention provides pharmaceutical compositions comprising a hybrid nuclease molecule together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

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

[0169] In some embodiments, acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations used. In some embodiments, the formulation materials are for subcutaneous and / or intravenous administration. In some embodiments, pharmaceutical compositions can include formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, flavor, sterility, stability, dissolution or release rate, adsorption, or permeability of the composition. In certain embodiments, suitable formulation ingredients include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrate, phosphate, 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 immunoglobulins); colors, flavors, and diluents; emulsifiers; hydrophilic polymers (such as poly vinylpyrrolidone, etc.); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapol, etc.); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol, etc.); delivery vehicles; diluents; excipients, and / or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company (1995). In some embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and / or 10 mM NAOAC, pH 5.2, 9% sucrose.

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

[0171] In certain embodiments, the optimal pharmaceutical composition will be determined by one of skill in the art depending on, e.g., the intended route of administration, delivery mode, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the antibodies of the invention.

[0172] In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier can be water for injection, physiological saline, or artificial cerebrospinal fluid, optionally supplemented with other ingredients common in compositions for parenteral administration. In some embodiments, saline includes isotonic phosphate-buffered saline. In certain embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, a pharmaceutical composition comprises a Tris buffer having a pH of about 7.0-8.5 or an acetate buffer having a pH of about 4.0-5.5, which may further contain sorbitol or a suitable substitute. In certain embodiments, a composition comprising a hybrid nuclease molecule, with or without at least one additional therapeutic agent, can be prepared for storage as a lyophilized cake or in the form of an aqueous solution by mixing the selected composition having the desired purity with optional formulating agents (Remington's Pharmaceutical Sciences, supra). Additionally, in certain embodiments, compositions comprising hybrid nuclease molecules, with or without at least one additional therapeutic agent, can be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0173] In some embodiments, pharmaceutical compositions can be selected for parenteral delivery. In some embodiments, compositions can be selected for inhalation or delivery through the digestive tract, such as oral administration. The preparation of such pharmaceutically acceptable compositions is within the capabilities of one skilled in the art.

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

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

[0176] In some embodiments, the pharmaceutical composition can be formulated for inhalation. In some embodiments, the hybrid nuclease molecule can be formulated as a dry powder for inhalation, with or without at least one additional therapeutic agent. In some embodiments, an inhalable solution comprising the hybrid nuclease molecule, with or without at least one additional therapeutic agent, can be formulated with a propellant for aerosol delivery. In some embodiments, the solution can also be nebulized. Pulmonary administration is further described in PCT Application No. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0177] In some embodiments, it is contemplated that the formulation may be administered orally. In some embodiments, the hybrid nuclease molecule administered in this manner may be formulated with or without at least one additional therapeutic agent, with or without carriers typically used in compounding solid dosage forms such as tablets and capsules. In some embodiments, the capsule may be designed to release the active portion of the formulation at a point 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 absorption of the hybrid nuclease molecule and / or any additional therapeutic agent. In some embodiments, diluents, flavoring agents, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

[0178] In some embodiments, pharmaceutical compositions can comprise an effective amount of hybrid nuclease molecule, with or without at least one additional therapeutic agent, in a mixture with non-toxic excipients suitable for tablet manufacture.In some embodiments, tablets can be prepared in unit dose form by dissolving in sterile water or another suitable medium to form a solution.In some embodiments, 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 will be apparent to those skilled in the art, including formulations comprising hybrid nuclease molecules, with or without at least one additional therapeutic agent, in formulations for sustained or controlled delivery. In certain embodiments, techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. See, for example, PCT Application No. PCT / US93 / 00829, which describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. In certain embodiments, sustained release formulations may include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained-release matrices can include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., supra), or poly-D(-)-3-hydroxybutyric acid (EP 133,988). In some embodiments, sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

[0180] Pharmaceutical compositions for in vivo administration are typically sterile.In some embodiments, this can be achieved by filtration through sterile filtration membranes.In some embodiments, when the composition is freeze-dried, sterilization using this method can be carried out either before or after freeze-drying and reconstitution.In some embodiments, the composition for parenteral administration can be stored in freeze-dried form or as a solution.In some embodiments, parenteral compositions are generally placed into a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

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

[0182] In some embodiments, kits for producing single-dose administration units are provided. In some embodiments, the kits can include both a first container with a dried protein and a second container with an aqueous formulation. In some embodiments, kits containing single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes) are also included.

[0183] In some embodiments, the effective amount of a pharmaceutical composition comprising a hybrid nuclease molecule, with or without at least one additional therapeutic agent, used therapeutically will depend, for example, on the context and purpose of the treatment. Those skilled in the art will understand that appropriate dosage levels for treatment will vary, in part, depending on the indication for use of the hybrid nuclease molecule, with or without at least one additional therapeutic agent, according to certain embodiments, and therefore on the patient's size (weight, body surface area, or organ size) and / or condition (age and general health) when the molecule is delivered. In some embodiments, a clinician can titrate the dosage and modify the route of administration to achieve optimal therapeutic effect. In some embodiments, typical dosages can range from about 0.1 μg / kg up to about 100 mg / kg or more, depending on the factors discussed above. In certain embodiments, dosages can range from 0.1 μg / kg up to about 100 mg / kg; or from 1 μg / kg up to about 100 mg / kg; or from 5 μg / kg up to about 100 mg / kg.

[0184] In some embodiments, the frequency of dosing will take into account the pharmacokinetic parameters of the hybrid nuclease molecule and / or any additional therapeutic agent in the formulation used. In some embodiments, the clinician administers the composition until a dosage is reached that achieves the desired effect. In some embodiments, the composition can thus 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 continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routine and within the scope of routine practice by those skilled in the art. In some embodiments, the appropriate dosage can be determined by using appropriate dose-response data.

[0185] In some embodiments, the route of administration of the pharmaceutical composition is according to known methods, for example, orally, through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal or intralesional routes; by sustained release system, or by implantation device, etc. In some embodiments, the composition may be administered by bolus injection, or continuously by infusion, or by implantation device.

[0186] In some embodiments, the compositions can be administered locally via implantation of a membrane, sponge, or another suitable material onto which the desired molecule is adsorbed or encapsulated. In some embodiments, when an implantation device is used, the device can be implanted in any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, sustained release bolus, or continuous administration.

[0187] In certain embodiments, it may be desirable to use a pharmaceutical composition comprising a hybrid nuclease molecule, with or without at least one additional therapeutic agent, in an ex vivo manner, in which cells, tissues, and / or organs removed from a patient are exposed to a pharmaceutical composition comprising a hybrid nuclease molecule, with or without at least one additional therapeutic agent, and then the cells, tissues, and / or organs are transplanted back into the patient.

[0188] In certain embodiments, the hybrid nuclease molecule and / or any additional therapeutic agent can be delivered by implanting certain cells that have been genetically engineered to express and secrete these polypeptides using methods such as those described herein. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In certain embodiments, the cells can be encapsulated to prevent infiltration of surrounding tissues, in order to reduce the likelihood of an immunological response. In certain embodiments, the encapsulating material is typically a biocompatible, semipermeable polymeric enclosure or membrane that allows release of the protein product but prevents destruction of the cells by the patient's immune system or other harmful factors from the surrounding tissues.

[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 foreign and self-antigens. In yet another embodiment, the polypeptides of the present invention are useful 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, early-onset diabetes mellitus, Goodpasture's syndrome, pemphigus vulgaris, , pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, cryptogenic liver cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid lupus erythematosus, systemic lupus erythematosus, or connective tissue disease.

[0190] kit The kits may include the hybrid nuclease molecules disclosed herein and instructions for use. The kits may include, in suitable containers, 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 into which the hybrid nuclease molecule may be placed and optionally suitably divided. If additional components are provided, the kit may include additional containers into which the components may be placed. The kit may also include means for tightly containing the containers of the hybrid nuclease molecule and any other reagents for sale. Such containers may include injection- or blow-molded plastic containers that hold the desired vials therein. The containers and / or kits may include labels with instructions and / or precautions for use. [Example]

[0192] Below are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0193] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art. Such techniques are explained fully in the literature, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundherg, Advanced Organic Chemistry 3 rd Ed (Plenum Press) Vols. A and B (1992).

[0194] Example 1. General approach to generating hybrid nuclease molecules Hybrid nuclease molecules were designed as modular cassettes with compatible restriction enzyme sites for shuttling and domain swapping so that desired structures and functional activities of mono- or multi-enzyme structures could be incorporated. Schematic structures of various embodiments of hybrid nuclease molecules are depicted 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 × 10e6 cells) using the QIAgen RNAeasy kit (Valencia, CA) and the QIAshredder kit (Qiagen, Valencia, CA) to homogenize the 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 x 10e6 splenocytes using the QIAgen RNAeasy kit (Valencia, CA). Cells were pelleted from the medium by centrifugation, and 5 x 10e6 cells were used for RNA preparation. RNA was isolated from the cells using the QIAGEN RNAeasy kit (Valencia, CA) for total RNA isolation and a QIAGEN QIAshredder according to the manufacturer's instructions. One to two micrograms (1-2 μg) of total RNA was used as a template for preparing cDNA by reverse transcription. The RNA, 300 ng of random primers, 500 ng of Oligo dT(12-18), and 1 μl of 25 mM dNTPs were combined and denatured at 80°C for 5 minutes before adding the enzyme. Superscript III reverse transcriptase (Invitrogen, Life Technologies) was added to the RNA + primer mixture in a total volume of 25 μl in the presence of 5× second-strand buffer and 0.1 M DTT provided with the enzyme. The reverse transcription reaction proceeded for 1 hour at 50°C.

[0197] 10–100 ng of cDNA was used in PCR amplification reactions using primers specific for the nuclease gene of interest (e.g., 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 Qiagen QIAquick columns to remove nucleotides, primers, and unwanted amplification products. Purified fragments were cloned into the pCR2.1 TOPO cloning vector (Invitrogen, Carlsbad, CA) and transformed into TOP10 competent bacteria. Isolated colonies were picked, placed in Luria Broth containing 50 μg / ml carbenicillin, and grown overnight for plasmid isolation. TOPO clones were screened for inserts of the correct size by digestion with EcoRI (NEB, Ipswich, MA) restriction enzyme and agarose gel electrophoresis of the digested fragments. DNA sequence analysis of positive clones was performed using ABI Ready Reaction Mix v3.1 and analyzed using an ABI 3730 XL DNA sequencer. Once correct clones were obtained, PCR reactions were performed to design additional sequence modifications and generate the desired alleles or expression cassettes. Truncated products and alleles were generated by PCR mutagenesis using overlapping primers to introduce mutations at specific positions in the gene. Linkers were synthesized by overlapping PCR using internal overlapping primers and sequential PCR to attach additional sequences to each end. Hybrid nuclease molecules were assembled as a concatenation of several interchangeable cassettes.The molecules of preferred embodiments contain a predefined leader peptide, a nuclease cassette, an optional cassette encoding one of several different polypeptide linkers, an Ig Fc domain cassette with either a stop codon or a linker at the carboxyl terminus of the CH3 domain, and, in the case of resolvICase-type molecules, 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 transferred into the appropriate mammalian expression plasmid, pDG, for transient expression in COS7 or other cells and stable expression in CHO DG44 cells using selection for DHFR with methotrexate.

[0198] Transient expression of hybrid nuclease molecules COS-7 cells were transiently transfected with the expression vector pDG containing a hybrid nuclease molecule gene insert. The day before transfection, cells were seeded at 4 x 10e5 cells per 60 mm dish in 4 ml DMEM (ThermoFisher / Mediatech cell gro) + 10% FBS tissue culture medium. DMEM basal medium was supplemented with 4.5 g / L glucose, sodium pyruvate, 4 mM L-glutamine, and non-essential amino acids. Fetal bovine serum (Hyclone, Logan, UT ThermoFisher Scientific) was added to the medium at a final volume of 10%. Cells were incubated overnight at 37°C and 5% CO2 until they reached approximately 40–80% confluency on the day of transfection. Plasmid DNA was prepared using a 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, using 2.5 μg of plasmid DNA per 60 mm dish and 15 μl of Polyfect reagent in 150 μl of serum-free DMEM transfection cocktail. After complex formation, the reaction mixture was diluted in 1 ml of cell growth medium containing serum and all additives and added dropwise to plates containing 3 ml of fresh DMEM complete medium. Transient transfections were incubated for 48–72 hours, after which culture supernatants were harvested for further analysis.

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

[0200] Supernatants were collected from CHO cells expressing the hybrid nuclease molecule, 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 wash buffer (90 mM Tris base, 150 mM NaCl, 0.05% sodium azide, pH 8.7), and bound protein was eluted with 0.1 M citrate buffer, pH 3.0. Fractions were collected, and protein concentrations were determined at 280 nM using a Nanodrop (Wilmington, DE) microsample spectrophotometer. A blank was also run using 0.1 M citrate buffer, pH 3.0. Fractions containing hybrid nuclease molecules were pooled and buffer exchanged to reduce the risk of endotoxin contamination by successive spins in PBS using a centricon concentrator followed by filtration through a 0.2 μm filter device.

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

[0202] After isolating the full-length clones, primers were designed to create fusion genes with the Fc domain of mouse IgG2a or human IgG1 (SEQ ID NO:40). Two primers were designed for the 5' sequence to fuse at the amino terminus of the Fc tail; the first incorporated the native leader peptide from mouse (or human) RNase, while the second attached an AgeI site to the amino terminus of the RNase at the predicted signal peptide cleavage site in order to fuse the RNase to the human VKIII leader peptide, which we had previously cloned and used for other expression studies. For mouse RNase, the sequence of the first primer was: mribNL5' 30mer (RNase 5' with native leader and HindIII + Kozak) TIFF2024167368000001.tif7159

[0203] The second primer creates a gene fusion junction between the existing leader sequence and the 5' mature sequence of the RNase at or near the predicted leader peptide cleavage site. 27mer (RNase 5' mature sequence (leaderless, with AgeI site) TIFF2024167368000002.tif7157

[0204] The sequence of the 3' primer for fusion to mouse IgG2a at the carboxy terminus of RNase and the amino terminus of the Fc tail is as follows: mrib3NH2 28mer (RNase 3' end with XhoI site for fusion with mIgG2a) TIFF2024167368000003.tif7158

[0205] Two additional oligos were designed to create an -Ig-RNase fusion gene in which the -Ig tail was amino-terminal to the RNase enzyme domain. mrib5X A 36-mer RNase 5' end with a linker aa and an XbaI site for fusion to the carboxy terminus of the Fc domain TIFF2024167368000004.tif14136mrib3X A 31-mer RNase 3' end with two stop codons and an XbaI site for fusion with the carboxy terminus of an Fc domain TIFF2024167368000005.tif7169

[0206] Example 3: Isolation of human and mouse Fc domains and introduction of mutations into the coding sequences For isolation of mouse and human Fc domains (SEQ ID NO:40), RNA was obtained from mouse or human tissues as follows: Single-cell suspensions were made in RPMI medium from mouse spleens. Alternatively, human PBMCs were isolated from fresh whole blood using Lymphocyte Separation Media (LSM) Organon Teknika (Durham, NC). Buffy coats were harvested according to the manufacturer's instructions, and cells were washed three times in PBS before use. Cells were pelleted from the medium by centrifugation and 2 × 10 7RNA was isolated from cells using a QIAGEN RNAeasy kit (Valencia, Calif.) total RNA isolation kit and QIAGEN QIAshredder columns according to the manufacturer's instructions. One microgram (4 μg) of total RNA was used as a template for preparing cDNA 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 combined and denatured at 80°C for 5 minutes before adding the enzyme. Superscript III reverse transcriptase (Invitrogen, Life Technologies) was added to the RNA + primer mixture in a total volume of 25 μl in the presence of the second-strand buffer provided with the enzyme and 0.1 M DTT. The reverse transcription reaction proceeded for 1 hour at 50°C. The cDNA was purified using a QIAquick (QIAGEN) PCR purification column and eluted in 40 μl of EB buffer before use in PCR reactions.

[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 the initial amplification of the wild-type sequence, with the desired mutational changes incorporated into the hinge domain: mahIgG1CH2M:47mer TIFF2024167368000006.tif7128hIgG1-5scc:49mer TIFF2024167368000007.tif7128mahIgG1S:51mer TIFF2024167368000008.tif7129muIgG2aCH2:58mer TIFF2024167368000009.tif7140mIgG2a-5scc:47mer TIFF2024167368000010.tif7128mIgG2a3S:48mer TIFF2024167368000011.tif6128

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

[0209] DNA from the correct clone was used as a template in overlap extension PCR to introduce mutations at the desired positions in the coding sequence of mouse IgG2a or human IgG1. PCR reactions were set up using a full-length wild-type clone (1 microliter) as template, 50 pmol of 5' and 3' primers to PCR each portion of the Fc domain up to and including the desired mutation site, and PCR hi fidelity Supermix (Invitrogen, Carlsbad, CA) in a 50 microliter reaction volume, with short amplification cycles. As an example of overlap 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 the full-length wild-type clone as template, where the 5' primer was hIgG1-5scc: TIFF2024167368000012.tif6128, while the 3' primer was P331AS: The 3' partial fragment was amplified using the full-length wild-type clone as template, where the 5' primer was P331S: TIFF2024167368000014.tif6128, while the 3' primer was mahIgG1S: The file was TIFF2024167368000015.tif7130.

[0211] The partial fragments were amplified and isolated by agarose gel electrophoresis. They were then purified on a QIAquick gel purification column and eluted in 30 microliters of EB buffer according to the manufacturer's instructions. Two PCR rounds were then performed, using the two partial fragments as overlapping templates in new reactions. The cycler was paused, and 5' (hIgG1-5scc, see above) and 3' (mahIgG1S, see above) flanking primers were added to the reaction (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. Fragments from clones with the correct sequence were then subcloned into expression vectors to generate the various hybrid nuclease molecules described herein.

[0212] Example 4: Quantification of RSLV-124 protein and RNase enzyme activity in mouse serum In vivo stability analysis of RSLV-124 construct (SEQ ID NO:106) in mice Four mice (C220, C221, C222, and C223) were intravenously injected with a single injection of RSLV-124 at time zero. Blood samples were collected at various time points postinjection and analyzed for the presence of RSLV-124 protein (human wild-type RNase (SEQ ID NO:106) linked to a wild-type human IgG1 Fc domain) and RNase enzymatic activity. To detect RSLV-124 compounds in mouse serum, an ELISA was developed that captures human Fc from mouse serum followed by detection of human RNase. ELISA was performed on blood samples from four mice, and RSLV-124 protein was detected at 38 μg / ml to 55 μg / ml 5 minutes after a single intravenous injection of 150 μg (Figure 2). One day after injection, RSLV-124 concentrations rapidly decreased to 8 μg / ml to 12 μg / ml. Drug blood concentrations remained relatively stable over the 7-day analysis period, with drug blood levels remaining approximately 5 μg / ml. The same blood samples used to measure RSLV-124 protein by ELISA were used to quantify the drug's RNase enzyme activity. The RNaseAlert QC system (catalog no. AM1966) from Ambion was used with minor modifications to measure the enzymatic reaction rate of RSLV-124 protein in mouse blood samples. Drug compounds were captured from mouse serum onto an RNaseAlert assay plate using a human anti-Fc monoclonal antibody and quantified by fluorescence measurement according to the Ambion kit instructions. Analysis of the relative fluorescence units (RFU) of RSLV-124 molecules showed 80,000–140,000 RFU at 5 min after injection (Figure 3). RFU rapidly decreased in parallel with protein concentration and then remained relatively stable between 18,000–40,000 RFU until day 7. The RNaseAlert QC system was used to generate a standard curve with known amounts of protein, from which the RFU of RSLV-124 in the blood samples was used to extrapolate the RSLV-124 protein concentration. This analysis revealed that the protein concentrations present in mouse blood over the 7-day experiment, calculated using the RNase enzyme activity assay, were very similar to those measured using ELISA (Figure 4).From these experiments, it was concluded that the RSLV-124 compound was stable in vivo in the mouse circulation for 7 days and retained its enzymatic activity. This suggests that the compound is not subject to degradation in vivo in mice, as nearly 100% enzymatic activity was maintained in the mouse circulation for 7 days. Because Fc fusion proteins are often susceptible to degradation in the circulation, this finding further supports the use of RNase-Fc fusion proteins as beneficial drugs.

[0213] Example 5: Phenotype of TLR7.1 x RNaseA double transgenic mice We generated mice overexpressing RNase A (RNase Tg). This nuclease is expressed at high levels in RNase Tg mice. We developed a single radial diffusion (SRED) assay (Figure 5) and a much more quantitative ELISA (see Figure 6) to quantify serum RNase levels. Both assays demonstrate a significant increase in RNase activity in RNase Tg mice. Quantification of RNase levels in Figure 6 showed an approximately 10-fold increase in RNase activity in RNase Tg mice compared with wild-type B6 mice. We crossed RNase A Tg mice with TLR7.1 Tg mice to generate double Tg mice (DTg). TLR7.1 mice have 8–16 copies of TLR7 and develop a highly aggressive, rapidly progressive lupus-like disease, beginning to die at 3 months of age with a median survival of 6 months. In preliminary analyses, we bled DTg and littermate controls to 3 months of age to determine whether DTg mice would show signs of improvement. As shown in Figure 5, DTg mice had very high levels of RNase in their serum (equivalent to >13 U / ml of RNase, based on our standard with a specific activity of 993 U / mg). RNase A concentrations in Tg and DTg mice were also measured by ELISA assay, as shown in Figure 6. RNase A Tg mice and TLR7.1 × RNase A Dtg mice had serum concentrations of 1–2 ng / ml of RNase A.

[0214] Detailed Methods for RNase A ELISA 1. Coat plates with anti-RNaseA Abcam Ab (ab6610): 2.5~10ug / ml O / N in 4C. 2. Wash the plate three times with 0.05% Tween / 1x PBS. 3. Block with 1% BSA in PBS for at least 1 hour. 4. Wash the plate three times with 0.05% Tween / 1×PBS. 5. Load the sample. The sample dilution is 1:50. 6. Incubate at room temperature for 2 hours. 7. Wash the plate three times with 0.05% Tween / 1×PBS. 8. Prepare a 1:4500 (2.2 μg / ml) dilution of biotinylated anti-RNase Ab. Allow to 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) 1:2500. Cover with foil and let stand at room temperature for 25-30 minutes. 11. Wash 6 times, allowing the liquid to sit in the wells for at least 30 seconds between washes. 12. Add BD OptEIA Substrate A+B in a 1:1 ratio. Allow 5-10 minutes for color to reach maximum. Ensure the standard in the top well does not exceed 1.0. Add 80ul. (Cat. Nos. 51-2606KC; Reagent A, 51-2607KC; Reagent B) 13. Add 40 ul of 1M sulfuric acid to stop the reaction.

[0215] Product / Reagent Information: RNase A Ab: ab6610 (90 mg / ml) ELISA buffer: 1% BSA in PBS ELISA washing buffer: 0.05% Tween / 1×PBS Anti-RNase A biotin-conjugated 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 curves of TLR7.1 transgenic mouse lines A highly significant difference in survival was observed between DTg and TLR7.1 littermate controls. As shown in Figure 7, at 10 months, 61% of TLR7.1 mice died, compared with 31% of DTg mice. These data indicate that RNase A overexpression exerted a strong therapeutic effect. The reasons for the early death of TLR7.1 mice are not entirely clear, but severe anemia, thrombocytopenia, and glomerulonephritis may have played a role. To determine whether red blood cell and platelet counts were favorably affected by RNase A expression in DTg mice, we performed complete blood counts (CBCs) and found no differences between TLR7.1 and DTg mice. In contrast, DTg mice showed significant improvement in renal histopathology. We observed decreased IgG and C3 deposition in DTg mice. PAS staining, reflecting inflammation in the mesangium, was also reduced in DTg mice compared with TLR7.1 littermate controls. In this study, we compared renal macrophage infiltration using anti-MAC-2 (galectin 3) antibodies (Lyoda et al. Nephrol Dial Transpiat 22: 3451, 2007) and found that MAC-2-positive cells were significantly fewer in the glomeruli of DTg mice. Calculated using 20 glomeruli per mouse with five mice per group, the mean ± SE for isolated Tg and DTg mice was 3.8 ± 1.1 and 1.4 ± 0.2, respectively, p = .05. In addition, we quantified glomerular tuft size and observed a significant decrease in glomerular tuft size in DTg mice (179 ± 41 and 128 ± 16.8 μm2 for isolated Tg and DTg mice, respectively, p = .037). In summary, TLR7.1 × RNaseA DTg mice survived longer than their single Tg TLR7.1 littermates and had less inflammation and injury in their kidneys. This finding indicates that clearing RNA immune complexes in this mouse model significantly improved overall mortality and reduced the kidney damage and overall inflammation associated with this lupus-like condition.

[0217] Example 7. Analysis of IRGs in the spleens of TLR Tg mice Analysis of interferon-responsive genes (IRGs) in the spleens of TLR7.1 Tg mice and TLR7.1 × RNaseA DTg mice showed that the expression of the IRF7 gene (interferon regulatory factor 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 with Tg mice, but the difference was not 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). After DNase treatment using Turbo DNA-free (Applied Biosystems, Foster City, CA, USA), randomly primed first-strand cDNA was generated using the RNA-to-cDNA kit (Applied Biosystems). The 260 / 280 values ​​for the isolated RNA were measured using a NanoDrop (Thermo Scientific, Waltham, MA, USA) and ranged from 1.7 to 2.0. cDNA was diluted to the equivalent of 1 ng / ul total RNA, with 8 ul per reaction. Primers for the reference gene (18s) and gene of interest (GOI) were synthesized (IDT, Coralville, Iowa, USA) and diluted to the appropriate concentration for qPCR using molecular-grade water. BLAST results for the primers indicated specific sequence homology to the reference gene or GOI only. Duplicate reactions (20 μl) were run on an ABI Fast7500 system using a 1:1 mixture of template and primers to SensiMix SYBR low-ROX master mix (Bioline, London, UK). Age-matched wild-type B6 mice were used as a baseline for determining fold changes for each GOI.-ddCT Relative quantification was calculated using the qRT-PCR method. Dissociation curves for the reactions showed a single melting peak for each gene. Standard curves showed comparable amplification efficiency for each gene, and template concentrations were within the linear dynamic range for each primer set.

[0218] Example 8. Construction and expression of DNase1-Ig single and dual enzyme hybrid nuclease molecules Naturally occurring alleles of human DNase 1 or DNase 1-like molecules have been reported. It has previously been reported that the A114F mutation is present in naturally occurring variants of human DNase 1-like enzymes and that it confers actin resistance to enzymes containing this sequence change. 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 or all populations and 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). Allelic variants at this position resulted in DNase 1 isoforms that possessed higher activity compared to the wild-type. Another naturally occurring polymorphic mutation (R21S) has also been reported to confer higher activity (Yasuda, supra).

[0220] It has been reported that SLE patients have significantly lower levels of DNase1 activity (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, naturally occurring enzyme variants may be less immunogenic when administered to patients due to the presence of these isoforms in the human population. We reasoned that combining the actin-resistant properties of an allele similar to A114F with the increased enzyme activity of an allele such as G105R would create a novel allelic variant of human DNase 1 that may exhibit improved clinical activity in vitro and in vivo. To our knowledge, our report is the first to describe this new mutant form of DNase 1 created by the combination of two naturally occurring variants, G105R and A114F.

[0222] Human DNase 1 was isolated from human pancreatic RNA (Ambion) by random-primed cDNA reverse transcription and PCR using the following primer set, as previously described: 5'hDNase1-age: TIFF2024167368000016.tif71475'hDNase1-bx: TIFF2024167368000017.tif7156

[0223] Alternatively, the 3'DNase cassette was amplified by PCR using the following primer pair: 3'hDNase1-RV: TIFF2024167368000018.tif71473'hDNase1-termination: TIFF2024167368000019.tif7165

[0224] PCR reactions were 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, with an amplification profile of 94°C for 30 seconds; 55°C for 30 seconds; and 68°C for 90 seconds (35 cycles).

[0225] The wild-type gene was amplified by PCR, the fragment was subjected to gel electrophoresis, and the 850-bp fragment was purified by QIAquick column purification. As described for other constructs, the fragment was cloned into pCR2.1 and transformed by TOPO cloning according to the manufacturer's instructions. After sequence verification, PCR primers were used to generate subfragments containing native alleles of DNase 1, which have been reported to improve specific activity and resistance to the inhibitory activity of actin. These subfragments contained overlapping sequences, allowing the amplification of complete DNase 1 subclones containing the desired allelic mutations. COS7 cells were transiently transfected in 60-mm dishes 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 a Nanodrop™, and an aliquot equivalent to 2.5 μg of plasmid DNA was used for each transfection reaction. Each DNaseIg or RNase-Ig-DNase expression cassette was inserted into the mammalian expression vector pDG, a derivative of pcDNA3.1. Transfected cells were incubated at 37°C and 5% CO2 for 72 hours, after which the culture supernatant was harvested for further analysis. The culture supernatant was harvested, the remaining cells were spun down from the solution, and the liquid was transferred to a new tube.

[0226] COS-7 cells were transiently transfected with plasmids containing wild-type or naturally occurring mutant alleles of human DNase 1 (G105R and / or A114F) fused to the wild-type human IgG1 Fc domain. The hinge-CH2-CH3 cassette contains a single C→S mutation in the hinge region that eliminates the first cysteine ​​in this domain, rendering it unpairable due to the absence of its pairing partner in the antibody light chain. In addition, more complex multinuclease fusion proteins were also expressed by transient transfection of COS cells.

[0227] Example 9: Isolation of human Ig tails, introduction of mutations into the coding sequence, and construction of mutant nuclease molecules For the isolation of mutant human Ig Fc domains, RNA was obtained from human PBMCs isolated from fresh whole blood using Lymphocyte Separation Media (LSM) from Organon Teknika (Durham, NC). Buffy coats were harvested according to the manufacturer's instructions, and cells were washed three times in PBS before use. Cells were pelleted from the medium by centrifugation and collected at 2 × 10 7RNA was isolated from cells using a QIAGEN RNAeasy kit (Valencia, Calif.) total RNA isolation kit and QIAGEN QIAshredder columns according to the manufacturer's instructions. One microgram (4 μg) of total RNA was used as a template for preparing cDNA 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 before adding the enzyme. Superscript III reverse transcriptase (Invitrogen, Life Technologies) was added to the RNA plus primer mixture in the presence of the second-strand buffer provided with the enzyme and 0.1 M DTT, for a total volume of 25 μl. The reverse transcription reaction proceeded at 50°C for 1 hour. The 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 in 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 PCR-directed mutagenesis using appropriate PCR primers containing the desired mutations and the wild-type cassette as a template. PCR reactions were performed using a C1000 thermal cycler (BioRad, Hercules, CA). The reaction included an initial denaturation step at 95°C for 2 minutes, followed by 34 cycles of 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 isolation of the full-length mutant tail, the fragment was TOPO cloned into the pCR2.1 vector. DNA was prepared using a QIAGEN spin plasmid miniprep kit according to the manufacturer's instructions, and clones were sequenced using the ABI Dye Terminator v3.1 sequencing reaction according to the manufacturer's instructions.

[0229] Recombinant molecules were generated by PCR mutagenesis using overlap extension PCR with mutagenic oligonucleotides.

[0230] To obtain these molecules, the following oligonucleotides were used: CS-P238S 5-1: TIFF2024167368000020.tif14128SSSH-5-2: TIFF2024167368000021.tif14128P331S-S: TIFF2024167368000022.tif14128P331S-AS: TIFF2024167368000023.tif14128hIgG1-3'WTnogt: TIFF2024167368000024.tif15128

[0231] The P238S mutation and the SCC to SSS substitution were introduced by PCR mutagenesis using two overlapping 5' oligos in sequential PCR reactions. The first PCR reaction contained the following 5' primer, which incorporates the P238S mutation into its sequence: CS-P238S 5-1: TIFF2024167368000025.tif14129

[0232] The second PCR reaction contained the following 5' primer, which overlaps with the first primer and adds the mutant hinge residue to the P238S mutant: SSSH-5-2: TIFF2024167368000026.tif14166

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

[0234] The 5' partial fragment was amplified using the full-length wild-type clone as template, where the 5' primer was SSSH-5-2: TIFF2024167368000027.tif14128, while the 3' primer was P331S-AS: The file was TIFF2024167368000028.tif14138.

[0235] The 3' partial fragment was amplified using the full-length wild-type clone as template, where the 5' primer was P331S-S: TIFF2024167368000029.tif6166, while the 3' primer was hIgG1-3'WTnogt: The file was TIFF2024167368000030.tif14166.

[0236] The partial fragments were amplified and isolated by agarose gel electrophoresis. They were then purified on a QIAquick gel purification column and eluted in 30 microliters of EB buffer according to the manufacturer's instructions. Two PCR rounds were then performed, using the two partial fragments as overlapping templates in new reactions. The cycler was paused, and 5' and 3' flanking primers (50 pmol each) were added to the reaction. 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. Fragments from clones with the correct sequence were then subcloned into expression vectors to generate the various nuclease molecules described herein.

[0237] For the multispecific nuclease molecule, PCR reactions were performed using alternative primers to the 3' end of the Fc domain. The stop codon was removed and an NLG linker and EcoRV restriction site were added to facilitate fusion of the remainder of the cassette. Primer sequences are listed below. TIFF2024167368000031.tif7139

[0238] RSLV mutant nucleases were constructed by fusing a mutated human Ig tail to a wild-type RNase domain with or without a linker separating the two domains. In RSLV125 and RSLV126, the human RNase is fused to a mutant hinge and an IgG1 Fc domain. RSLV125 contains no linker, while RSLV126 contains a (gly4ser)4 linker as a (BglII-XhoI) fragment inserted between the nuclease domain and hinge region. RSLV-125 incorporates a wild-type RNase cassette directly fused to an SSS-type (rather than CCC or wild-type) human IgG1 hinge and a P238S, P331S mutant human IgG1 Fc domain (SEQ ID NOs: 61-62).

[0239] RSLV126 incorporates a wild-type RNase cassette fused to a (gly4ser)4 linker domain, followed by an SSS mutant hinge and a P238S-P331S double mutant Fc domain (SEQ ID NOs: 63-64).

[0240] RSLV-127 is a multi-nuclease fusion construct incorporating an amino-terminal human DNase (G105R / A114F) fused to a (gly4ser)4 linker domain, followed by an SSS mutant hinge and a P238S-P331S double mutant Fc domain, which is fused to an NLG linker domain, followed by a C-terminal wild-type RNase domain (SEQ ID NOs: 65-66).

[0241] RSLV-128 is a multi-nuclease fusion construct incorporating an amino-terminal wild-type human RNase domain fused to a (gly4ser)4 linker domain, followed by an SSS mutant hinge and a P238S-P331S double mutant Fc domain, which is fused to an NLG linker domain, followed by a C-terminal mutant DNase (G105R / A114F) domain (SEQ ID NOs: 67-68).

[0242] RSLV-129 is a multispecific fusion construct incorporating an amino-terminal wild-type human RNase domain fused to an SSS mutant hinge and a P238S-P331S double mutant Fc domain, which is fused to an NLG linker domain followed by a C-terminal mutant DNase (G105R / A114F) domain (SEQ ID NOs: 69-70).

[0243] RSLV-132 incorporates a wild-type RNase cassette directly fused to an SCC-type human IgG1 hinge and a P238S, P331S mutant human IgG1 Fc domain (SEQ ID NOs: 91-92 and 95-96).

[0244] RSLV-133 is a multispecific fusion construct incorporating an amino-terminal wild-type human RNase domain fused to an SCC mutant hinge and a P238S-P331S double mutant Fc domain, which is fused to an NLG linker domain followed by a C-terminal mutant DNase (G105R / A114F) domain (SEQ ID NOs: 93-94 and 97-98).

[0245] Other forms of RSLV-125 to RSLV-129 with SCC hinges are shown in Table 1 as RSLV-125-2 (SEQ ID NOs: 77-78), RSLV-126-2 (SEQ ID NOs: 79-80), RSLV-127-2 (SEQ ID NOs: 81-82), RSLV-128-2 (SEQ ID NOs: 83-84), and RSLV-129-2 (SEQ ID NOs: 85-86).

[0246] Example 10: Western blot of RSLV 125-129 fusion protein 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 supernatants were harvested 48 hours later. 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 transfectants of COS cells. Western blot analysis was performed on supernatants from transient transfectants. The molecules shown in Figure 9 contain either human RNase 1 fused to the human SSS IgG1 hinge and IgG1 P238S-P331S Fc domain, or human RNase 1 (wild-type) fused to 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 the mutant G105R-A114F allele of human DNase 1 at the carboxy terminus of the molecule. In addition, RSLV-127 encodes the above human DNase 1 mutant at the amino terminus of the mutant-Ig tail and RNase 1 WT at the carboxy terminus. COS supernatants were harvested after 72 hours, and 0.5 ml samples were immunoprecipitated with 100 μl of protein A-agarose beads overnight at 4°C. Protein A beads were centrifuged and washed twice in PBS before being resuspended in SDS-PAGE loading buffer or, for NuPAGE gels, in reducing or non-reducing LDS sample buffer (Invitrogen, Carlsbad, CA). Samples were heated according to the manufacturer's instructions, the Protein A beads were pelleted by centrifugation, and the sample buffer was loaded onto a 5-12% NuPAGE gradient gel. Samples were electrophoresed 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% nonfat 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% Tween 20, and then developed with ECL reagent. The results demonstrate that the nuclease-Fc fusion proteins were successfully constructed and readily expressed from COS cells. Furthermore, analysis of the reducing and nonreducing profiles of these nuclease-Fc fusion proteins confirms that the DNA constructs encode proteins of the appropriate molecular weight. Nonreducing SDS-PAGE patterns confirm that the disulfide bond characteristics of the proteins are consistent with the expected behavior of the constructs.

[0247] Example 11: SRED analysis of affinity purified proteins from COS7 transfectants Figure 10 shows the SRED analysis comparing aliquots of Protein A-purified proteins from RSLV-transfected COS supernatants 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. A 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. The mixture was immediately poured onto a glass plate. Sampling wells were created by punching holes in the gel. 2 μl each of control, serum sample, or affinity-purified RSLV protein was loaded into the wells, and the gel was incubated at 37°C for 4 hours in a humidified chamber. The gels were 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 gels were taken on a UV transilluminator using a Kodak digital camera DC290 system equipped with an ethidium bromide filter and analyzed using Kodak Molecular Imaging software. RNase enzyme activity assay results indicated that all constructs contained catalytically active RNase moieties.

[0248] Example 12: In-gel DNase activity of RSLV nuclease molecules Figure 11 shows the results of a DNase nuclease activity assay performed on Protein A-purified proteins from COS7 supernatants transfected with the RSLV fusion plasmid described in Example 10. Figure 11 shows five panels (11a, 11b, and 11c), with each gel panel showing the digestion patterns of decreasing amounts of the indicated fusion protein and 1 microgram of plasmid DNA. Each protein was serially diluted in nuclease-free water in two-fold increments, from 500 ng to 4 ng of enzyme. 1 μg of PDG plasmid DNA was added to each sample and incubated at 37°C for 30 minutes. One-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 an in-gel DNase enzyme activity assay using commercially available DNase 1 (Biolabs, Inc.). The rightmost lane is a negative control of DNA alone with no enzyme, and the lane to its left is another negative control, this time an RNase-Ig molecule with RNase activity but not DNase activity. As expected, in both cases the plasmid DNA remained intact and was not digested. These results demonstrate that the commercially available DNase 1 enzyme is highly active, digesting all of the DNA at most of the concentrations tested. The results in Figures 11a and 11b show the DNase activity of four nuclease-Fc fusion constructs. The top panel in Figure 11a shows the ability of the DNase-Ig fusion protein to digest plasmid DNA; as can be seen from the gel, the enzyme digested all of the DNA at all concentrations tested with activity equal to or greater than that of commercially available DNase 1. The bottom panel of Figure 11a shows a bispecific nuclease Fc fusion protein (SEQ IDs 65-66) with DNase at the amino terminus of the Fc, which also has robust DNase enzymatic activity, although somewhat weaker than the DNase-Ig in the top panel of Figure 11a. The top panel of Figure 11b shows the DNase enzymatic activity of another bispecific nuclease, this Fc fusion protein, with DNase tethered to the Fc at its C-terminus via a specially engineered NLG linker (SEQ IDs 67-68).As can be seen from the data, this enzyme also possesses robust DNase activity, appearing to be more active than the other bispecific nucleases examined here. The bottom panel of Figure 11b shows the DNase activity of another bispecific nuclease molecule (SEQ ID 69-70) that lacks the (G4S)4 linker connecting the RNase module to the Fc. This bispecific nuclease also possesses good DNase activity, although it appears to be somewhat weaker than the other two bispecific nuclease-Fc fusion proteins presented in this study. The data suggest that all of the bispecific nucleases possess good DNase activity, which is unexpected given previous work by others in this field (Dwyer et al. JBC; Vol. 271, No. 14; pp. 9738-9743). Furthermore, the location of the DNase in the construct, as well as the length and composition of the linker connecting the DNase to the Fc, are critically important in generating a highly active DNase enzyme in the context of a bispecific nuclease-Fc fusion protein.

[0249] Example 13: Enzyme kinetic analysis Figures 12-13 show the results of a kinetic fluorescent enzyme activity assay comparing the RNase enzyme activity of recombinant RNase A (Ambion), RSLV-125, RSLV-126, hRNase WT-SCCH-WThIgG1, and hRNaseG88D-SCCH-(P238S / K322S / P331S)hIgG1. To further characterize the functional properties of the bivalent mRNase-Ig fusion proteins, we investigated the enzymatic kinetics of various nuclease fusion proteins using RNase Alert Substrate (Ambion / IDT) and quantified fluorescence using a Biotek Synergy2 microplate reader. Data were analyzed using Gen5 software (Biotek Instruments, Inc., Winooski, Vermont). Relative fluorescence units as a function of time were assayed every minute over the course of a 45-minute experiment, incubated at 37°C according to the manufacturer's instructions, with enzyme concentrations starting at 10 pg / ul and serially diluted in 0.67-fold increments down to 0.1 pg / ul. Each sample contained a fixed concentration of RNase Alert substrate (200 nM) in 1x RNase Alert reaction buffer.

[0250] Figure 12 shows the RFU (relative fluorescence units) versus time for each protein at equimolar concentrations in the presence of 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.

[0251] Figure 13 shows Lineweaver-Burk plots for various molecules. To estimate Vmax and Km, RNase Alert kinetic fluorometric assays were 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 finely adjusted in this series of experiments. The data show Lineweaver-Burk plots generated for various fusion proteins under these conditions. Taken together, the data in Figures 12 and 13 demonstrate that the RNase moiety in the three RNase Fc fusion protein constructs constructed and tested herein is highly active.

[0252] Example 14: Evaluation of in vitro cytotoxicity against human THP-1 cell line Figures 14-15 show the results of in vitro studies analyzing the effect of RNaseIg fusion proteins containing wild-type or mutant (including SCC, P238S, and P331S) IgG Fc domains on the viability of the human monocytic cell line THP1. THP1 cells were maintained in logarithmic growth in RPMI / 10% FBS and then harvested for assay. Cell viability exceeded 98% before use in the cytotoxicity assay. THP1 cells were plated at 1 x 10e6 cells / ml or 100,000 cells per well in 96-well plates. Hybrid nuclease protein was added to consecutive wells using a two-fold serial dilution series starting at 5 micrograms / ml and ending with 0.01 micrograms / ml of fusion protein per reaction. In this experiment, an RNase-Fc fusion protein with a wild-type IgG1 Fc (wtRNasewtIgG) was compared with an RNase-Fc with a mutant Fc (P238S, P331S) that exhibits significantly reduced Fc receptor binding and internalization (mtRNasemgIgG) for its ability to induce cytotoxicity in cultured THP1 cells. Reactions were incubated in 96-well plates at 37°C under 5% CO2 for 3 days, after which cells were harvested and analyzed. After 3 days, cells were harvested by centrifugation at 1000 rpm, washed with PBS / 2% FBS, and incubated with FITC Annexin V Apoptosis Detection Kit Reagent (#556547, Becton Dickinson / Pharmingen) according to the manufacturer's instructions. Cells were washed with 100 microliters of chilled binding buffer provided with the kit, and Annexin V-FITC / propidium iodide (PI) was added at a 1:100 concentration in 100 μl of binding buffer. Samples were incubated on ice for 20 minutes, after which 400 μl of additional binding buffer was added to each sample. Stained samples were analyzed by flow cytometry using a FACS Canto (Becton Dickinson), and data were 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, assessed by two methods: Annexin V binding (top panel) and propidium iodide binding (bottom panel), both of which are sensitive measures of cell death. This experiment demonstrates that the RNase fusion protein with mutant Fc (P238S, P331S) exhibits reduced binding to Fc receptors on the surface of THP1 cells, as well as reduced subsequent internalization of the protein. The results demonstrate that the RNase-Fc mutant significantly reduces cell death compared with the RNase-wild-type Fc fusion protein (e.g., approximately 3-fold reduction at 1.25 μg / ml of protein). Figure 15 presents the results of a fluorescence-activated cell sorting (FACS) experiment to examine the cytotoxic effects of RNase Fc fusion constructs with wild-type or mutant Fc domains (RNase-wtIgG or RNase-mtIgG, respectively). The data demonstrate a significant reduction in the number of dead cells when THP1 cells were incubated with RNase Fc constructs containing mutant Fc regions (the peak size is smaller for the RNase Fc mutants on the right side of the graph compared to RNase-wtIgG). These data indicate that the RNase Fc mutants reduce cell death by approximately 3-5-fold compared to wild-type. These and other experiments examining Fc receptor binding clearly demonstrate that in the presence of Fc receptor-bearing cells, RNase Fc constructs with mutated Fc regions exhibit reduced Fc receptor binding and reduced cellular internalization, resulting in less cell death due to the construct's RNase activity. Such constructs are particularly useful in the treatment of autoimmune diseases, as it may be undesirable to use protein therapeutics that are cytotoxic to Fc receptor-bearing cells.

[0254] Example 15: Addition of RSLV-132 to in vitro cultures inhibits IFN-α production by human PBMCs Addition of RSLV132 abolished the induction of interferon-α from human peripheral blood mononuclear cells stimulated with immune complexes formed with serum from three SLE patients plus necrotic cell extract (NCE) (Figure 16). To measure the ability of RSLV-132 to bind to and degrade RNA contained in immune complexes from lupus patients, an in vitro bioassay was developed. This experiment involved the formation of immune complexes in vitro using autoantibodies from lupus patients and NCE 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 an interferon-α ELISA. Reporter cells were obtained from normal volunteers by Ficoll density gradient centrifugation. Serum from lupus patients or healthy normal volunteers was obtained in accordance with University of Washington Institutional Review Board #HSD No. 3971. Serum was diluted 1 / 1000 and added to necrotic cell extract (NCE) obtained from cultured U937 cells as described above at 10% (v / v). Diluted lupus patient or normal volunteer serum was incubated with NCE for 15 minutes at room temperature, and the resulting IC was incubated with or without various doses of RSLV-132, RSLV-124, or wild-type RNase for 15 minutes. Subsequently, the amount of IFN secreted by the PBMC cultures was measured in the presence of 500 U / mL Universal IFN for 20 hours. Serum was obtained from three different lupus patients with disease activity ranging from mild to active. NCEs were incubated with either lupus patient serum or healthy normal volunteer serum for 15 minutes at room temperature, followed by a 20 hour incubation with PBMCs.IFN-α was quantified by ELISA, where IFN-α was captured with a mouse MAb (MMHA-11) against human IFN-α [PBL Biomedical Laboratories, product #2112-1] and detected with a rabbit polyclonal antibody against IFN-α [PBL Biomedical Laboratories, product #31101-1], followed by development with anti-rabbit HRP [Jackson Immuno Research, product #711-035-152] and TMB substrate. In some cases, the analyte (RSLV-124 or RSLV-132) was added to the NCE at concentrations of 0.16, 0.5, 1.6, and 5.0 μg / mL, or RNase was added at equimolar concentrations of 0.05, 0.16, 0.5, and 1.6 μg / mL, prior to addition of the NCE to PBMCs. The ability of lupus patient serum to stimulate IFN production from PBMCs was reduced by approximately 50% by the addition of 5.0 μg / mL RSLV-124. This inhibition mirrored that of an equimolar amount of RNase. The addition of the same concentration of huRSLV-132 was equally or more effective in inhibiting IFN than RSLV-124, and the addition of 5.0 μg / mL huRSLV-132 almost completely abolished IFN production. When combined with NCE, lupus patient anti-RNA / DNA antibodies potently induced IFN from freshly isolated PBMCs. Serum from normal volunteers did not have the same ability to induce IFN production from reporter cells. This data indicates that circulating autoantibodies in lupus patient serum can form immune complexes that likely trigger TLR7 and subsequent IFN production. The exact type and subtype of IFN were not analyzed. These data indicate that RSLV-132 binds to and potently degrades its molecular target, RNA associated with lupus ICs, thereby preventing IFN stimulation by PBMCs (Figure 16). RSLV-132 appears to be more active than RSLV-124 in this assay.

[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 circulating RNA in mice, a pharmacodynamic model was developed using the RNA mimetic polyinosinic:polycytidylic acid (poly I:C), a 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 cytidylic acid. It is known to interact with toll-like receptor 3 (TLR3), which is expressed on the membranes of B cells, macrophages, and dendritic cells. Poly(I:C) is available from Invitrogen. The effects of poly I:C can be quantified by measuring the expression levels of interferon-stimulated genes in the spleens of mice after administration. On day 0, ten 3-month-old B6 mice were administered either RSLV-132 (250 μg per mouse) or intravenous immunoglobulin (IVIG) (Privigen, Behring) (250 μg per mouse) as a control, both administered intraperitoneally. Twenty hours after injection of RSLV-132 or IVIG, the mice were intraperitoneally injected with poly(I:C) at 200 μg per mouse. Two hours later, the mice were sacrificed by CO exposure, and the spleens were collected into RNAlater (Qiagen) and stored at -80°C for later testing of interferon-stimulated gene (ISG) expression. Spleen samples were subjected to qPCR testing for expression of ISGs, including Ifit1 (interferon-inducible protein with tetratricopeptide repeats 1 (UniProt Q64282)), Irf7 (interferon regulatory factor 7 (UniProt P70434)), and Mx1 genes. Results from these experiments demonstrate that intraperitoneal injection of RSLV-132 results in serum concentrations of RSLV-132 capable of binding to circulating poly(I:C) and effectively degrading this RNA mimic, thereby effectively disrupting the interferon pathway and stimulation of the three monitored ISGs (Figure 17).

[0256] Example 17. Enzyme kinetic analysis for 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 RNaseAlert QC kit from Ambion (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. The results demonstrate that RSLV-132 is a highly active RNase enzyme and has higher RNase activity than other RNase Fc fusion constructs, such as RSLV-124, and wild-type RNases. For example, using the same amount (400 pM) of RSLV-132 and RSLV-124, RSLV-132 produces more than twice the RFU compared to RSLV-124 (80,000 vs. 35,000). In addition, two production lots were tested for their stability at 4C. RSLV-132.1 was stored at 4 C for 8 weeks before this experiment, while RSLV-132.2 was stored at -80 C and thawed immediately before testing, demonstrating that the protein is stable for up to 2 months at 4 C. Drug stability and increased catalytic activity may lead to improved efficacy in therapeutic settings.

[0257] Figure 19 shows RNase enzyme activity (as RFU) over time, comparing the amount of RNase activity of the bispecific RSLV-133 molecule with that of the monospecific RSLV-132 and wild-type RNase. As demonstrated in Figure 19, the RSLV-133 molecule had significantly higher RNase activity than the monospecific RSLV-124 molecule, the earlier bispecific nuclease Fc RSLV-123, or wild-type RNase, yielding more than twice the RFU with the same amount of protein. Figure 20 shows the results of a DNase enzyme activity assay of the RLSV-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. Cleavage of the DNA substrate produced fluorescent light, which was 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, or wild-type DNase. The results demonstrate that RSLV-133 has higher DNase activity than wild-type DNase and the earlier bispecific nuclease molecule, RSLV-123, resulting in more than three-fold higher DNase activity in the linear range of the experiment. Figure 21 demonstrates the ability of the RSLV-133 molecule to digest DNA in an in-gel digestion experiment. The results indicate that RSLV-133 can digest DNA as effectively as wild-type DNase in this assay (compare lanes 5 and 7). Given the relative molecular weights of RSLV-133 and wild-type DNase, RSLV-133 appears to be more effective at digesting DNA in this assay as well.

[0258] Example 18: RSLV-132 demonstrates reduced binding to Fc receptors To examine 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-bearing human myeloid cell line THP1, and specific binding to the cells was quantified by fluorescence-activated cell sorting (FACS) analysis. RLSV-124 and RSLV-132 were fluorescently labeled with Alexa Fluor dye AF-647 (Cat. No. A20006) from Invitrogen. After dialysis of the RNase Fc fusion proteins to remove unbound dye, various amounts of labeled protein were incubated with THP1 cells for 1 hour. The cells were extensively washed to remove unbound RNase Fc fusion proteins, and specifically bound protein was quantified by FACS, measuring mean fluorescence intensity. The results in Figure 22 demonstrate that the RSLV-132 protein with the mutant Fc domain binds to Fc receptors significantly less than RSLV-124 with the wild-type Fc domain, with binding to Fc receptors reduced by more than 4-fold. This finding is consistent with our previous findings that an RNase Fc fusion protein with a mutant Fc domain (P238S / P331S) has 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 Examples above. Optionally, the hybrid nuclease molecule is a polypeptide. Optionally, the hybrid nuclease molecule comprises one or more sequences from Table 1. Optionally, the hybrid nuclease molecule comprises a nuclease domain linked to a mutant Fc domain. Optionally, the hybrid nuclease molecule comprises a mutant Fc domain. Optionally, the mutant Fc domain comprises a mutation in the hinge domain, CH2 domain, and / or CH3 domain. Optionally, the mutant Fc domain comprises P238S and / or P331S and may comprise a mutation in one or more of the three cysteines in the hinge region. Optionally, the mutant Fc domain comprises P238S and / or P331S, and / or a mutation in the three cysteines in the hinge region. In some cases, the mutant Fc domain comprises P238S and / or P331S, and / or mutations at the three cysteines in the hinge region to SSS. In some cases, the mutant Fc domain comprises P238S and P331S, and mutations at the three cysteines in the hinge region. In some cases, the mutant Fc domain comprises P238S, P331S, and SSS. In some cases, the mutant Fc domain is set forth in SEQ ID NOs: 59, 60, and 61. In some cases, the hybrid nuclease molecule is set forth in SEQ ID NOs. Various linker domains (e.g., those described herein) can be used to link the Fc domain to the nuclease domain. For example, a linker domain that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more amino acids in length can be used.Molecules are assayed for in vitro nuclease specific activity using a qualitative assay to ensure that they have the desired nuclease function.Then, specific activity is generally determined by a fluorescence-based kinetic assay that utilizes a substrate such as RNase or DNase Alert Kit reagent and a fluorescent plate reader that is set to read as a function of time.In addition, protein solutions are 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 0.06EU / ml.Then, molecules are assayed for biological activity using various in vitro assays.

[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 the presence or absence of the molecules in culture. Normal or patient PBMCs (approximately 1 x 10e6 cells) are cultured for 24, 48, or 96 hours, depending on the assay. PBMCs are 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 is measured using commercially available reagents, such as antibody pair kits from Biolegend (San Diego, CA) 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 in vitro cultures are collected at 24, 48 hours, or later. IFN-α production is measured using anti-human IFN-α antibodies and standard curve reagents available, for example, from PBL Interferon Suppliers (Piscataway, NJ). Similar assays are performed using subpopulations of human lymphocytes (isolated monocytes, B cells, pDCs, T cells, etc.); purified using commercially available magnetic bead-based isolation kits available, for example, from Miltenyi Biotech (Auburn, CA).

[0261] Additionally, the effect of the molecules on the expression of lymphocyte activation receptors, such as CD5, CD23, CD69, CD80, CD86, and CD25, will be assessed at various time points after stimulation. To determine 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 set of assays will measure the effects of these molecules on the proliferation of various lymphocyte subpopulations in vitro. These assays utilize, for example, CFDA-SE staining of human PBMCs (Invitrogen, Carlsbad, CA) prior to stimulation. 5 mM CFSE is diluted 1:3000 in PBS / 0.5% BSA with 10e7-10e8 PBMCs or purified cell subsets, and the labeling reaction is incubated at 37°C for 3-4 minutes before being washed several times in RPMI / 10% FBS to remove residual CFSE. CFSE-labeled cells are then incubated in coculture reactions with various stimuli (TLR ligands, costimulatory antibodies, etc.) and molecules for 4 days, after which cell proliferation is analyzed by flow cytometry using dye-conjugated antibodies specific for the cell subpopulations.

[0263] Another assay measures the cytotoxic effect of one or more molecules. In this assay, Annexin 5 staining (e.g., Annexin 5-FITC) is used to measure toxicity. Interesting cells (e.g., monocytes or monocyte cell lines) are contacted with the hybrid nuclease molecule of interest (e.g., the hybrid nuclease molecule with mutant Fc domain) or one or more controls. At various times after contact, the cells are separated from the culture and stained with Annexin 5. The number of apoptotic or dead cells is then counted, for example, by flow cytometry or fluorescence microscopy. The cells contacted with the hybrid nuclease molecule of interest show a lower number of cells that are positively stained for Annexin 5 compared to the positive control.

[0264] The effect of these molecules on the in vitro maturation of monocytes into DCs and macrophages is also assessed using both normal and patient PBMC samples.

[0265] The effectiveness of a hybrid nuclease molecule is demonstrated by comparing the results of an assay using cells treated with the hybrid nuclease molecule disclosed herein with the results of an assay using cells treated with a control formulation. After treatment, the levels of various markers (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 hybrid nuclease molecules to a mammal in need thereof The test involves the use of a mammal (e.g., mouse, rat, rodent, human, guinea pig). The mammal is administered (e.g., intravenously) one or more hybrid nuclease molecules comprising one or more sequences from Table 1, or a control. Optionally, the hybrid nuclease molecule is a polypeptide. Optionally, the hybrid nuclease molecule comprises one or more sequences from Table 1. Optionally, the hybrid nuclease molecule comprises a nuclease domain linked to a mutant Fc domain. Optionally, the hybrid nuclease molecule comprises a mutant Fc domain. Optionally, the mutant Fc domain comprises a mutation in the hinge domain, CH2 domain, and / or CH3 domain. Optionally, the mutant Fc domain comprises P238S and / or P331S, and optionally a mutation in one or more of the three cysteines in the hinge region. In some cases, the mutant Fc domain comprises a P238S and / or P331S mutation, and / or a mutation at the three cysteines in the hinge region. In some cases, the mutant Fc domain comprises a P238S and / or P331S mutation, and / or a mutation at the three cysteines in the hinge region to SSS. In some cases, the mutant Fc domain comprises P238S and P331S mutations, and a mutation at the three cysteines in the hinge region. In some cases, the mutant Fc domain comprises P238S and P331S and SSS. In some cases, the mutant Fc domain is set forth in SEQ ID NOs: 59, 60, and 61. In some cases, the hybrid nuclease molecule is set forth in SEQ ID NOs. Various linker domains (e.g., those described herein) can be used to link the Fc domain to the nuclease domain. For example, a linker domain that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more amino acids in length can be used.In some cases, the hybrid nuclease molecule is formulated with a pharmaceutically acceptable carrier.In some cases, the molecule is formulated as a pharmaceutical composition, as described in the pharmaceutical composition section above.The hybrid nuclease molecule targets RNase and / or DNase.

[0267] Multiple doses are used if deemed useful. The effects on IFN-α levels, IFN-α-responsive gene levels, autoantibody titers, renal function and pathology, and / or circulating immune complex levels are monitored in mammals. Similar studies are performed using different treatment protocols and administration routes (e.g., intramuscular administration, etc.). The efficacy of the hybrid nuclease molecules is demonstrated by comparing IFN-α levels, IFN-α-responsive gene levels, autoantibody titers, renal function and pathology, and / or circulating immune complex levels in mammals treated with the hybrid nuclease molecules disclosed herein with mammals treated with a control formulation.

[0268] In one example, select or identify a human subject who needs treatment.The subject may, for example, need to reduce the cause or symptoms of SLE.The subject can be identified in a clinical setting, or in other places, for example, at the subject's home, by the subject using a self-test kit.

[0269] At time zero, the subject is administered with a suitable initial dose of hybrid nuclease molecule.The hybrid nuclease molecule is formulated as described herein.After a certain period of time from the initial administration, for example, 7 days, 14 days and 21 days, the subject's condition is evaluated by measuring, for example, IFN-α level, IFN-α responsive gene level, autoantibody titer, renal function and pathology, and / or circulating immune complex level.Other relevant criteria can also be measured.The number and intensity of administration are adjusted according to the needs of the patient.

[0270] Following treatment, the subject's IFN-α levels, IFN-α responsive gene levels, autoantibody titers, kidney function and pathology, 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 in need of treatment is selected or identified. Identification of the subject may be in a laboratory setting or elsewhere.

[0272] At time zero, administer a suitable initial dose of hybrid nuclease molecule to the subject.The hybrid nuclease molecule is formulated as described herein.After a certain period of time from the initial administration, for example, 7 days, 14 days and 21 days, evaluate the condition of the subject by measuring, for example, IFN-α level, IFN-α responsive gene level, autoantibody titer, kidney function and pathology, and / or circulating immune complex level.Other relevant criteria can also be measured.The number and intensity of administration are adjusted according to the needs of the subject.

[0273] Following treatment, the subject's IFN-α levels, IFN-α responsive gene levels, autoantibody titers, kidney function and pathology, 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 shown and described in detail with reference to preferred and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0275] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

[0276] [Table 1] TIFF2024167368000033.tif241168TIFF2024167368000034.tif239168TIFF2024167368000035.tif239168TIFF2024167368000036.tif241168TIFF2024167368000037.tif240168TIFF2024167368000038.tif240168TIFF2024167368000039.tif240168TIFF2024167368000040.tif241168TIFF2024167368000041.tif241168TIFF2024167368000042.tif240168TIFF2024167368000043.tif240168TIFF2024167368000044.tif241168TIFF2024167368000045.tif240168TIFF2024167368000046.tif241168TIFF2024167368000047.tif243168TIFF2024167368000048.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 gatggggcgac 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 accctgaggt 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 aggagttttg 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 Ala Val Glu 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 ggctcccga 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 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 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 Convert Gl Glu Lys Val Thr To Cys Lys And Gly Gln Gly To 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 cacgcctcccc 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 Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr Lys Met Ser 405 410 415 Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser Arg Tyr Asp 420 425 430 Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr Ala Val Gly 435 440 445 Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp Thr Tyr His Tyr 450 455 460 Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu Arg Tyr Leu 465 470 475 480 Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp Ser Tyr Tyr Tyr 485 490 495 Asp Asp Gly Cys Glu Pro Cys Arg Asn Asp Thr Phe Asn Arg Glu Pro 500 505 510 Phe Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg Glu Phe Ala 515 520 525 Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala Glu Ile Asp 530 535 540 Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp Gly Leu Glu 545 550 555 560 Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val Arg 565,570,575 Pro Ser Gln Trp Ser ...

Claims

1. A nuclease-containing composition effective to degrade immune complexes containing RNA, DNA, or both RNA and DNA, comprising a pharma- ceutically 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% identical to the amino acid sequence set forth in SEQ ID NO: 96, 92, 62, or 78 and retains the activity of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 96, 92, 62, or 78; or (ii) an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 98 or 94 and retains the activity of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 98 or 94. and wherein the composition is formulated for intravenous administration.

2. A composition containing the nuclease described in claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:96 and retains the activity of a polypeptide containing the amino acid sequence set forth in SEQ ID NO:

96.

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

98.

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

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

6. A composition containing a nuclease described in any one of claims 1 to 5, wherein the modified Fc domain has been modified to reduce binding to an Fcγ receptor, a complement protein, or both.

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

8. A composition containing a nuclease described in 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 a nuclease described in any one of claims 1 to 5, wherein the modified Fc domain comprises an amino acid sequence having mutations SCC or SSS, P238S, and P331S.

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

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

12. A composition containing a nuclease described in any one of claims 1 to 5, wherein the activity of the RNase is at least 9-fold lower than the activity of a control RNase molecule.

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

14. A composition containing a nuclease described in any one of claims 1 to 5, wherein the polypeptide contains DNase and the activity of the DNase is at least 9-fold lower than the activity of a control DNase molecule.

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

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

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

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

19. 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, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes mellitus, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, hydrocephalus ... The pharmaceutical composition of claim 18, wherein the disease is selected from the group consisting of crystalloid uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerative colitis, Sjogren'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 a nuclease described in any one of claims 1 to 5.

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

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

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

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

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

26. The pharmaceutical composition described in claim 25, wherein the dimeric polypeptide is a homodimer.

27. ​​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, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes mellitus, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, lenticular disease, The pharmaceutical composition according to any one of claims 24 to 26, wherein the disease is selected from the group consisting of uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, idiopathic liver cirrhosis, ulcerative colitis, Sjogren'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 described in any one of claims 24 to 27, wherein the autoimmune disease is SLE.

29. The pharmaceutical composition described in 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 of any one of claims 24 to 27, wherein the autoimmune disease is Sjogren's syndrome.

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