Therapeutic nuclease compositions and methods

Abstract

Hybrid nuclease molecules for treating immune-related diseases or disorders in mammals and pharmaceutical compositions for treating immune-related diseases in mammals are provided. The present invention provides a hybrid nuclease molecule comprising a first nuclease domain and an Fc domain, wherein the first nuclease domain is operably connected to the Fc domain, and further comprising a first linker domain, wherein the first nuclease domain is operably connected to the Fc domain by the first linker domain.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of 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 which 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.

[0003] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically as a text file named XXXXXPCT_sequencelisting.txt, created in X month of 20XX and having a size of X bytes. This Sequence Listing is incorporated by reference. [Background technology]

[0004] 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 coengagement 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

[0005] overview Disclosed herein is a hybrid nuclease molecule comprising a first nuclease domain and an Fc domain, wherein the first nuclease domain is operably connected to the Fc domain. In some embodiments, the hybrid nuclease molecule further comprises a first linker domain, wherein the first nuclease domain is operably connected to the Fc domain by the first linker domain.

[0006] 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 wild-type IgG1 Fc domain; and the first linker domain is connected to the C-terminus of the first nuclease domain and the N-terminus of the Fc domain. In some embodiments, the hybrid nuclease molecule is a polypeptide comprising or consisting of a sequence set forth in Table 2. In some embodiments, the hybrid nuclease molecule is a polypeptide comprising SEQ ID NO: 149. In some embodiments, the hybrid nuclease molecule is a polypeptide comprising SEQ ID NO: 145. In some embodiments, the hybrid nuclease molecule is a polypeptide comprising SEQ ID NO: 161. In some embodiments, the hybrid nuclease molecule is a polypeptide comprising SEQ ID NO: 162. In some embodiments, the hybrid nuclease molecule is a polypeptide comprising SEQ ID NO:163.

[0007] In some embodiments, the hybrid nuclease molecule comprises wild-type human DNase 1 linked to wild-type human IgG1. In some embodiments, the hybrid nuclease molecule comprises human DNase 1 G105R A114F linked to a wild-type human IgG1 Fc domain by a (gly4ser)n linker 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 wild-type human IgG1 linked to wild-type human DNase 1. In some embodiments, the hybrid nuclease molecule comprises wild-type human RNase 1 linked to wild-type human IgG1 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 an NLG peptide 5-32 amino acids in length, the amino acid sequence of the Fc domain comprises the amino acid sequence of a wild-type 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.

[0008] In some embodiments, the Fc domain binds to an Fc receptor on a human cell. In some embodiments, the serum half-life of the molecule is significantly longer than the serum half-life of the first nuclease domain alone. In some embodiments, the nuclease activity of the first nuclease domain of the molecule is the same as or greater than the nuclease domain alone. In some embodiments, administration of the molecule to a mouse increases the survival rate of the mouse as measured by a mouse lupus model assay.

[0009] In some embodiments, the hybrid nuclease molecule comprises a leader sequence, ie, a human VK3LP peptide from the human kappa light chain family, connected to the N-terminus of the first nuclease domain.

[0010] In some embodiments, the molecule is a polypeptide. In some embodiments, the molecule is a polynucleotide.

[0011] 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% similar to the amino acid sequence of an RNase listed in Table 2. In some embodiments, the RNase is a member of the human RNase A family. In some embodiments, the RNase is human pancreatic RNase 1.

[0012] 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% similar to the amino acid sequence of a DNase listed in Table 2. In some embodiments, the DNase is selected from the group consisting of human DNase I, TREX1, and human DNase 1L3.

[0013] In some embodiments, the Fc domain is a human Fc domain. In some embodiments, the Fc domain is a wild-type Fc domain. In some embodiments, the Fc domain is a mutant Fc domain. 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% similar to the amino acid sequence of an Fc domain listed in Table 2.

[0014] 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 is of the formula (Gly4Ser)n, where 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. In some embodiments, the first linker domain comprises an N-linked glycosylation site.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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. [The present invention 1001] A hybrid nuclease molecule comprising a first nuclease domain and an Fc domain, wherein the first nuclease domain is operably connected to the Fc domain. [The present invention 1002] 1001. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide, the amino acid sequence of the first nuclease domain comprises the amino acid sequence of a human wild-type RNase as set forth in SEQ ID NO:149, and the amino acid sequence of the Fc domain comprises the amino acid sequence of a human wild-type IgG1 Fc domain as set forth in SEQ ID NO:145. [The present invention 1003] 1001. The hybrid nuclease molecule of the present invention, which is a polypeptide consisting of SEQ ID NO:163. [The present invention 1004] 1001. A hybrid nuclease molecule of the present invention comprising wild-type human DNase 1 linked to wild-type human IgG1. [The present invention 1005] 1001. A hybrid nuclease molecule of the present invention comprising human DNase 1 G105R A114F linked to wild-type human IgG1. [The present invention 1006] 1001. A hybrid nuclease molecule of the present invention comprising wild-type human RNase 1 linked to wild-type human IgG1 linked to wild-type human DNase 1. [The present invention 1007] 1001. A hybrid nuclease molecule of the present invention comprising wild-type human RNase 1 linked to wild-type human IgG1 linked to human DNase 1 G105R A114F. [The present invention 1008] 1001. A hybrid nuclease molecule of the present invention, wherein the Fc domain binds to an Fc receptor on a human cell. [The present invention 1009] 1001. The hybrid nuclease molecule of the invention, wherein the serum half-life of said molecule is significantly longer than the serum half-life of the first nuclease domain alone. [The present invention 1010] 1001. The hybrid nuclease molecule of the present invention, wherein the nuclease activity of the first nuclease domain of said molecule is equal to or greater than that of the nuclease domain alone. [The present invention 1011] 1001. The hybrid nuclease molecule of the present invention, wherein administration of said molecule to a mouse increases the survival rate of said mouse as measured by an assay in a mouse lupus model. [The present invention 1012] 1001. The hybrid nuclease molecule of the present invention, wherein said molecule further comprises a first linker domain, and wherein the first nuclease domain is operably connected to the Fc domain by the first linker domain. [The present invention 1013] A hybrid nuclease molecule of the present invention 1012, wherein the hybrid nuclease molecule is a polypeptide, the amino acid sequence of the first nuclease domain comprises the amino acid sequence of an RNase, the first linker domain is 5 to 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. [The present invention 1014] A hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide, the amino acid sequence of the first nuclease domain comprises the amino acid sequence of human RNase, the first linker domain is an NLG peptide of 5 to 32 amino acids in length, the amino acid sequence of the Fc domain comprises the amino acid sequence of a human wild-type 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. [The present invention 1015] 1001. The hybrid nuclease molecule of the present invention, further comprising a leader sequence. [The present invention 1016] 1015. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide, the leader sequence is a human VK3LP peptide, and the leader sequence is connected to the N-terminus of the first nuclease domain. [The present invention 1017] 1001. The hybrid nuclease molecule of the present invention, which is a polypeptide. [The present invention 1018] The hybrid nuclease molecule of the present invention 1001, which is a polynucleotide. [The present invention 1019] 1001. The hybrid nuclease molecule of the present invention, wherein the first nuclease domain comprises an RNase. [The present invention 1020] The hybrid nuclease molecule of the invention 1019, wherein the RNase is a human RNase. [The present invention 1021] The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the RNase is a polypeptide comprising an amino acid sequence that is at least 90% similar to the amino acid sequence of an RNase listed in Table 2. [The present invention 1022] The hybrid nuclease molecule of the present invention, wherein the RNase is human pancreatic RNase 1. [The present invention 1023] 1001. The hybrid nuclease molecule of the present invention, wherein the first nuclease domain comprises a DNase. [The present invention 1024] The hybrid nuclease molecule of the present invention 1023, wherein the DNase is human DNase. [The present invention 1025] The hybrid nuclease molecule of the present invention 1023, wherein the hybrid nuclease molecule is a polypeptide and the DNase is a polypeptide comprising an amino acid sequence that is at least 90% similar to the amino acid sequence of a DNase listed in Table 2. [The present invention 1026] The hybrid nuclease molecule of the present invention 1023, wherein the DNase is selected from the group consisting of human DNase I, TREX1 and human DNase 1L3. [The present invention 1027] 1001. The hybrid nuclease molecule of the present invention, wherein the Fc domain is a human Fc domain. [The present invention 1028] 1001. The hybrid nuclease molecule of the present invention, wherein the Fc domain is a wild-type Fc domain. [The present invention 1029] 1001. The hybrid nuclease molecule of the present invention, wherein the Fc domain is a mutated Fc domain. [The present invention 1030] 1001. The hybrid nuclease molecule of the present invention, wherein the Fc domain is a human IgG1 Fc domain. [The present invention 1031] 1001. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the Fc domain is a polypeptide comprising an amino acid sequence that is at least 90% similar to the amino acid sequence of an Fc domain listed in Table 2. [The present invention 1032] 1012. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the first linker domain has a length of about 1 to about 50 amino acids. [The present invention 1033] 1012. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the first linker domain has a length of about 5 to about 32 amino acids. [The present invention 1034] 1012. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the first linker domain has a length of about 15 to about 25 amino acids. [This invention 1035] 1012. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the first linker domain has a length of about 20 to about 32 amino acids. [The present invention 1036] 1012. The hybrid nuclease molecule of the invention, wherein the hybrid nuclease molecule is a polypeptide and the first linker domain has a length of about 20 amino acids. [This invention 1037] 1012. The hybrid nuclease molecule of the invention, wherein the hybrid nuclease molecule is a polypeptide and the first linker domain has a length of about 25 amino acids. [The present invention 1038] 1012. The hybrid nuclease molecule of the invention, wherein the hybrid nuclease molecule is a polypeptide and the first linker domain has a length of about 18 amino acids. [This invention 1039] 1012. The hybrid nuclease molecule of the invention, wherein the hybrid nuclease molecule is a polypeptide and the first linker domain comprises a gly / ser peptide. [The present invention 1040] 1039. The hybrid nuclease molecule of the present invention, wherein the gly / ser peptide is of the formula (Gly4Ser)n, where n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. [The present invention 1041] 1039. The hybrid nuclease molecule of the present invention, wherein the gly / ser peptide comprises (Gly4Ser)3, (Gly4Ser)4 or (Gly4Ser)5. [The present invention 1042] 1012. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the first linker domain comprises an NLG peptide. [This invention 1043] The hybrid nuclease molecule of the invention 1012, wherein the hybrid nuclease molecule is a polypeptide and the first linker domain comprises an N-linked glycosylation site. [This invention 1044] 1001. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the first nuclease domain is linked to the N-terminus of the Fc domain. [This invention 1045] 1001. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the first nuclease domain is linked to the C-terminus of the Fc domain. [The present invention 1046] The hybrid nuclease molecule of the present invention 1001 further comprising a second nuclease domain. [This invention 1047] The hybrid nuclease molecule of the invention 1046, wherein the first and second nuclease domains are different nuclease domains. [This invention 1048] The hybrid nuclease molecule of the invention 1046, wherein the first and second nuclease domains are the same nuclease domain. [This invention 1049] 1046. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the second nuclease domain is linked to the C-terminus of the Fc domain. [The present invention 1050] 1046. The hybrid nuclease molecule of the invention, wherein the hybrid nuclease molecule is a polypeptide and the second nuclease domain is linked to the N-terminus of the Fc domain. [This invention 1051] 1046. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the second nuclease domain is linked to the C-terminus of the first nuclease domain. [This invention 1052] 1046. The hybrid nuclease molecule of the present invention, wherein the hybrid nuclease molecule is a polypeptide and the second nuclease domain is linked to the N-terminus of the first nuclease domain. [This invention 1053] 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 connected to the Fc domain. [This invention 1054] The dimeric polypeptide of the present invention 1053, wherein 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 connected to the second Fc domain. [This invention 1055] A pharmaceutical composition comprising at least one hybrid nuclease molecule and / or at least one dimeric polypeptide of any of claims 1001 to 1054 of the present invention, and a pharmaceutically acceptable excipient. [This invention 1056] A nucleic acid molecule encoding a hybrid nuclease molecule of the present invention. [This invention 1057] A recombinant expression vector comprising a nucleic acid molecule of the present invention. [This invention 1058] A host cell transformed with a recombinant expression vector of the present invention 1057. [This invention 1059] A method for producing the hybrid nuclease molecule of the present invention, 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. [The present invention 1060] A method 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 of the present invention. [The present invention 1061] The method of claim 1060, wherein the condition is an autoimmune disease. [This invention 1062] Autoimmune diseases include insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes mellitus, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, and cross-sectional area. 1061. The method of the present invention, wherein the inflammatory bowel disease is selected from the group consisting of: inflammatory bowel disease, phacopathic 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. [The present invention 1063] The method of claim 1061, wherein the autoimmune disease is SLE. [Brief explanation of the drawings]

[0022] These and other features, aspects and advantages of the present invention will become better understood in connection with the following description and accompanying drawings.

[0023] [Figure 1-1] The nucleotide and amino acid sequences of mRNase-mIgG2a with mutations at P238S, K322S, and P331S are shown, and are listed in the Sequence Listing as huVK3LP+mrib1+mIgG2A-C-2S (SEQ ID NO:114). [Figure 1-2] The nucleotide and amino acid sequences of mRNase-mIgG2a with mutations at P238S, K322S, and P331S are shown, and are listed in the Sequence Listing as huVK3LP+mrib1+mIgG2A-C-2S (SEQ ID NO:114). [Figure 1-3]The nucleotide and amino acid sequences of mRNase-mIgG2a with mutations at P238S, K322S, and P331S are shown, and are listed in the Sequence Listing as huVK3LP+mrib1+mIgG2A-C-2S (SEQ ID NO:114). [Figure 2] 1 shows a schematic diagram of some embodiments of the hybrid nuclease molecules described herein. [Figure 3] 1 shows an SDS-PAGE gel analysis of mRNase-mIgG2a-c under both reducing and non-reducing conditions. [Figure 4] 1 shows gel immunoprecipitation analysis of mRNase mIg2a-c. [Figure 5] Figure 1 shows ELISA titers of anti-RNA antibodies in mouse 410 before and after intravenous injection of RNase-Ig hybrid nuclease molecules. The data show that injection of RNase-Ig caused a decrease in anti-RNA antibody titers that persisted over 3 weeks. [Figure 6] We show that the induction of interferon-α from human peripheral blood mononuclear cells stimulated with immune complexes formed by serum and nuclear extract (NE) from an SLE patient (J11) was abolished by the addition of RNase-Ig. The titer of anti-RNA antibodies was reduced after injection of RNase-Ig. [Figure 7] We show that the induction of interferon-α from human peripheral blood mononuclear cells stimulated with immune complexes formed by serum plus nuclear extract from an SLE patient (J11) was abolished by the addition of RNase-Ig. [Figure 8] Single radial enzyme diffusion (SRED) analysis of serum from two RNase transgenic (Tg) mice compared with normal B6 mice is shown. [Figure 9] 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 10]Survival of TLR7.1 Tg mice and TLR7.1×RNaseA DTg mice is shown. [Figure 11] Quantitative PCR of IRGs in the spleens of Tg and DTg mice is shown. [Figure 12] 1 shows prototype structures for creating various embodiments of hybrid nuclease molecules. [Figure 13] 1 shows the enzymatic kinetics of the hRNase1-G88D-hIgG1 SCCH-P238S-K322S-P331S hybrid nuclease molecule measured using RNase Alert Substrate™. [Figure 14] Binding of hRNase1-WT-hIgG1-WT to the human monocytic cell lines U937 and THP1 is shown. The left peak in both plots is the control, and the right peak in both plots is hRNase1-WT-hIgG1-WT. [Figure 15] 1 shows the blocking activity of human IVIg against the binding of hRNase1-WT-hIgG1-WT to U937 cells and THP-1 cells. [Figure 16] 1 shows the results of a DNA digestion assay with alternative forms of Trex1-(g4s)n-mIgG. [Figure 17] This shows the results of Western blotting for the culture supernatants of trex1-(Gly4S)4-Ig and trex1-(Gly4S)5-Ig cells obtained by transient transfection of COS-7 cells. [Figure 18] 1 shows DNA digestion patterns by different stably transfected CHO DG44 clones, designated 2A3, 3A5 and 8H8, expressing the DNase1L3-mIgG2a-c hybrid nuclease molecule. [Figure 19] 1 shows DNA digestion patterns with decreasing amounts of DNase1L3-Ig hybrid nuclease molecules after various incubation times in the presence and absence of heparin as an enzyme inhibitor. [Figure 20]1 shows a Western blot of immunoprecipitated fusion proteins from transiently transfected COS cells expressing various forms of hRNase 1-Ig-hDNase 1 or hDNase 1-Ig hybrid nuclease molecules. [Figure 21] 1 shows an SRED assay to assess the presence of RNase activity in COS supernatants expressing various embodiments of hRNase 1-Ig-hDNase 1 or hDNase 1-Ig hybrid nuclease molecules. [Figure 22] 21 shows a composite diagram displaying the results of a DNase nuclease activity assay performed on COS supernatants from transfected cells. The numbering designations from FIG. 21 (e.g., 090210-8 and 091210-8) also apply to this figure. [Figure 23] Enzyme kinetics was assayed using RNase Alert Substrate (Ambion / IDT) and fluorescence was quantified using a Spectramax M2 microplate reader. Data were analyzed using Softmax Pro software (Molecular Devices). Reaction rates at various substrate concentrations were measured, and the data are presented as Lineweaver-Burk plots. The apparent Km, corrected for volume, is 280 nM. [Figure 24] 1 shows the levels of anti-RNA antibodies in mouse sera from H564 and H564RNaseA double transgenic mice at successive intervals as the transgenic mice age. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description 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.

[0025] "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.

[0026] 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.

[0027] 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.

[0028] "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.

[0029] "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.

[0030] 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.

[0031] 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. SEQ ID NO: 161 is an example of a hybrid nuclease molecule. Other examples are described in more detail below. In one embodiment, the hybrid nuclease molecule of the present invention can comprise further modifications. In another embodiment, the hybrid nuclease molecule can be modified to include a functional moiety (e.g., PEG, a drug, or a label) attached thereto.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] As used herein, the term "Fc domain" refers to a portion of a single immunoglobulin (Ig) heavy chain. Therefore, an Fc domain can also be referred to as "Ig" or "IgG." In some embodiments, an Fc domain begins at the hinge region immediately upstream of the papain cleavage site of an 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, an 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, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In one embodiment, an Fc domain comprises a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In another embodiment, an 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, the Fc domain of the present invention comprises at least a portion of an Fc molecule known in the art to be necessary for Protein 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.

[0037] 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).

[0038] 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.

[0039] 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 that is 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] In one embodiment, a polypeptide of the invention consists of, consists essentially of, or comprises an amino acid sequence selected from Table 2, and functionally active variants thereof. In one embodiment, a polypeptide comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence set forth in Table 2. In one embodiment, a polypeptide comprises a contiguous amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a contiguous amino acid sequence set forth in Table 2. 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 2.

[0044] 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 sequence of the present invention comprises, consists of, or consists essentially of a nucleotide sequence selected from Table 2. In one embodiment, the nucleotide sequence comprises a 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 nucleotide sequence set forth in Table 2. 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 2. 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 2.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] The term "in situ" refers to a process that occurs in living cells growing away from the organism, for example, in tissue culture.

[0050] The term "in vivo" refers to a process that takes place in a living organism.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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).

[0055] 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.

[0056] 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.

[0057] 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."

[0058] 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.

[0059] 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.

[0060] 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 some embodiments, the hybrid nuclease molecule will comprise a stop codon. In some embodiments, the stop codon will be at the C-terminus of the Fc domain.

[0061] 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 12 shows at least one embodiment of a hybrid nuclease molecule. In some embodiments, the hybrid nuclease molecule comprises a sequence set forth in Table 2.

[0062] 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 hinge cysteines. In some such aspects, the mutation in one or more of the three hinge cysteines 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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, enabling 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.

[0068] In some embodiments, hybrid nuclease molecules comprising a wt Fc domain are used for the treatment of autoimmune diseases, such as SLE.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] In some embodiments, the hybrid nuclease molecule includes a DNase-Ig hybrid nuclease molecule and / or a hybrid DNase-RNase hybrid nuclease molecule.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] In some embodiments, the hybrid nuclease molecule comprises TREX1-linker1-Ig-linker2-RNase.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Fc domain In some embodiments, the hybrid nuclease molecule comprises an Fc domain. 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, rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates (e.g., chimpanzees, macaques). 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.

[0087] 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 to have 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 derived 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 invention. It will be further understood that the scope of the present invention encompasses alleles, variants, and mutations of constant region DNA sequences.

[0088] 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).

[0089] 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.

[0090] 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).

[0091] 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).

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] Changes to Fc amino acids In some embodiments, the Fc domain used in the hybrid nuclease molecules of the present invention is modified, for example, by amino acid mutation (e.g., addition, deletion, or substitution). As used herein, the term "Fc domain variant" refers to an Fc domain that has at least one amino acid 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 contains at least one amino acid mutation (e.g., substitution) compared to the wild-type amino acid at the corresponding position of the human IgG1 Fc region.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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 for 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.

[0104] 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.

[0105] 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.

[0106] 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 analogs thereof can then be conjugated to a functional domain (e.g., conjugated with a thiol-reactive heterobifunctional linker) using art-recognized techniques.

[0107] 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).

[0108] 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.

[0109] 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.

[0110] 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).

[0111] 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.

[0112] 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).

[0113] 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.

[0114] 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).

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] In some embodiments, the nuclease domain is a DNase. In some embodiments, the DNase is a type I secretory DNase. In some embodiments, the DNase is DNase 1 and / or DNase 1-like (DNaseL) enzymes 1-3. In some embodiments, the DNase is TREX1.

[0123] In some embodiments, the nuclease domain is an RNase. In some embodiments, the RNase is an extracellular or secreted RNase of the RNase A superfamily, e.g., RNase A.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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).

[0132] 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.

[0133] 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.

[0134] 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.

[0135] Any of a large number of available and well-known host cells can be used in practicing 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.

[0136] The transformed host is 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.

[0137] Compounds can also be produced by synthetic methods. For example, solid phase synthesis techniques can be used. Suitable methods 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.

[0138] Other methods for the expression / synthesis of molecules are generally known to those skilled in the art.

[0139] 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.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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. [Example]

[0164] 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.

[0165] 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).

[0166] Example 1: 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).

[0167] Once a full-length clone was isolated, primers were designed to create a fusion gene with the Fc domain of mouse IgG2a (SEQ ID NO:114) or human IgG1 (SEQ ID NO:110). 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) TIFF0007657249000001.tif13141

[0168] 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) TIFF0007657249000002.tif13138

[0169] 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). TIFF0007657249000003.tif13141

[0170] 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 TIFF0007657249000004.tif13135mrib3X A 31-mer RNase 3' end with two stop codons and an XbaI site for fusion with the carboxy terminus of an Fc domain TIFF0007657249000005.tif13141

[0171] Example 2: Isolation of anti-RNA or anti-DNA scFv from hybridomas expressing monoclonal antibodies An anti-RNA hybridoma designated H564 was used to isolate RNA-specific V regions. Prior to harvest, H564 anti-RNA hybridoma cells were maintained in logarithmic growth for several days in RPMI 1640 medium (Invitrogen / Life Technologies, Gaithersburg, Md.) supplemented with glutamine, pyruvate, DMEM non-essential amino acids, and penicillin-streptomycin. Cells were pelleted from the medium by centrifugation and 2 × 10 7RNA was isolated from hybridoma cells using a QIAGEN RNAeasy kit (Valencia, Calif.) for total RNA isolation and a QIAGEN QIAshredder according to the manufacturer's instructions. Four micrograms (4 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 the second-strand buffer and 0.1 M DTT provided with the enzyme. The reverse transcription reaction proceeded for 1 hour at 50°C.

[0172] The cDNA generated by the reverse transcriptase reaction was purified using a QIAquick PCR purification kit (QIAGEN, Valencia, CA) and tailed with a poly-G sequence using terminal transferase (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The tailed cDNA was purified again using QIAquick PCR purification and eluted in 30 μl of the elution buffer (EB buffer) provided with the kit. To amplify the variable regions for the light and heavy chains of the H564 antibody by PCR, 2 μl of the tailed cDNA was used as a template with an anchor-tail 5' primer containing a poly-C domain and a degenerate 3' primer specific for the constant region. The two variable chains were designed with restriction enzyme sites so that, after amplification and restriction enzyme digestion, scFvs could be assembled by three-way ligation of the two V regions to a linker sequence.

[0173] The (gly4ser) 4-peptide linker inserted between the two V regions was incorporated by amplifying this linker sequence by overlap-extension PCR using overlapping primers encoding each half of the molecule. PCR fragments were isolated by agarose gel electrophoresis, the fragments were isolated by excising the appropriate band from the gel, and the amplified DNA was purified using a QIAquick gel extraction kit (QIAGEN, Valencia, CA). scFv derivatives from the H564 hybridoma were assembled as VH-linker-VL fusion genes that could be attached to either end of larger Ig fusion genes. The V.sub.H domain was amplified without a leader peptide but included a 5' AgeI restriction site for fusion with V.sub.L and a BglII restriction site at the 3' end for fusion with the linker domain.

[0174] The scFv-Ig was assembled by inserting the scFv HindIII-XhoI fragment into pDG containing the human IgG1 hinge, CH2, and CH3 regions, which was digested with HindIII and XhoI restriction enzymes. After ligation, the ligation product was transformed into DH5-α bacteria. The scFv-Ig cDNA was cycle sequenced in a PE 9700 thermocycler using a 25-cycle program consisting of denaturation at 96°C for 10 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 4 minutes. The sequencing primers were the pDG forward and reverse primers and an internal primer annealing to the human CH2 domain within the IgG constant region. Sequencing reactions were performed using Big Dye Terminator Ready Sequencing Mix v3.1 (PE-Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions. Samples were then purified using an Autoseq G25 column (GE Healthcare), and the eluate was dried in a Savant vacuum oven, denatured in Template Suppression Reagent (PE-ABI), and analyzed on an ABI 310 Genetic Analyzer (PE-Applied Biosystems). Sequences were edited, converted, and analyzed using Vector Nti version 10.0 (Informax / Invitrogen, North Bethesda, Md.).

[0175] Construction of human RNaseI-hIgG1 (SEQ ID NO: 125-127) fusion gene Human RNase 1 (SEQ ID NO: 113) was isolated by PCR amplification from human pancreatic total RNA obtained from Ambion / Applied Biosystems (Austin, TX). Four micrograms (4 μg) of total RNA was used as a template to prepare 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 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 reaction was further purified using a QIAquick PCR purification column, and the cDNA was eluted in 40 μL of EB buffer prior to PCR. Two microliters of the cDNA eluate was added to a PCR reaction containing 50 pmol of 5' and 3' primers specific for human RNase 1, and 45 microliters of PCR high fidelity supermix (Invitrogen, Carlsbad, CA) was added to a 0.2 ml PCR reaction tube. PCR reactions were performed using a C1000 thermal cycler (BioRad, Hercules, CA). The reaction 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 68°C for 1 minute, followed by a final 4-minute extension at 72°C. After isolating the wild-type 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. Plasmid DNA was sequenced using the ABI Dye Terminator v3.1 ready reaction mix according to the manufacturer's instructions.

[0176] Example 3: Isolation of human and mouse Fc domains and introduction of mutations into the coding sequences For isolation of the mouse Fc domain (SEQ ID NO:114) and human Fc domain (SEQ ID NO:110), RNA was derived 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). The buffy coat was collected according to the manufacturer's instructions and used after washing the cells three times in PBS. Cells were pelleted from the medium by centrifugation and 2 × 10 7 RNA was isolated from the 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 the PCR reaction.

[0177] 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: TIFF0007657249000006.tif82131

[0178] 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.

[0179] 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 the 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, using 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:

[0180] The 5' subfragment was amplified using the full-length wild-type clone as template, where the 5' primer was hIgG1-5scc: TIFF0007657249000007.tif6128, while the 3' primer was P331AS: The 3' subfragment was amplified using the full-length wild-type clone as template, where the 5' primer was P331S: TIFF0007657249000009.tif6128, while the 3' primer was mahIgG1S: The file name was TIFF0007657249000010.tif6131.

[0181] Subfragments 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 subfragments 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.

[0182] Example 4: Expression of RNase-Ig (SEQ ID NOs: 124, 125, 126, 127, 174 (nucleotides) or 160, 161, 162, 163, 175 (amino acids)), DNase-Ig (SEQ ID NOs: 118, 119, 120, 121, 122, 123, 186 (nucleotides) or SEQ ID NOs: 154, 155, 156, 157, 158, 159, 187 (amino acids)), multi-subunit Ig fusion constructs (SEQ ID NOs: 115, 116, 117, 172, 176, 178, 180 (nucleotides) or SEQ ID NOs: 151, 152, 153, 173, 177, 179, 181 (amino acids)) and H564 scFv-Ig fusion proteins in stable CHO cell lines This example illustrates the expression of various Ig fusion genes described herein in eukaryotic cell lines and the characterization of the expressed fusion proteins by SDS-PAGE and IgG sandwich ELISA.

[0183] The -Ig fusion gene fragment with the correct sequence was inserted into the mammalian expression vector pDG, and DNA from positive clones was amplified using a QIAGEN plasmid preparation kit (QIAGEN, Valencia, Calif.). The recombinant plasmid DNA (100 μg) was then linearized in the nonessential region by digestion with AscI, purified by phenol extraction, and resuspended in tissue culture medium Excell 302 (catalog no. 14312-79P, JRH Biosciences, Lenexa, Kans. / SAFC). CHO DG44 cells, used for transfection, were maintained in logarithmic growth and were incubated for 10 min. 7 100 cells were harvested for each transfection reaction. Linearized DNA was added to CHO cells in a total volume of 0.8 ml for electroporation.

[0184] Stable production of the RNase-Ig fusion protein was achieved by electroporation of the selectable, amplifiable plasmid pDG, containing the RNase-Ig cDNA under the control of the 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 to prepare 100-μg RNase-Ig fusion proteins. 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 -Ig fusion protein 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) in PBS. Plates were blocked in PBS / 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. 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 T75 flasks to obtain sufficient numbers of cells for freezing and for scale-up of fusion protein production. Production levels in cultures from the top four excellent clones were further increased by progressive amplification in methotrexate-containing medium. With each successive passage of the cells, increasing concentrations of methotrexate were included in Excell 302 complete medium, ensuring that only cells in which the DHFR plasmid had been amplified would survive. Production levels in the top four unamplified master wells from RNaseIg CHO transfectants ranged from 30 to 50 micrograms / ml of culture. Amplified cultures are currently being assayed to determine production levels.

[0185] Supernatants from CHO cells expressing RNase-Ig were collected and filtered through a 0.2 μm PES express filter (Nalgene, Rochester, NY) before being 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, along with a blank using 0.1 M citrate buffer, pH 3.0. Fractions containing the fusion protein were pooled and buffer exchanged to reduce the risk of endotoxin contamination by sequential spins in PBS using a centricon concentrator followed by filtration through a 0.2 μm filter device. Using the protein analysis tool in the Vector Nti Version 10.0 software package (Informax, North Bethesda, Md.) and the predicted cleavage site from the online ExPasy protein analysis tool, the extinction coefficient was determined to be 1.05.

[0186] Example 5: SDS-PAGE analysis of RNaseIg fusion proteins Purified RNase-Ig (SEQ ID NO:115) was analyzed by electrophoresis on an SDS-polyacrylamide gel. Fusion protein samples were boiled in SDS sample buffer with and without disulfide bond reduction and applied to an SDS 10% Tris-BIS gel (catalog no. NP0301, Novex, Carlsbad, Calif.). Five micrograms of each purified protein were loaded onto the gel. After electrophoresis, proteins were visualized by Coomassie blue staining (Pierce Gel Code Blue Stain Reagent, catalog no. 24590, Pierce, Rockford, Ill.) and destaining in distilled water. Molecular weight markers were included on the same gel (Kaleidoscope Prestained Standards, catalog no. 161-0324, Bio-Rad, Hercules, Calif.). Other samples were run as follows: RNase-Ig fusion protein in sampling buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, with and without 5% 2-mercaptoethanol) was loaded onto a 4-12% precast gel (Bio-RAD). The gel was run at 100 volts until the dye had run off the gel. The gel was stained in GelCode Blue (Thermo Scientific) overnight at room temperature, followed by a water wash.

[0187] Figure 3 shows the RNase-Ig fusion protein compared to mouse IgG. RNase-Ig was purified from the supernatant of transfected CHO cells by binding to and elution from Protein A Sepharose. SDS-PAGE gels show that RNase-Ig is approximately 50 kDa when reduced and approximately 110 kDa when not reduced.

[0188] Example 6: Detection of RNase-Ig in mouse serum SRED assay A 2% agarose gel was prepared using distilled water. Poly-IC (Sigma) was dissolved in distilled water at 3 mg / ml, and 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 of each serum sample was loaded into the well, and the gel was incubated at 37°C for 4 hours in a humidified chamber. The gel was then incubated on ice in buffer (20 mM sodium acetate pH 5.2, 20 mg / ml ethidium bromide) for 30 minutes, and read under UV light.

[0189] Figure 4 shows RNase activity in three mice (410, 413, and 418) after intravenous injection of RNase-Ig fusion protein (SEQ ID NO:150) (purified in this experiment from the supernatant of transfected COS cells by binding to and elution from Protein A Sepharose). Standards were used in the top row. Note the second injection two weeks later for mouse 410 (see arrow). Two microliters of serum from each of the three mice was loaded onto a 1% agarose gel containing 0.5 mg / ml poly-C. The gel was incubated at 37°C for 4 hours in a humidified chamber and then immersed in a buffer containing 20 mM sodium acetate and 20 μg / ml ethidium bromide for 30 minutes. RNase activity is reflected by the size and intensity around the central well. This data indicates that the RNase-Ig fusion protein has an extended half-life in mouse serum.

[0190] Example 7: Use of anti-RNA ELISA to measure RNA-specific antibodies in mouse serum 96-well plates (Nunc, Thermal Fisher Scientific) were coated overnight with 50 μg / ml poly-L-lysine (Sigma). After washing five times with 0.05% Tween in PBS, the plates were coated overnight at 4°C with 10 μg / ml yeast RNA in PBS. After washing five times, the plates were blocked with 1% BSA in PBS at room temperature for 2 hours. Serum samples diluted 1:50 were added to the plates and incubated overnight at 4°C. Hybridoma H564 (anti-RNA) medium was used as a standard, using two-fold serial dilutions starting from 1:300. The detection antibody was alkaline phosphatase-conjugated anti-mouse IgG (Jackson Lab), which was added to the plates at 1:5000 and left at room temperature for 1 hour. Phosphatase substrate (Sigma) was dissolved in developing buffer (ThermoFisher Scientific) and added to the plates at 50 μl / well. Samples were read at 405 nm using a Spectramax Plus plate reader (Microdevices, Sunnyvale, CA).

[0191] Figure 5 shows the results of ELISA titers of anti-RNA antibodies in mouse 410 before and after intravenous injection of RNase-Ig fusion protein (SEQ ID NO:150). Pre-coated poly-L-lysine (50 μg / ml) plates were coated with 10 μg / ml yeast RNA. Serum (1:50) was loaded onto the plates and incubated overnight at 4°C. The detection antibody was anti-mouse IgG-alkaline phosphatase (Jackson Labs) at 1:5000 for 1 hour at room temperature, followed by addition of phosphatase substrate and reading at 405 nm. The data show that injection of RNase-Ig caused a decrease in anti-RNA antibody titers that persisted over three weeks.

[0192] Figure 6 shows the results of ELISA titers of anti-RNA antibodies in mouse 413 within three weeks before and after injection of RNase-Ig fusion protein (SEQ ID NO:150). The experiment was performed as described for mouse 410. The titers of anti-RNA antibodies decreased after injection of RNase-Ig.

[0193] Example 8: IFN-α production by human PBMC is inhibited by the addition of RNase Ig to in vitro cultures Addition of RNase-Ig (SEQ ID NO:150) abolished the induction of interferon-α from human peripheral blood mononuclear cells stimulated with immune complexes formed by serum plus nuclear extract (NE) from an SLE patient (J11). Briefly, ELISA plates were coated with 50 microliters of 1:2500 capture antibody (anti-IFNα, PBL 21112-1, Piscataway, NJ) and incubated overnight at 4°C. Plates were washed with PBS / 0.05% Tween 20, blocked in PBS / 1% BSA for 2 hours at room temperature, washed with PBS / 0.05% Tween-20, and incubated with a standard dilution of IFN-α or serial dilutions of serum samples for 2 hours at room temperature. Plates were washed and incubated with 1:2000 detection antibody (PBL 31101-2, Piscataway, NJ) in PBS / 1% BSA. Plates were washed with PBS / 0.05% Tween-20 and incubated with 50 microliters of donkey anti-rabbit HRP (Jackson Immunoresearch, Westgrove, PA) at 1:12,000 in PBS / 1% BSA. Plates were washed five times, followed by the addition of TMB substrate. The reaction was stopped by the addition of 1 / 2 volume of 2N H2SO4, and samples were read at 450 nm on a Spectramax Pro plate reader (MicroDevices, Sunnyvale, CA). The results, shown in Figure 7, demonstrate that the addition of RNase-Ig abolished the induction of interferon-α from human peripheral blood mononuclear cells stimulated with immune complexes formed by serum plus nuclear extract from an SLE patient (J11).

[0194] Example 9: 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 (see Figure 8). We developed a single radial diffusion (SRED) assay (left panel) to quantify RNase in serum, as well as a much more quantitative ELISA (see Figure 9). We crossed RNase A Tg mice with TLR7.1 Tg mice to generate double Tg (DTg) mice. 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 mice and littermate controls to 3 months of age to determine whether DTg mice showed signs of improvement. As shown in Figure 8, 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). RNaseA concentrations in Tg and DTg mice were also measured by ELISA assay, as shown in Figure 9. RNaseA Tg mice and TLR7.1 x RNaseA Dtg mice had serum concentrations of 1-2 ng / ml of RNaseA.

[0195] Detailed Method for RNase A ELISA (Example 9, Figure 9) 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.

[0196] 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

[0197] Example 10. 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 10, at 10 months, 61% of TLR7.1 mice died, compared with 31% of DTg mice. This data indicates 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 deposition of IgG and C3 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 live longer than their single Tg TLR7.1 littermates and have less inflammation and injury in their kidneys.

[0198] Example 11. Analysis of IRG in the spleen 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 significantly lower expression of the IRF7 gene in DTg mice (p = 0.03). Several other IRGs, including MX1 and VIG1, were lower in DTg mice compared to Tg mice, but the difference was not significant (see Figure 11). Quantitative PCR was performed as follows: total RNA was isolated from mouse spleens using the RNeasy mini kit (Qiagen, Valencia, CA, USA), DNase-treated using Turbo DNA-free (Applied Biosystems, Foster City, CA, USA), and first-strand cDNA was generated using random primers with the RNA-to-cDNA kit (Applied Biosystems). The 260 / 280 values ​​for the isolated RNA were between 1.7 and 2.0, as measured using a NanoDrop (Thermo Scientific, Waltham, MA, USA). cDNA was diluted to the equivalent of 1 ng / ul total RNA, and 8 ul was used 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 of the primers showed specific sequence homology to the reference gene or GOI alone. Duplicate reactions (20 ul) were run on an ABI Fast7500 system using a 1:1 mixture of template and primers in 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. -ddCTRelative 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.

[0199] Example 12. Constructs for creating 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 12. Primers are listed in Table 1. Nucleotide and amino acid sequences of representative hybrid nuclease molecules are listed in Table 2.

[0200] A general approach for the construction of hybrid nuclease molecules. 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).

[0201] Mouse spleen RNA was isolated from approximately 5 × 10e6 splenocytes using the QIAgen RNAeasy kit (Valencia, CA). Cells were pelleted from the medium by centrifugation, and 5 × 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.

[0202] 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 12 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.

[0203] 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.

[0204] 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 to prepare 10 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.

[0205] 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.

[0206] Example 13. Enzyme kinetic analysis for hRNase1-G88D-hIgG1[SCCH-P238S-K322S-P331S] Human RNase 1 sequences were isolated from human pancreatic RNA by random-primed cDNA and PCR amplification as described for the nuclease molecule in Example 12. The following primers from the primer set listed in the PCR primer table were used at 50 pmol per reaction: TIFF0007657249000011.tif13129

[0207] A mutant form of human RNase G88D was created using the following two primers in a PCR reaction and overlap PCR reaction to introduce a mutation at position 88 that alters the resistance of the enzyme to cytoplasmic inhibitors: TIFF0007657249000012.tif13128

[0208] Both wild-type and mutant human RNase 1 were isolated and cloned as described above for the hybrid nuclease molecules. The wild-type sequence was cloned using the first two primers listed above. The RNase fragment was TOPO cloned and sequenced, and the AgeI-XhoI cassette was transferred into a pDG expression vector already containing the human VK3LP insert and human IgG1-WT cassette. The constructs were verified by digestion, and plasmid DNA was prepared for transient transfection. After confirming functionality in small-scale transient transfections, these molecules were stably transfected into CHO DG44 cells for expression in sufficient quantities for further in vitro analysis. The wild-type human RNase 1 fusion protein is shown in Table 2 and is hVK3LP-hRNase 1-WT-hIgG1-WT (SEQ ID NO:163). Similarly, wild-type human RNase 1 was also expressed as a fusion gene with a (gly4ser)4 (SEQ ID NO:125 or SEQ ID NO:161) or (gly4ser)5 (SEQ ID NO:126 or SEQ ID NO:162) linker domain inserted between the hRNase cassette and the hIgG1 Fc domain. As listed in Table 2, the G88D mutant of human RNase 1 was also expressed as a fusion gene designated hVK3LP-hRNase-G88D-hIgG1-WT (SEQ ID NO:124 or 160) or hIgG1-SCCH-P238S-K322S-P331S (SEQ ID NO:174 or 175).

[0209] A Lineweaver-Burk plot of the enzyme kinetics for mutant hRNase 1-G88D-hIgG1 [SCCH-P238S-K322S-P331S] (SEQ ID NO: 175) is shown in Figure 13. To further characterize the functional properties of this bivalent RNase-Ig fusion protein, we performed a preliminary determination of the Michaelis constant, Km. The enzyme kinetics of purified human RNase 1-Ig fusion protein was assayed using RNase Alert Substrate (Ambion / IDT, San Diego, CA) according to the manufacturer's instructions, and fluorescence was assayed using a Spectramax M2 microplate reader (Molecular Devices, Sunnyvale, CA). Fluorescence data were collected at 30-second intervals over the course of a 30-minute incubation and analyzed using SoftmaxPro software (Molecular Devices). Reaction rates at various substrate concentrations were measured, and the data are presented in the form of a Lineweaver-Burk plot.

[0210] Example 14. Analysis of hRNase1-hIgG binding to human monocyte lines To assess FcR-mediated binding of wild-type or mutant Fc-containing molecules, the protein A-purified hybrid nuclease molecule hRNase1-hIgG1-WT was incubated with the human monocytic cell lines THP-1 and U937. Figure 14 shows the binding pattern of hRNase1-WT-hIgG1-WT (SEQ ID NO:161) to these two cell lines. Cells were incubated with 5 μg / ml of purified fusion protein in PBS / 2% FBS for 45 minutes on ice, washed three times in PBS / 2% FBS, and incubated with 1:200 FITC-goat anti-human IgG (Fc-specific) (Jackson Immunoresearch, West Grove, PA) for 45 minutes on ice. Cells were washed twice in PBS / 2% FBS and analyzed by flow cytometry using a FACS Canto (BD, Franklin Lakes, NJ) flow cytometer and FlowJo software (TreeStar, Ashland, OR).

[0211] Example 15. Blockade of hRNase1-hIG1 binding to a human monocyte line by IVIg THP-1 or U937 cells were preincubated with IVIg in 10-fold serial dilutions across the wells of a 96-well plate, starting at 10 mg / ml. Cells (approximately 1 x 10e6 per well) were incubated on ice for 45 minutes. Prebound cells were washed twice, and AF750-conjugated hRNase1-WT-hIgG1-WT (SEQ ID NO: 161) was added to each well at approximately 5 μg / ml. Binding reactions were incubated on ice for 45 minutes, washed twice in PBS / 2% FBS, and analyzed by flow cytometry as described above. IVIg was able to partially block binding of the labeled nuclease fusion protein; however, even at 10 mg / ml, residual binding above background was still detectable. Figure 15 shows the blocking activity of human IVIg against hRNase1-WT-hIgG1-WT (SEQ ID NO: 161) binding to U937 and THP-1 cells.

[0212] Example 16. Trex1-Ig ​​activity assay Mouse Trex1 was cloned from mouse cDNA using the primers listed below: TIFF0007657249000013.tif13133

[0213] PCR reactions were performed using 50 pmol of each primer in a total volume of 50 μl, with an amplification profile of 35 cycles of 94°C for 30 seconds; 50°C for 60 seconds; and 68°C for 90 seconds. PCR products were cloned into the pCR2.1 vector and TOPO clones were screened as described above for the cloning of prototype nuclease fusion genes. After sequence verification, the cassettes were subcloned into the pDG expression vector fused with mIgG tails or cocloned with one of the (g4s)n linkers to construct Trex1-lnk molecules with various linker lengths. Plasmid isolates were transiently transfected into COS cells as described above, and stable CHO transfectants were generated, as described for the prototype nuclease fusion genes.

[0214] Fusion genes encoding Trex1Ig were constructed as follows: these genes incorporate the human VK3 leader peptide fused to a COOH-terminally truncated mouse Trex1 by 72 amino acids (to remove the intracellular nuclear targeting sequence), fused to a (gly4ser)4 (SEQ ID NO:130) or (gly4ser)5 linker (SEQ ID NO:131), and fused to a mouse IgG2a / c allele that incorporates several changes compared to the IgGc sequence of the Balb / c IgG2a allele.

[0215] The exonuclease activity of Trex1-Ig ​​was measured in a 30 μl reaction mixture containing 20 mM Tris (pH 7.5), 5 mM MgCl2, and 2 mM DTT using a 36-mer oligonucleotide as a substrate. The incubation reaction proceeded for 20–30 minutes at 37°C. Samples were electrophoresed overnight on a 23% polyacrylamide DNA gel. The gel was incubated in TBE buffer containing 0.5 μg / ml ethidium bromide. DNA was visualized using a UV transilluminator, and photographs were taken using a Kodak EDAS 290 digital camera equipped with an ethidium bromide filter and analyzed using Kodak Molecular Imaging software. The results of the trex1 activity assay for COS that produced mTrex1-(g4s)4-mIgG2a-c (SEQ ID NO:166) and mTrex1-(g4s)5-mIgG2a-c (SEQ ID NO:167) are shown in FIG. 16.

[0216] Example 17. Western blot of mTrex1-Ig ​​single hybrid nuclease molecules produced by transient transfection of COS-7 COS-7 cells were transiently transfected with a plasmid containing a hybrid nuclease molecule encoding Trex1-Ig: these genes incorporate a human VK3 leader peptide fused to a 72-amino acid COOH-terminal truncation of mouse Trex1 (to remove the nuclear targeting sequence), fused to a (gly4ser)4 or (gly4ser)5 linker, and fused to a mouse IgG2a / c allele that incorporates several changes compared to the IgGc sequence of the Balb / c IgG2a allele. COS supernatants were harvested 72 hours later, and 0.5–1.0 ml samples (depending on the experiment) were immunoprecipitated with 100 μl of protein A-agarose beads overnight at 4°C. The protein A beads were centrifuged, washed twice in PBS, and resuspended in reducing SDS-PAGE loading buffer. Samples were heat-treated at 100°C for 5 minutes, the Protein A beads were pelleted by centrifugation, and the sample buffer was loaded onto a 10% SDS-PAGE 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. Blots were incubated with 1:2500 HRP (horseradish peroxidase)-conjugated goat anti-mouse IgG2a / c (Fc specific, KPL) at room temperature for 1.5 hours, washed five or more times in PBS / 0.5% Tween 20, and then developed with ECL reagent. FIG. 17 shows a Western blot of immunoprecipitates from COS7 culture supernatants expressing mTrex1-(g4s)4 (SEQ ID NO: 166) or (g4s)5-mIgG2a-c (SEQ ID NO: 167) fusion proteins.

[0217] Example 18. Exonuclease activity of DNase1L3Ig CHO-derived fusion protein DNase1L3 was cloned from mouse spleen cDNA using the following primer pair to clone mDNase1L3 including its native leader peptide sequence: TIFF0007657249000014.tif28136

[0218] Alternatively, a PCR reaction was set up using the following primer pair to attach the human VK3 leader peptide in place of the native leader: TIFF0007657249000015.tif28137

[0219] PCR reactions were performed using 50 pmol of each primer in a total volume of 50 μl, with an amplification profile of 35 cycles of amplification at 94°C for 30 seconds; 50°C for 60 seconds; and 68°C for 90 seconds. PCR products were cloned into the pCR2.1 vector and TOPO clones were screened as described above for the cloning of prototype nuclease fusion genes. After sequence verification, the cassette was subcloned into the pDG expression vector fused to an mIgG tail. Plasmid isolates were transiently transfected into COS cells as described above, and stable CHO transfectants were generated, as described for the prototype nuclease fusion genes.

[0220] Exonuclease activity in protein extracts from the DNase1L3Ig (SEQ ID NO:185) CHO clone was measured in 30 μl reactions containing 20 mM Tris (pH 7.5), 5 mM MgCl2, 2 mM DTT, and substrate. Incubation was at 37°C for 20–30 minutes. Samples were then run overnight on an agarose DNA gel. The gel was incubated in TBE buffer containing ethidium bromide. DNA was visualized under UV light. The results of the chromatin digestion analysis are shown in Figure 18.

[0221] Example 19. Dose titration of exonuclease activity with increasing volumes of CHO supernatant Figure 19 shows a titration analysis of exonuclease digestion patterns obtained from COS supernatants expressing DNase1L3Ig fusion proteins (SEQ ID NO: 183 or 185). Nuclear DNA degradation assays were performed as follows: HeLa cells were cultured in DMEM medium, and nuclei were isolated from 10e5 cells using NP-40 lysis. Nuclei were diluted in 200 μl of reaction buffer containing 10 mM Hepes (pH 7.0), 50 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, and 40 mM b-glycerophosphate. Nuclei were incubated in the indicated volume of culture supernatant from COS cells transfected with DNase1L3 for 3 hours at 37°C. Nuclear DNA was isolated using a QiAmp blood DNA minikit. DNA was analyzed by 1.5% agarose gel electrophoresis. For control reactions, heparin was used at 250 i.u. / ml to inhibit nuclease activity.

[0222] Example 20. 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.

[0223] 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).

[0224] 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).

[0225] 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.

[0226] 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: TIFF0007657249000016.tif28137

[0227] Alternatively, the 3'DNase cassette was amplified by PCR using the following primer pair: TIFF0007657249000017.tif28136

[0228] 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).

[0229] 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 (SEQ ID NOS: 118, 119, 120, 121, 122, or 123) or RNase-Ig-DNase (SEQ ID NOS: 115, 116, or 117) 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, and the liquid was transferred to a new tube.

[0230] COS-7 cells were transiently transfected with plasmids containing either wild-type human DNase 1 (SEQ ID NO: 118) or naturally occurring DNase 1 mutant alleles (G105R and / or A114F) (SEQ ID NOs: 115, 116, or 117) fused to the wild-type human IgG1 Fc domain. This hinge-CH2-CH3 cassette contains a single C→S mutation in the hinge region that eliminates the first cysteine ​​in this domain, 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. Western blot analysis was performed on supernatants from transient transfectants. The molecules shown in Figure 20 contain either human DNase 1 fused to the human IgG1 wild-type Fc domain (SEQ ID NO: 154, 155, 156, or 159), or human RNase 1 (wild-type) fused to the SCC hinge-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 either wild-type (SEQ ID NO: 153) or mutant allele (SEQ ID NO: 151 or 152) human DNase 1 at the carboxy terminus of the molecule. COS supernatants were harvested after 72 hours, and 0.5-1.0 ml samples (depending on the experiment) were immunoprecipitated with 100 μl of protein A-agarose beads overnight at 4°C. Protein A beads were centrifuged, washed twice in PBS, and resuspended in SDS-PAGE loading buffer for NuPAGE gels, either reducing or non-reducing LDS sample buffer. 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.Blots were incubated with 1:2500 HRP (horseradish peroxidase)-conjugated goat anti-human IgG (Fc-specific, Jackson Immunoresearch) or goat anti-mouse IgG at room temperature for 1.5 hours, washed five or more times in PBS / 0.5% Tween 20, and developed with ECL reagent.

[0231] Example 22. Screening of COS supernatants for nuclease enzyme activity FIG. 21 shows the results of SRED-mediated RNase activity assay (SRED) analysis on harvested COS supernatants expressing hDNase 1Ig and hRNase 1-Ig-hDNase 1 fusion protein.

[0232] COS supernatants from transient transfections of hDNaseIg mono- or multispecific nucleases were assayed for nuclease activity as follows. 2% agarose gels were prepared using distilled water. Poly-C (Sigma) was dissolved in distilled water at 3 mg / ml, and gel plates were 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-C, and 0.5 ml of water were placed in a tube and maintained at 50°C for 5 minutes. 3 ml of agarose (kept 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. Approximately 2 μl of each sample was loaded, and the gel was incubated at 37°C for 4 hours in a humidified chamber. The gel was then incubated on ice for 30 minutes in buffer (20 mM sodium acetate pH 5.2, 20 mg / ml ethidium bromide). Photographs of the gel on a UV transilluminator were taken using a Kodak DC290 digital camera equipped with an ethidium bromide filter and analyzed using Kodak Molecular Imaging software.

[0233] Figure 22 shows a composite diagram displaying the results of a DNase nuclease activity assay performed on COS supernatants from transfected cells. Culture supernatants were harvested 72 hours after transfection with the following clones of DNase 1 wild-type and mutant Ig fusion proteins: (1) 090210-8 = hDNase 1-WT-hIgG1 WT (SEQ ID NO: 154); (2) 090210-9 = hDNase 1-G105R;A114F-hIgG1 WT (SEQ ID NO: 159); (3) 091210-8 = hRNase 1-WT-hIgG1-WT-DNase 1-G105R;A114F (SEQ ID NO: 151), and (4) 091210-14 = hRNase-WT-hIgG1-WT-DNase 1-A114F (SEQ ID NO: 152).

[0234] To facilitate binding of the expressed -Ig fusion protein to Protein A agarose beads, the pH of the supernatant was adjusted to 0.0 with bicarbonate buffer. Panel A of Figure 23 shows a gel electrophoresis analysis of the plasmid DNA digestion: Protein A agarose slurries (50 μl per sample) were washed in PBS and incubated overnight at 4°C with 100 μl of culture supernatant for immunoprecipitation of -Ig fusion proteins. The immunoprecipitates were washed 4-5 times in 750 μl of PBS and centrifuged at approximately 3500 rpm, after which the PBS was aspirated. The final Protein A precipitate was resuspended in 50 μl of reaction buffer containing 20 mM Tris pH 7.5, 2 mM CaCl2, and 2 mM MgCl2, containing 1.5 μg of plasmid DNA (pDG expression vector). The reactions were incubated at 37°C for 30 minutes, heated to 65°C for 5 minutes, and the DNA present in the reactions was analyzed by agarose gel electrophoresis on a 1.5% TBE-agarose gel.

[0235] Panel B shows the results of a nuclease activity assay performed on the same culture supernatant using the DNase Alert Kit (IDT / Ambion). A reaction tube containing lyophilized DNase Alert Substrate (50 pmol) was resuspended with 5 μl of nuclease-free ddH2O and 5 μl of 10x DNase Alert buffer provided with the kit, and 40 μl of Protein A slurry was immunoprecipitated as follows: For these immunoprecipitations, 50 μl of Protein A agarose beads were incubated overnight with 50 μl of culture supernatant. The samples were then washed five times with 0.75 ml of PBS. The final Protein A precipitate was resuspended in 80 μl of nuclease-free ddH2O, and 40 μl of the slurry (half of the precipitate) was transferred to the reaction tube. Negative controls using mock-transfected IP and ddH2O were also set up. A positive control was also set up, containing DNase 1 (2 units) provided with the kit. The reactions were incubated at 37° C. for 1 hour and exposed to short-wave UV transillumination to visualize fluorescence. The relative amount of DNA digestion is indicated by the degree of fluorescence.

[0236] Example 22. Examination of mac-2 positive cells in DTg mice Death from early lupus usually results from nephritis or infections resulting from immunosuppression used to treat nephritis. Therefore, a critical outcome for any new treatment is amelioration of nephritis. While human studies are limited to quantification of proteinuria and creatinine, accurate assessment of renal inflammation and damage can be obtained in mice through histology and immunocytochemistry. We have reported that TLR7.1 × RNase double transgenic (DTg) mice exhibit fewer anti-RNA antibodies, reduced B cell activation, fewer immune deposits, and fewer glomeruli staining positively with PAS. We further compared renal macrophage infiltration using anti-Mac-2 (galectin 3) antibodies (Iyoda et al. Nephrol Dial Transplant 22: 3451, 2007). Frozen sections from kidneys obtained from single-transgenic and double-transgenic mice were examined for the number of Mac-2-positive macrophages and glomerular size as previously described (Iyoda et al.). Twenty randomly selected glomeruli (from the outside to the inside of the kidney) were counted for positive cells. MAC-2-positive staining cells in glomeruli were significantly fewer in DTg mice compared with single Tg mice (data not shown). Results of a pilot study with n = 4-5 in each group, counting 20 glomeruli per mouse, revealed a mean ± SE of 3.8 ± 1.1 and 1.4 ± 0.2 for single Tg and DTg, respectively, p = 0.05. In addition, we quantified glomerular tuft size and observed a significant decrease in glomerular tuft size in DTg mice (179.4 ± 41 and 128 ± 16.8 μm2 for single Tg and DTg, respectively, p = 0.037).

[0237] Example 23. Km of purified mouse RNase A-Ig fusion protein To further characterize the functional properties of the bivalent RNase-Ig fusion protein (SEQ ID NO:150), we determined the Michaelis constant (Km). As shown in Figure 23, the enzyme has high affinity, with a tentative Km of 280 nM (compared to the Km of 34 nM for RNase A using polyC as a substrate (delCardayre et al., Prot Eng 8:261, 1995)). Figure 23 shows the enzyme kinetics assayed with RNase Alert Substrate (Ambion / IDT) and quantified using a Spectramax M2 microplate reader. Data were analyzed using Softmax Pro software (Molecular Devices). Reaction rates were measured at various substrate concentrations, and the data are presented as Lineweaver-Burk plots. The apparent Km, corrected for volume, is 280 nM.

[0238] Example 24. Analysis of 564Igi Tg mice for anti-RNA antibodies 564 Igi Tg mice: Dr. Imanishi-Kara rearranged the VDJ genes from the H564 hybridoma within the endogenous Igh and Igk loci to generate 564Igi mice on a B6 background. Serum from these mice stained the cytoplasm and nucleoli of fixed cells, indicating a pronounced anti-RNA specificity. Consistent with this finding, and particularly relevant to this patent application, rendering these mice TRL7 deficient suppressed antibody production, indicating that RNA is indeed the stimulator of antibody production. This mouse strain develops late-onset glomerulonephritis. We analyzed the expression of anti-RNA antibodies in mice transgenic for H564, as well as in double transgenic mice coexpressing the 564Ig transgene and the RNase transgene. Figure 24 compares the levels of anti-RNA antibodies in mouse serum at successive intervals as these transgenic mice age.

[0239] Gavalchin , J. , Seder RA , and SK Datta . 1987. The NZB X SWR model of lupus nephritis. I. Cross-reactive idiotypes of monoclonal anti-DNA antibodies in relation to antigenic specificity, charge, and allotype. Identification of interconnected idiotype families inherited from normal SWR and autoimmune NZB parents. J. Immunol. 138:128-137;and Berland, R., L. Fernandez, E. Kari, JH Han, I. Lomakin, S. Akira, HH Wortis, JF Kearney, AA Ucci, and T. Imanishi-Kari. 2006. Toll-like receptor 7-dependent loss of B cell tolerance in pathogenic autoantibody knockin mice. Immunity 25:429–440.

[0240] Example 25. In vitro evaluation of the biological activity of hybrid nuclease molecules The one or more hybrid nuclease molecules are purified, for example, by affinity chromatography or ion exchange chromatography, as previously described in the Examples above. In some cases, the hybrid nuclease molecule is a polypeptide. In some cases, the hybrid nuclease molecule comprises one or more sequences from Table 2. In some cases, the molecule is SEQ ID NO: 161, 162, or 163. In some cases, the molecule comprises SEQ ID NO: 145 and SEQ ID NO: 149. In some cases, the molecule is SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 166, 167, 169, 170, 171, 173, 175, 177, 179, 181, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205 or 207. The hybrid nuclease molecule can be any of those disclosed herein and any that can be constructed from the sequences disclosed herein (see Table 2), e.g., by taking a nuclease domain and linking it to an Fc domain; or by taking a nuclease domain and linking it to an Fc domain with a linker domain, e.g., Various linker domains (e.g., those described herein) can be used to link the Fc domain and / or nuclease domain. For example, linker domains that are 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 confirm that they have the desired nuclease function. Specific activity is then typically determined by a fluorescence-based kinetic assay using a substrate, such as an RNase or DNase Alert Kit reagent, and a fluorescent plate reader configured to read as a function of time.In addition, protein solutions are typically tested for endotoxin contamination using commercially available kits, such as the Pyrotell Limulus Amebocyte Lysate (LAL) kit from Cape Cod, Inc. (E. Palmouth, MA), which has a detection limit of 0.06 EU / ml. Molecules are then assayed using a variety of in vitro assays for biological activity.

[0241] 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).

[0242] 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.

[0243] 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% PBS 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.

[0244] 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.

[0245] 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 described above (e.g., cytokines, cell surface receptors, proliferation) generally improve in the group treated with the effective molecule compared to the marker levels present before treatment or compared to the levels measured in the control group.

[0246] Example 26: Administration of hybrid nuclease molecules to a mammal in need thereof A mammal (e.g., mouse, rat, rodent, human, guinea pig) is used in the test. The mammal is administered (e.g., intravenously) one or more hybrid nuclease molecules comprising one or more sequences from Table 2, or a control. In some cases, the molecule is SEQ ID NO: 161, 162, or 163. In some cases, the molecule comprises SEQ ID NO: 145 and SEQ ID NO: 149. In some cases, the molecule is SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 166, 167, 169, 170, 171, 173, 175, 177, 179, 181, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205 or 207. The hybrid nuclease molecule can be any of those disclosed herein and any that can be constructed from the sequences disclosed herein (see Table 2), e.g., by taking a nuclease domain and linking it to an Fc domain; or by taking a nuclease domain and linking it to an Fc domain with a linker domain, e.g., Various linker domains (e.g., those described herein) can be used to link the Fc domain and / or nuclease domain. For example, linker domains that are 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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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.

[0255] 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.

[0256] [Table 1] TIFF0007657249000019.tif241158TIFF0007657249000020.tif67158

[0257]

Table 2

[0258] Array information SEQUENCE LISTING <110> UNIVERSITY OF WASHINGTON <120> THERAPEUTIC NUCLEASE COMPOSITIONS AND METHODS <150> US 61 / 370,752 <151> 2010-08-04 <150> US 61 / 257,458 <151> 2009-11-02 <160> 223 <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 oligonucleotide <400> 4 aaatctagac ctcaaccagg tagggaatct gcagcacaga agtttcag 48 <210> 5 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <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> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 16 accggtaagg aatcccgggc caagaaattc c 31 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 17 ctcgagatct gtagagtcct ccacagaagc atcaaagtgg 40 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 18 agactgccgc ctgacaaacg actccaggta ccc 33 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 19 gggtacctgg agtcgtttgt caggcggcag tct 33 <210> 20 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 20 accggtatgg gctcacagac cctgccccat ggtcaca 37 <210> 21 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 21 ctcgagatct gttgttccag tggtagccgg agtgccgtac atg 43 <210> 22 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 22 gttaagcttg ccaccatgtc cctgcaccca gcttccccac gcctg 45 <210> 23 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 23 ctcgagatct gaggagcgat tgcctttttt tctctttttg agag 44 <210> 24 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 24 accggtctaa ggctctgctc cttcaatgtg aggtcctttg ga 42 <210> 25 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 25 ctcgagatct gaggagcgat tgcctttttt tctctttttg agag 44 <210> 26 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 26 gttaccggtc tgaagatcgc agccttcaac atccag 36 <210> 27 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 27 gttctcgaga tctttcagca tcacctccac tggatagtg 39 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 28 gttgatatcc tgaagatcgc agccttcaac atccag 36 <210> 29 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 29 gtttctagat tatcacttca gcatcacctc cactggatag tg 42 <210> 30 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 30 tgtccaccgt gtccagcacc tgaactcctg ggtggatcgt cagtcttcc 49 <210> 31 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 31 agatctcgag cccaaatctt ctgacaaaac tcacacatgt ccaccgtgt 49 <210> 32 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 32 gaagatctcg agcccaaatc ttctgacaaa actcacacat gt 42 <210> 33 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 33 gttagatctc gagcccaaat cttctgacaa aactcacaca tct 43 <210> 34 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 34 tctagattat catttacccg gagacagaga gaggctcttc tgcgtgtagt g 51 <210> 35 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 35 aaggtctcca acaaagccct cccagcctcc atcgagaaaa caatctcc 48 <210> 36 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 36 gttttctcga tggaggctgg gagggctttg ttggagacc 39 <210> 37 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 37 aagcttgcca ccatggctct ggagaagtct cttgtccggc tcc 43 <210> 38 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 38 ctcgagatct gtagagtcct ccacagaagc atcaaagtgg 40 <210> 39 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 39 accggtaagg aatcccgggc caagaaattc c 31 <210> 40 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 40 gatatccctt ccctgggcaa ggaatcccgg gccaagaaat tccag 45 <210> 41 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 41 gtttctagat tattaggtag agtcctccac agaagcatca aagtg 45 <210> 42 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 42 ggtaagcttg ccaccatgtc acgggagctg gccccactgc tgctt 45 <210> 43 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 43 ctcgagatct gaggagcgtt tgctctttgt tttcttcctt ag 42 <210> 44 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 44 accggtatga ggatctgctc cttcaacgtc aggtcctttg g 41 <210> 45 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 45 gttaccggtc tgaagatcgc agccttcaac atccag 36 <210> 46 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 46 gttctcgaga tctttcagca tcacctccac tggatagtg 39 <210> 47 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 47 gttgatatcc tgaagatcgc agccttcaac atccag 36 <210> 48 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 48 gtttctagat tatcacttca gcatcacctc cactggatag tg 42 <210> 49 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 49 gatggctgcg agccctgcag gaacgacacc ttcaaccgag agccattcat tgtcaggttc 60 <210> 50 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 50 gaacctgaca atgaatggct ctcggttgaa ggtgtcgttc ctgcagggct cgcagccatc 60 <210> 51 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 51 ggagaagaac ctgacaatga atggctctcg gttgaaggt 39 <210> 52 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 52 accttcaacc gagagccatt cattgtcagg ttcttctcc 39 <210> 53 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 53 accggtatgg gccctggagc tcgcagacag ggcag 35 <210> 54 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 54 ctcgagatct ttggtcctag cagaggctgt gacc 34 <210> 55 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 55 accggtctcg agatgggccc tggagctcgc agacagg 37 <210> 56 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 56 ctcgagtttg gtcctagcag aggctgtgac c 31 <210> 57 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 57 accggtatgg gctcacagac cctgccccat ggtcaca 37 <210> 58 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 58 ctcgagatct gttgttccag tggtagccgg agtgccgtac atg 43 <210> 59 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 59 gttaagcttg ccaccatgtc cctgcaccca gcttccccac gcctg 45 <210> 60 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 60 ctcgagatct gaggagcgat tgcctttttt tctctttttg agag 44 <210> 61 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 61 gttaagcttg ccaccatggg tctggagaag tccctcattc tg 42 <210> 62 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 62 ggctcgagca cagtagcatc aaagtggact ggtacgtagg 40 <210> 63 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 63 cctccatgca aatgcccagc acctaacctc ttgggtggat catccgtctt catcttcc 58 <210> 64 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 64 agatctcgag cccagaggtc ccacaatcaa gccctctcct ccatgcaaat gcc 53 <210> 65 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 65 gaagatctcg agcccagagg tcccacaatc aagccctctc ctcca 45 <210> 66 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 66 atcaagccct ctcctccatc taaatcccca gcacctaac 39 <210> 67 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 67 agtggcaagg agttcaaatg ctcggtcaag aagaaagacc tcccagcgtc catcgag 57 <210> 68 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 68 ggttctctcg atggacgctg ggaggtcttt gttgttgacc gagcatttga actcc 55 <210> 69 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 69 gtttctagat tatcatttac ccggagtccg agagaagctc ttagtcgt 48 <210> 70 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 70 gctagctccg tcgactttac ccggagacag agagagg 37 <210> 71 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 71 gactggctga atggcaagga gtacaagtgc tcggtctcca acaaagccct c 51 <210> 72 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 72 gagggctttg ttggagaccg agcacttgta agacttgcca ttcagccagt c 51 <210> 73 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 73 ccgcgggagg agcagtacag cagcacgtac cgtgtggtca gcgtc 45 <210> 74 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 74 gacgctgacc acacggtacg tgctgctgta ctgctcctcc cgcgg 45 <210> 75 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 75 gatatctcta gatttacccg gagtccgaga gaagctctta gtcgt 45 <210> 76 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 76 gatatctccg gagtcgactt tacccggagt ccgagagaag ctcttag 47 <210> 77 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 77 cacaaaccca tagagaggat tacagcagta ctctccgggt ggtc 44 <210> 78 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primary <400> 78 gaccacccgg agagtactgc tgtaatcctc tctatgggtt tgag 44 <210> 79 <400> 79 000 <210> 80 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 80 gatatcaccg gtagaaccac ctccaccact cccacctcct ccagtgcctc c 51 <210> 81 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 81 gtcgactccg gaggaggtgg ctcaggtggt ggaggcagtg gaggaggtgg 50 <210> 82 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 82 aaagtcgacg gagctagcag ccccgtgaac gtgagcagcc ccagcgtg 48 <210> 83 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 83 cccatgatat cctgcacgct ggggctgctc 30 <210> 84 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 84 accggtatga ggatctgctc cttcaacgtc aggtcctttg g 41 <210> 85 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 85 agatctttat caggagcgtt tgctctttgt tttcttcctt ag 42 <210> 86 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 86 tctagattat caggagcgat tgcctttttt tctctttttg agag 44 <210> 87 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 87 accggtctaa ggctctgctc cttcaatgtg aggtcctttg ga 42 <210> 88 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 88 gataccaccg gtagggaatc tgcagcacag aagtttcag 39 <210> 89 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 89 aaatctagac ctcaaccagg tagggaatct gcagcacaga agtttcag 48 <210> 90 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 90 tctagactat cacacagtag catcaaagtg gactggtacg ta 42 <210> 91 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 91 agactgccgc ctgacaaacg actccaggta ccc 33 <210> 92 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 92 gggtacctgg agtcgtttgt caggcggcag tct 33 <210> 93 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 93 ggctcaggtg gtggaggatc tggaggaggt ggctcaggtg gtggaggatc tg 52 <210> 94 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 94 gttagatctc tccggaggag gtggctcagg tggtggagga tctgga 46 <210> 95 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 95 ctcgagactc ccacctcctc cagatcctcc accacctgag ccacct 46 <210> 96 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 96 aaagatctct ccggaggagg tggctcaggt ggtggaggat ctggaggagg 50 <210> 97 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 97 ctcgagaccg gtagaaccac ctccaccact cccacctcct ccagatcctc 50 <210> 98 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 98 gttagatctc tccggaggag gtggctca 28 <210> 99 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 99 accggtctcg agactcccac ctcctccaga tc 32 <210> 100 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 100 agatctctcc ggaggaggtg gctcaggtgg tggaggatct ggaggaggtg ggagtggtgg 60 aggtggttct accggtctcg ag 82 <210> 101 <211> 97 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 101 agatctctcc ggaggaggtg gctcaggtgg tggaggatct ggaggaggtg gctcaggtgg 60 tggaggatct ggaggaggtg ggagtaccgg tctcgag 97 <210> 102 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 102 agatctctcc ggaggaggtg gctcaggtgg tggaggatct ggaggaggtg gctcaggtgg 60 tggaggatct ggaggaggtg ggagtctcga g 91 <210> 103 <211> 462 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 103 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 taataatcta ga 462 <210> 104 <211> 798 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 104 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 agccctgcag gaacgacacc ttcaaccgag agccattcat tgtcaggttc 360 ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 420 gacgcagtag ccgagatcga cgctctctat gacgtctacc tggatgtcca agagaaatgg 480 ggcttggagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 540 tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 600 agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 660 atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 720 tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 780 ctgaagtgat aatctaga 798 <210> 105 <211> 798 <212> DNA <213> Homo sapiens <400> 105 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 agccagccat tgtcaggttc 360 ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 420 gacgcagtag ccgagatcga cgctctctat gacgtctacc tggatgtcca agagaaatgg 480 ggcttggagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 540 tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 600 agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 660 atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 720 tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 780 ctgaaatgat aatctaga 798 <210> 106 <211> 798 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 106 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 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 ctgaagtgat aatctaga 798 <210> 107 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 107 accggtctga agatcgcagc cttcaacatc cagacatttg gggagaccaa gatgtccaat 60 gccaccctcg tcagctacat tgtgcagatc ctgagccgct atgacatcgc cctggtccag 120 gaggtcagag acagccacct gactgccgtg gggaagctgc tggacaacct caatcaggat 180 gcaccagaca cctatcacta cgtggtcagt gagccactgg gacggaacag ctataaggag 240 cgctacctgt tcgtgtacag gcctgaccag gtgtctgcgg tggacagcta ctactacgat 300 gatggctgcg agccctgcag gaacgacacc ttcaaccgag agccattcat tgtcaggttc 360 ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 420 gacgcagtag ccgagatcga cgctctctat gacgtctacc tggatgtcca agagaaatgg 480 ggcttggagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 540 tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 600 agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 660 atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 720 tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 780 ctgaaagatc tcgag 795 <210> 108 <211> 795 <212> DNA <213> Homo sapiens <400> 108 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 agccctgcgg gaacgacacc ttcaaccgag agccagccat tgtcaggttc 360 ttctcccggt tcacagaggt cagggagttt gccattgttc ccctgcatgc ggccccgggg 420 gacgcagtag ccgagatcga cgctctctat gacgtctacc tggatgtcca agagaaatgg 480 ggcttggagg acgtcatgtt gatgggcgac ttcaatgcgg gctgcagcta tgtgagaccc 540 tcccagtggt catccatccg cctgtggaca agccccacct tccagtggct gatccccgac 600 agcgctgaca ccacagctac acccacgcac tgtgcctatg acaggatcgt ggttgcaggg 660 atgctgctcc gaggcgccgt tgttcccgac tcggctcttc cctttaactt ccaggctgcc 720 tatggcctga gtgaccaact ggcccaagcc atcagtgacc actatccagt ggaggtgatg 780 ctgaaagatc tcgag 795 <210> 109 <211> 795 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 109 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> 110 <211> 715 <212> DNA <213> Homo sapiens <400> 110 agatctcgag cccaaatctt ctgacaaaac tcacacatgt ccaccgtgcc cagcacctga 60 actcctgggg ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat 120 ctcccggacc cctgaggtca catgcgtggt ggtggacgtg agccacgaag acctgaggt 180 caagttcaac tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga 240 ggagcagtac aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg 300 gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga 360 gaaaaccatc tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc 420 atcccgggat gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta 480 tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac 540 cacgcctccc gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga 600 caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca 660 caaccactac acgcagaaga gcctctctct gtctccgggt aaatgataat ctaga 715 <210> 111 <211> 858 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 111 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> 112 <211> 918 <212> DNA <213> Homo sapiens <400> 112 atgtcacggg agctggcccc actgctgctt ctcctcctct ccatccacag cgccctggcc atgaggatct gctccttcaa cgtcaggtcc tttggggaa gcaagcagga agacaaga 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> 113 <211> 459 <212> DNA <213> Homo sapiens <400> 113 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> 114 <211> 1162 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 114 gttaagcttg ccaccatgga aaccccagcg cagcttctct tcctcctgct actctggctc ccagatacca ccggtaggga atctgcagca cagaagtttc agcggcagca catggatcca 180. gatggttcct ccatcaacag ccccacctac tgcaaccaaa tgatgaaacg ccgggatatg acaaatgggt catgcaagcc cgtgaacacc ttcgtgcatg agcccttggc agatgtccag 240 gccgtctgct cccaggaaaa tgtcacctgc aagaacagga agagcaactg ctacaagagc agctctgccc tgcacatcac tgactgccac ctgaagggca actccaagta tcccaactgt gactacaaga ccactcaata ccagaagcac atcattgtgg cctgtgaagg gaacccctac gtaccagtcc actttgatgc tactgtgctc gagcccagag gtctcacaat caagccctct 480 cctccatgca aatgcccagc acctaacctc ttggggtggat catccgtctt catcttccct 540 ccaaagatca aggatgtact catgatctcc ctgagcccca tggtcacatg tgtggtggtg gatgtgagcg aggatgaccc agacgtccag atcagctggt ttgtgaacaa cgtggaagta 660 cacacagctc agacacaaac ccatagagag gattacaaca gtactctccg ggtggtcagt 720 gccctcccca tccagcacca ggactggatg agtggcagg agttcaatg ctcggtcaac 780 aacaaagacc tcccagcgtc catcgagaga accatctca aacccagagg gccagtaaga 840 gctccacagg tatatgtctt gcctccacca gcagaagaga tgactaagaa agagttcagt 900 ctgacctgca tgatcacagg cttcttacct gccgaaattg ctgtggactg gaccagcaat 960 gggcgtacag agcaaacta caacaacc gcaacagtcc tggactctga tggttcttac 1020 ttcatgtaca gcaagctcag agtacaaag agcacttggg aagaggaag tctttcgcc 1080 tgctcagtgg tccacgaggg tctgcacaat caccttacga ctaagagctt ctctcggact 1140 cggggtaat gatatctag aa 1162 <210> 115 <211> 2004 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <220> <221> modified_base <222> (1921)..(1921) <223> a, c, t, g, unknown or other <400> 115 aagcttgccg ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtaaggaatc ccgggccaag aaattccagc ggcagcatat ggactcagac 120 agttccccca gcagcagctc cacctactgt aaccaaatga tgaggcgccg gaatatgaca 180 caggggcggt gcaaaccagt gaacaccttt gtgcacgagc ccctggtaga tgtccagaat 240 gtctgtttcc aggaaaaggt cacctgcaag aacgggcagg gcaactgcta caagagcaac 300 tccagcatgc acatcacaga ctgccgcctg acaaacggct ccaggtaccc caactgtgca 360 taccggacca gcccgaagga gagacacatc attgtggcct gtgaagggag cccatatgtg 420 ccagtccact ttgatgcttc tgtggaggac tctacagatc tcgagcccaa atcttctgac 480 aaaactcaca catgtccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 540 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 600 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 660 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 720 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 780 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 840 cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 900 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 960 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1020 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1080 gtcttctcat gctccgtgat gcatgagggt ctgcacaacc actacacgca gaagagcctc 1140 tctctgtctc cgggtaaagt cgacggtgct agcagccatg tgaatgtgag cagccctagc 1200 gtgcaggata tcctgaagat cgcagccttc aacatccaga catttgggga gaccaagatg 1260 tccaatgcca ccctcgtcag ctacattgtg cagatcctga gccgctatga catcgccctg 1320 gtccaggagg tcagagacag ccacctgact gccgtgggga agctgctgga caacctcaat 1380 caggatgcac cagacaccta tcactacgtg gtcagtgagc cactgggacg gaacagctat 1440 aagcgct acctgttcgt gtacaggcct gaccaggtgt ctgcggtgga cagctactac 1500 tacgatgatg gctcgagcc ctgcgggaac gacaccttca accgagagcc agccattgtc 1560 aggttttct cccggttcac agaggtcagg gagtttgcca ttgttcccct gcatgcggcc 1620 ccgggggacg footgccga gatcgacgct ctctatgacg tctacctgga tgtccaagag 1680 aaatggggct cgggaggacgt catgttgatg ggcgacttca atgcgggctg cagctatgtg 1740 agaccctccc agtggtcatc catccgcctg tggacaagcc ccaccttcca gtggctgatc 1800 cccgacagcg ctgacaccac agctacaccc acgcactgtg cctatgacag gatcgtggtt 1860 gcagggatgc tgctccgagg cgccgttgtt cccgactcgg ctcttccctt taacttccag 1920 nctgcctatg gcctgagtga ccaactggcc caagccatca gtgaccacta tccagtggag 1980 gtgatgctga agtgataatc taga 2004 <210> 116 <211> 2004 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 116 aagcttgccg ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtaaggaatc ccgggccaag aaattccagc ggcagcatat ggactcagac 120 agttccccca gcagcagctc cacctactgt aaccaaatga tgaggcgccg gaatatgaca 180 caggggcggt gcaaaccagt gaacaccttt gtgcacgagc ccctggtaga tgtccagaat 240 gtctgtttcc aggaaaaggt cacctgcaag aacgggcagg gcaactgcta caagagcaac 300 tccagcatgc acatcacaga ctgccgcctg acaaacggct ccaggtaccc caactgtgca 360 taccggacca gcccgaagga gagacacatc attgtggcct gtgaagggag cccatatgtg 420 ccagtccact ttgatgcttc tgtggaggac tctacagatc tcgagcccaa atcttctgac 480 aaaactcaca catgtccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 540 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 600 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 660 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 720 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 780 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 840 cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 900 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 960 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1020 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1080 gtcttctcat gctccgtgat gcatgagggt ctgcacaacc actacacgca gaagagcctc 1140 tctctgtctc cgggtaaagt cgacggtgct agcagccatg tgaatgtgag cagccctagc 1200 gtgcaggata tcctgaagat cgcagccttc aacatccaga catttgggga gaccaagatg 1260 tccaatgcca ccctcgtcag ctacattgtg cagatcctga gccgctatga catcgccctg 1320 gtccaggagg tcagagacag ccacctgact gccgtgggga agctgctgga caacctcaat 1380 caggatgcac cagacaccta tcactacgtg gtcagtgagc cactgggacg gaacagctat 1440 aagcgct acctgttcgt gtacaggcct gaccaggtgt ctgcggtgga cagctactac 1500 tacgatgatg gctcgagcc ctgcgggaac gacaccttca accgagagcc attcattgtc 1560 aggttttct cccggttcac agaggtcagg gagtttgcca ttgttcccct gcatgcggcc 1620 ccggggacg footgccga gatcgacgct ctctatgacg tctacctgga tgtccaagag 1680 aaatggggct tagaggacgt catgttgatg ggcgacttca atgcgggctg cagctatgtg 1740 agaccctccc agtggtcatc catccgcctg tggacaagcc ccaccttcca gtggctgatc 1800 cccgacagcg ctgacaccac agctacaccc acgcactgtg cctatgacag gatcgtggtt 1860 gcagggatgc tgctccgagg cgccgttgtt cccgactcgg ctcttccctt taacttccag 1920 gctgcctatg gcctgagtga ccaactggcc caagccatca gtgaccacta tccagtggag 1980 gtgatgctga agtgataatc taga 2004 <210> 117 <211> 2004 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 117 aagcttgccg ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtaaggaatc ccgggccaag aaattccagc ggcagcatat ggactcagac 120 agttccccca gcagcagctc cacctactgt aaccaaatga tgaggcgccg gaatatgaca 180 caggggcggt gcaaaccagt gaacaccttt gtgcacgagc ccctggtaga tgtccagaat 240 gtctgtttcc aggaaaaggt cacctgcaag aacgggcagg gcaactgcta caagagcaac 300 tccagcatgc acatcacaga ctgccgcctg acaaacggct ccaggtaccc caactgtgca 360 taccggacca gcccgaagga gagacacatc attgtggcct gtgaagggag cccatatgtg 420 ccagtccact ttgatgcttc tgtggaggac tctacagatc tcgagcccaa atcttctgac 480 aaaactcaca catgtccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc 540 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 600 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 660 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 720 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 780 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 840 cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 900 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 960 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1020 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1080 gtcttctcat gctccgtgat gcatgagggt ctgcacaacc actacacgca gaagagcctc 1140 tctctgtctc cgggtaaagt cgacggtgct agcagccatg tgaatgtgag cagccctagc 1200 gtgcaggata tcctgaagat cgcagccttc aacatccaga catttgggaga gaccaagatg 1260 tccaatgcca ccctcgtcag ctacattgtg cagatcctga gccgctatga catcgccctg 1320 gtccaggagg tcagagacag ccacctgact gccgtgggga agctgctgga caacctcaat 1380 caggatgcac cagacaccta tcactacgtg gtcagtgagc cactgggacg gaacagctat 1440 aagcgct acctgttcgt gtacaggcct gaccaggtgt ctgcggtgga cagctactac 1500 tacgatgatg gctcgagcc ctgcgggaac gacaccttca accgagagcc agccattgtc 1560 aggttttct cccggttcac agaggtcagg gagtttgcca ttgttcccct gcatgcggcc 1620 ccgggggacg footgccga gatcgacgct ctctatgacg tctacctgga tgtccaagag 1680 aaatggggct tggaggacgt catgttgatg ggcgacttca atgcgggctg cagctatgtg 1740 agaccctccc agtggtcatc catccgcctg tggacaagcc ccaccttcca gtggctgatc 1800 cccgacagcg ctgacaccac agctacaccc acgcactgtg cctatgacag gatcgtggtt 1860 gcagggatgc tgctccgagg cgccgttgtt cccgactcgg ctcttccctt taacttccag 1920 gctgcctatg gcctgagtga ccaactggcc caagccatca gtgaccacta tccagtggag gtgatgctga aatgataatc from <210> 118 <211> 1569 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 118 gttagcttg ccaccatgga aaccccagcg cagcttctct tcctcctgct actctggctc ccagatacca ccggtctgaa gatcgcagcc ttcaacatcc agacatttgg ggagaccaag atgtccaatg ccaccctcgt cagctacatt gtgcagatcc tgagccgcta tgacatcgcc 180 ctggtccagg aggtcagaga cagccacctg actgccgtgg ggagctgct ggacaacctc aatcaggatg caccagacac ctatcactac gtggtcagtg agccactggg acggaacagc tataaggagc gctacctgtt cgtgtacagg cctgaccagg tgtctgcggt ggacagctac 360 tactacgatg atggctgcga gccctgcggg aacgacacct tcaaccgaga gccagccatt 420 gtcaggttct tctcccggtt cacagaggtc agggagtttg ccattgttcc cctgcatgcg 480 gccccggggg acgcagtagc cgagatcgac gctctctatg acgtctacct ggatgtccaa 540 gagaaatggg gcttggagga cgtcatgttg atgggcgact tcaatgcggg ctgcagctat 600 gtgagaccct cccagtggtc atccatccgc ctgtggacaa gccccacctt ccagtggctg 660 atccccgaca gcgctgacac cacagctaca cccacgcact gtgcctatga caggatcgtg 720 gttgcaggga tgctgctccg aggcgccgtt gttcccgact cggctcttcc ctttaacttc 780 caggctgcct atggcctgag tgaccaactg gcccaagcca tcagtgacca ctatccagtg 840 gaggtgatgc tgaaagatct cgagcccaaa tcttctgaca aaactcacac atgtccaccg 900 tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 960 gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 1020 gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1080 acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1140 ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1200 ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1260 tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 1320 gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1380 aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1440 aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1500 catgaggctc tgcacaacca ctacacgcag aagagcctct ctctgtctcc gggtaaatga 1560 taatctaga 1569 <210> 119 <211> 1569 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 119 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 gccattcatt 420 gtcaggttct tctcccggtt cacagaggtc agggagtttg ccattgttcc cctgcatgcg 480 gccccggggg acgcagtagc cgagatcgac gctctctatg acgtctacct ggatgtccaa 540 gagaaatggg gcttagagga cgtcatgttg atgggcgact tcaatgcggg ctgcagctat 600 gtgagaccct cccagtggtc atccatccgc ctgtggacaa gccccacctt ccagtggctg 660 atccccgaca gcgctgacac cacagctaca cccacgcact gtgcctatga caggatcgtg 720 gttgcaggga tgctgctccg aggcgccgtt gttcccgact cggctcttcc ctttaacttc 780 caggctgcct atggcctgag tgaccaactg gcccaagcca tcagtgacca ctatccagtg 840 gaggtgatgc tgaaagatct cgagcccaaa tcttctgaca aaactcacac atgtccaccg 900 tgcccagcac ctgaactcct gggggaccg tcagtcttcc tcttcccccc aaaacccaag 960 gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 1020 gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1080 acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1140 ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1200 ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1260 tacaccctgc cccacatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 1320 gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1380 aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1440 aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1500 catgaggctc tgcacaacca ctacacgcag aagagcctct ctctgtctcc gggtaaatga 1560 taatctaga 1569 <210> 120 <211> 1641 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 120 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 gccctgcagg aacgacacct tcaaccgaga gccattcatt 420 gtcaggttct tctcccggtt cacagaggtc agggagtttg ccattgttcc cctgcatgcg 480 gccccggggg acgcagtagc cgagatcgac gctctctatg acgtctacct ggatgtccaa 540 gagaaatggg gcttggagga cgtcatgttg atgggcgact tcaatgcggg ctgcagctat 600 gtgagaccct cccagtggtc atccatccgc ctgtggacaa gccccacctt ccagtggctg 660 atccccgaca gcgctgacac cacagctaca cccacgcact gtgcctatga caggatcgtg 720 gttgcaggga tgctgctccg aggcgccgtt gttcccgact cggctcttcc ctttaacttc 780 caggctgcct atggcctgag tgaccaactg gcccaagcca tcagtgacca ctatccagtg 840 gaggtgatgc tgaaagatct ctccggagga ggtggctcag gtggtggagg atctggagga 900 ggtgggagtg gtggaggtgg ttctaccggt ctcgagccca aatcttctga caaaactcac 960 acatgtccac cgtgcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc 1020 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 1080 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1140 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1200 gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1260 aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 1320 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1380 ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1440 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1500 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1560 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctctctgtct 1620 ccgggtaaat gataatctag a 1641 <210> 121 <211> 1656 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 121 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 gccctgcagg aacgacacct tcaaccgaga gccattcatt 420 gtcaggttct tctcccggtt cacagaggtc agggagtttg ccattgttcc cctgcatgcg 480 gccccggggg acgcagtagc cgagatcgac gctctctatg acgtctacct ggatgtccaa 540 gagaaatggg gcttggagga cgtcatgttg atgggcgact tcaatgcggg ctgcagctat 600 gtgagaccct cccagtggtc atccatccgc ctgtggacaa gccccacctt ccagtggctg 660 atccccgaca gcgctgacac cacagctaca cccacgcact gtgcctatga caggatcgtg 720 gttgcaggga tgctgctccg aggcgccgtt gttcccgact cggctcttcc ctttaacttc 780 caggctgcct atggcctgag tgaccaactg gcccaagcca tcagtgacca ctatccagtg 840 gaggtgatgc tgaaagatct ctccggagga ggtggctcag gtggtggagg atctggagga 900 ggtggctcag gtggtggagg atctgggaga ggtgggagta ccggtctcga gcccaaatct 960 tctgacaaaa ctcacacatg tccaccgtgc ccagcacctg aactcctggg gggaccgtca 1020 gtttctctct tccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc 1080 acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 1140 gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg 1200 taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac 1260 aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat ctccaaagcc 1320 aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc 1380 aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga catcgccgtg 1440 gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac 1500 tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1560 gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag 1620 agcctctctc tgtctccggg taaatgataa tctaga 1656 <210> 122 <211> 1650 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 122 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 gccctgcagg aacgacacct tcaaccgaga gccattcatt 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 tgaagatct ctccggagga ggtggctcag gtggtggagg atctggagga 900 ggtggctcag gtggtggagg atctggagga ggtgggagtc tcgagcccaa atcttctgac 960 aaaactcaca catgtccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc ctcttcccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 1080. gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 1140 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 1200 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 1260 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1320 cagccccgag aaccacaggt gtacaccctg cccccatccc gggatgagct gaccaagaac 1380 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1440 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1500 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1560 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1620 tctctgtctc cgggtaaatg ataatctaga 1650 <210> 123 <211> 1569 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 123 gttaagcttg ccaccatgga aaccccagcg cagcttctct tcctcctgct actctggctc ccagatacca ccggtctgaa gatcgcagcc ttcaacatcc agacatttgg ggagaccaag atgtccaatg ccaccctcgt cagctacatt gtgcagatcc tgagccgcta tgacatcgcc 180 ctggtccagg aggtcagaga cagccacctg actgccgtgg ggagctgct ggacaacctc aatcaggatg caccagacac ctatcactac gtggtcagtg agccactggg acggaacagc tataaggagc gctacctgtt cgtgtacagg cctgaccagg tgtctgcggt ggacagctac 360 tactacgatg atggctgcga gccctgcagg aacgacacct tcaaccgaga gccattcatt 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 720 gttgcaggga tgctgctccg aggcgccgtt gttcccgact cggctcttcc ctttaacttc 780 caggctgcct atggcctgag tgaccaactg gcccaagcca tcagtgacca ctatccagtg 840 gaggtgatgc tgaaagatct cgagcccaaa tcttctgaca aaactcacac atgtccaccg 900 tgcccagcac ctgaactcct gggggaccg tcagtcttcc tcttcccccc aaaacccaag 960 gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 1020 gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1080 acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1140 ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1200 ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1260 tacaccctgc cccacatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 1320 gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1380 aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1440 aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1500 catgaggctc tgcacaacca ctacacgcag aagagcctct ctctgtctcc gggtaaatga 1560 taatctaga 1569 <210> 124 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 124 gttaagcttg ccaccatgga aaccccagcg cagcttctct tcctcctgct actctggctc 60 ccagatacca ccggtaagga atcccgggcc aagaaattcc agcggcagca tatggactca 120 gacagttccc ccagcagcag ctccacctac tgtaaccaaa tgatgaggcg ccggaatatg 180 acacaggggc ggtgcaaacc agtgaacacc tttgtgcacg agcccctggt agatgtccag 240 aatgtctgtt tccaggaaaa ggtcacctgc aagaacgggc agggcaactg ctacaagagc 300 aactccagca tgcacatcac agactgccgc ctgacaaacg actccaggta ccccaactgt 360 gcatccgga ccagcccgaa ggagagacac atcattgtgg cctgtgaagg gagcccatat 420 gtgccagtcc actttgatgc ttctgtggag gactctacag atctcgagcc caaatcttct 480 gacaaaactc acacatgtcc accgtgccca gcacctgaac tcctgggggg accgtcagtc 540 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 600 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 660 ggcgtggagg tgcataatgc caacaaag ccgcgggagg agcagtacaa cagcacgtac 720 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggatcaag 780 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 840 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 900 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 960 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1020 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1080 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1140 ctctctctgt ctccgggtaa atgataatct aga 1173 <210> 125 <211> 1245 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 125 gttaagcttg ccaccatgga aaccccagcg cagcttctct tcctcctgct actctggctc 60 ccagatacca ccggtaagga atcccgggcc aagaaattcc agcggcagca tatggactca 120 gacagttccc ccagcagcag ctccacctac tgtaaccaaa tgatgaggcg ccggaatatg 180 acacaggggc ggtgcaaacc agtgaacacc tttgtgcacg agcccctggt agatgtccag 240 aatgtctgtt tccaggaaaa ggtcacctgc aagaacgggc agggcaactg ctacaagagc 300 aactccagca tgcacatcac agactgccgc ctgacaaacg gctccaggta ccccaactgt 360 gcataccgga ccagcccga ggagagacac atcattgtgg cctgtgaagg gagcccatat 420 gtgccagtcc actttgatgc ttctgtggag gactctacag atctctccgg aggaggtggc 480 tcaggtggtg gaggatctgg aggaggtggg agtggtggag gtggttctac cggtctcgag 540 cccaaatctt ctgacaaaac tcacacatgt ccaccgtgcc cagcacctga actcctgggg 600 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 660 cctgaggtca catgcgtggt ggtggacgtg agccacgag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 780 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 840 aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1020. gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtggagag caatgggcag ccggagaca actacaagac cacgcctccc 1080. gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1140 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1200 acgcagaaga gcctctctct gtctccgggt aaatgataat ctaga 1245 <210> 126 <211> 1254 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 126 gttaagcttg ccaccatgga aaccccagcg cagcttctct tcctcctgct actctggctc 60 ccagatacca ccggtaagga atcccgggcc aagaaattcc agcggcagca tatggactca 120 gacagttccc ccagcagcag ctccacctac tgtaaccaaa tgatgaggcg ccggaatatg 180 acacaggggc ggtgcaaacc agtgaacacc tttgtgcacg agcccctggt agatgtccag 240 aatgtctgtt tccaggaaaa ggtcacctgc aagaacgggc agggcaactg ctacaagagc 300 aactccagca tgcacatcac agactgccgc ctgacaaacg gctccaggta ccccaactgt 360 gcataccgga ccagcccga ggagagacac atcattgtgg cctgtgaagg gagcccatat 420 gtgccagtcc actttgatgc ttctgtggag gactctacag atctctccgg aggaggtggc 480 tcaggtggtg gaggatctgg aggaggtggc tcaggtggtg gaggatctgg aggaggtggg 540 agtctcgagc ccaaatcttc tgacaaaact cacacatgtc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 660 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 720 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 780 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 840. ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagacaa ctacaagacc 1080 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1140 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1200 aaccactaca cgcagaagag cctctctctg tctccgggta aatgataatc taga 1254 <210> 127 <211> 1173 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 127 gttaagcttg ccaccatgga aaccccagcg cagcttctct tcctcctgct actctggctc 60 ccagatacca ccggtaagga atcccgggcc aagaaattcc agcggcagca tatggactca 120 gacagttccc ccagcagcag ctccacctac tgtaaccaaa tgatgaggcg ccggaatatg 180 acacaggggc ggtgcaaacc agtgaacacc tttgtgcacg agcccctggt agatgtccag 240 aatgtctgtt tccaggaaaa ggtcacctgc aagaacgggc agggcaactg ctacaagagc 300 aactccagca tgcacatcac agactgccgc ctgacaaacg gctccaggta ccccaactgt 360 gcatccgga ccagcccgaa ggagagacac atcattgtgg cctgtgaagg gagcccatat 420 gtgccagtcc actttgatgc ttctgtggag gactctacag atctcgagcc caaatcttct 480 gacaaaactc acacatgtcc accgtgccca gcacctgaac tcctgggggg accgtcagtc 540 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 600 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 660 ggcgtggagg tgcataatgc caacaaag ccgcgggagg agcagtacaa cagcacgtac 720 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggatcaag 780 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 840 gggcagcccc gagaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 900 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 960 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1020 gacggctcct tcttctctca cagcaagctc accgtggaca agagcaggtg gcagcagggg 1080 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1140 ctctctctgt ctccgggtaa atgataatct aga 1173 <210> 128 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 128 atgggctcac agaccctgcc ccatggtcac atgcagaccc tcatcttctt agacctggaa 60 gccactggcc tgccttcgtc tcggcccgaa gtcacagagc tgtgcctgct ggctgtccac 120 agacgtgctc tggagaacac ttccatttct cagggacatc cacctccagt gcccagaccg 180 ccccgtgtgg tggacaagct ctctctgtgc attgctccag ggaaagcctg tagccctggg 240 gccagtgaga tcacaggtct gagcaaagct gagctggaag tacaggggcg tcaacgcttc 300 gatgacaacc tggccatcct gctccgagcc ttcctgcagc gccagccaca gccttgctgc 360 cttgtggcac acaacggtga ccgctatgac tttcctctgc tccagacaga gcttgctagg 420 ctgagcactc ccagtcccct agatggtacc ttctgtgtgg acagcatcgc tgccctaaag 480 gccttggaac aagctagcag cccctcaggg aatggttcga ggaaaagcta cagcctgggc 540 agcatctaca cccgcctgta ctggcaagca ccgacagact cacatactgc tgaaggtgat 600 gttctaaccc tgctcagcat ctgtcagtgg aagccacagg ccctactgca gtgggtggac 660 gaacatgccc ggccctttag caccgtcaag cccatgtacg gcactccggc taccactgga 720 acaaccaacc taaggccaca tgctgccaca gctactacac ccctggccac agccaatgga 780 agtcccagca atggcaggag caggcgacct aagagtcctc ctccagagaa ggtcccagaa 840 gccccatcac aggaggggct gctggcccca ctgagcctgc tgaccctcct gaccttggca 900 atagccactc tgtatggact cttcctggcc tcacctgggc agtaa 945 <210> 129 <211> 735 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 129 atgggctcac agaccctgcc ccatggtcac atgcagaccc tcatcttctt agacctggaa 60 gccactggcc tgccttcgtc tcggcccgaa gtcacagagc tgtgcctgct ggctgtccac 120 agacgtgctc tggagaacac ttccatttct cagggacatc cacctccagt gcccagaccg 180 ccccgtgtgg tggacaagct ctctctgtgc attgctccag ggaaagcctg tagccctggg 240 gccagtgaga tcacaggtct gagcaaagct gagctggaag tacaggggcg tcaacgcttc 300 gatgacaacc tggccatcct gctccgagcc ttcctgcagc gccagccaca gccttgctgc 360 cttgtggcac acaacggtga ccgctatgac tttcctctgc tccagacaga gcttgctagg 420 ctgagcactc ccagtcccct agatggtacc ttctgtgtgg acagcatcgc tgccctaaag 480 gccttggaac aagctagcag cccctcaggg aatggttcga ggaaaagcta cagcctgggc 540 agcatctaca cccgcctgta ctggcaagca ccgacagact cacatactgc tgaaggtgat 600 gttctaaccc tgctcagcat ctgtcagtgg aagccacagg ccctactgca gtgggtggac 660 gaacatgccc ggccctttag caccgtcaag cccatgtacg gcactccggc taccactgga 720 acaacagatc tcgag 735 <210> 130 <211> 1593 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 130 aagcttgcca ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtatgggctc acagaccctg ccccatggtc acatgcagac cctcatcttc 120 ttagacctgg aagccactgg cctgccttcg tctcggcccg aagtcacaga gctgtgcctg 180 ctggctgtcc acagacgtgc tctggagaac acttccattt ctcagggaca tccacctcca 240 gtgcccagac cgccccgtgt ggtggacaag ctctctctgt gcattgctcc agggaaagcc 300 tgtagccctg gggccagtga gatcacaggt ctgagcaaag ctgagctgga agtacagggg 360 cgtcaacgct tcgatgacaa cctggccatc ctgctccgag ccttcctgca gcgccagcca 420 cagccttgct gccttgtggc acacaacggt gaccgctatg actttcctct gctccagaca 480 gagcttgcta ggctgagcac tcccagtccc ctagatggta ccttctgtgt ggacagcatc 540 gctgccctaa aggccttgga acaagctagc agcccctcag ggaatggttc gaggaaaagc 600 tacagcctgg gcagcatcta cacccgcctg tactggcaag caccgacaga ctcacatact 660 gctgaaggtg atgttctaac cctgctcagc atctgtcagt ggaagccaca ggccctactg 720 cagtgggtgg acgaacatgc ccggcccttt agcaccgtca agcccatgta cggcactccg 780 gctaccactg gaacaacaga tctctccgga ggaggtggct caggtggtgg aggatctgga 840 ggaggtggct cagggagtgg tggaggtggt tctaccggtc tcgagcccag aggtcccaca 900 atcaagccct ctcctccatg caaatgccca gcacctaacc tcttgggtgg atcatccgtc 960 ttcatcttcc ctccaaagat caaggatgta ctcatgatct ccctgagccc catggtcaca 1020 tgtgtggtgg tggatgtgag cgaggatgac ccagacgtcc agatcagctg gtttgtgaac 1080 aacgtggaag tacacacagc tcagacacaa acccatagag aggattacaa cagtactctc 1140 cgggtggtca gtgccctccc catccagcac caggactgga tgagtggcaa ggagttcaaa 1200 tgctcggtca acaacaaaga cctcccagcg tccatcgaga gaaccatctc aaaacccaga 1260 gggccagtaa gagctccaca ggtatatgtc ttgcctccac cagcagaaga gatgactaag 1320 aaagagttca gtctgacctg catgatcaca ggcttcttac ctgccgaaat tgctgtggac 1380 tggaccagca atgggcgtac agagcaaaac tacaagaaca ccgcaacagt cctggactct 1440 gatggttctt acttcatgta cagcaagctc agagtacaaa agagcacttg ggaaagagga 1500 agtcttttcg cctgctcagt ggtccacgag ggtctgcaca atcaccttac gactaagagc 1560 ttctctcgga ctccgggtaa atgataatct aga 1593 <210> 131 <211> 1596 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 131 aagcttgcca ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtatgggctc acagaccctg ccccatggtc acatgcagac cctcatcttc 120 ttagacctgg aagccactgg cctgccttcg tctcggcccg aagtcacaga gctgtgcctg 180 ctggctgtcc acagacgtgc tctggagaac acttccattt ctcagggaca tccacctcca 240 gtgcccagac cgccccgtgt ggtggacaag ctctctctgt gcattgctcc agggaaagcc 300 tgtagccctg gggccagtga gatcacaggt ctgagcaaag ctgagctgga agtacagggg 360 cgtcaacgct tcgatgacaa cctggccatc ctgctccgag ccttcctgca gcgccagcca 420 cagccttgct gccttgtggc acacaacggt gaccgctatg actttcctct gctccagaca 480 gagcttgcta ggctgagcac tcccagtccc ctagatggta ccttctgtgt ggacagcatc 540 gctgccctaa aggccttgga acaagctagc agcccctcag ggaatggttc gaggaaaagc 600 tacagcctgg gcagcatcta cacccgcctg tactggcaag caccgacaga ctcacatact 660 gctgaaggtg atgttctaac cctgctcagc atctgtcagt ggaagccaca ggccctactg 720 cagtgggtgg acgaacatgc ccggcccttt agcaccgtca agcccatgta cggcactccg 780 gctaccactg gaacaacaga tctctccgga ggaggtggct caggtggtgg aggatctgga 840 ggaggtggct caggtggtgg aggatctgga ggaggtggga gtctcgagcc cagaggtccc 900 aaatcaagc cctctctccc atgcaaatgc ccagcaccta acctcttggg tggatcatcc 960 gtcttcatct tccctccaaa gatcaaggat gtactcatga tctccctgag cccccatggtc 1020 acatgtgtgg tggtggatgt gagcgaggat gacccagacg tccagatcag ctggtttgtg 1080 aacaacgtgg aagtacacac agctcagaca caaacccata gagagatta caacagtact 1140 ctccgggtgg tcagtgccct cccacatccag caccaggact ggatgagtgg caaggagttc 1200 aaatgctcgg tcaacaacaa agacctccca gcgtccatcg agagaaccat ctcaaaaccc 1260 agagggccag taagagctcc acaggtatat gtcttgcctc caccagcaga agagatgact 1320 aagaaagagt tcagtctgac ctgcatgatc acaggcttct tacctgccga aattgctgtg 1380 gactggacca gcaatgggcg tacagagcaa aactacaaga acaccgcaac agtcctggac 1440 tctgatggtt cttattcat gtacagcaag ctcagagtac aaaagagcac ttgggaaaga 1500 ggaagtcttt tcgcctgctc agtggtccac gagggtctgc aaatcacct tacgactaag 1560 agcttctctc ggactccggg taaatgataa tctaga 1596 <210> 132 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 132 gtcgacggcg cggccgccag ccccgtgaac gtgagcagcc ccagcgtgca ggatatc 57 <210> 133 <211> 2328 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 133 aagcttgcca ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtatgggctc acagaccctg ccccatggtc acatgcagac cctcatcttc 120 ttagacctgg aagccactgg cctgccttcg tctcggcccg aagtcacaga gctgtgcctg 180 ctggctgtcc acagacgtgc tctggagaac acttccattt ctcagggaca tccacctcca 240 gtgcccagac cgccccgtgt ggtggacaag ctctctctgt gcattgctcc agggaaagcc 300 tgtagccctg gggccagtga gatcacaggt ctgagcaaag ctgagctgga agtacagggg 360 cgtcaacgct tcgatgacaa cctggccatc ctgctccgag ccttcctgca gcgccagcca 420 cagccttgct gccttgtggc acacaacggt gaccgctatg actttcctct gctccagaca 480 gagcttgcta ggctgagcac tcccagtccc ctagatggta ccttctgtgt ggacagcatc 540 gctgccctaa aggccttgga acaagctagc agcccctcag ggaatggttc gaggaaaagc 600 tacagcctgg gcagcatcta cacccgcctg tactggcaag caccgacaga ctcacatact 660 gctgaaggtg atgttctaac cctgctcagc atctgtcagt ggaagccaca ggccctactg 720 cagtgggtgg acgaacatgc ccggcccttt agcaccgtca agcccatgta cggcactccg 780 gctaccactg gaacaacaga tctcatgggc tcacagaccc tgccccatgg tcacatgcag 840 accctcatct tcttagacct ggaagccact ggcctgcctt cgtctcggcc cgaagtcaca 900 gagctgtgcc tgctggctgt ccacagacgt gctctggaga acacttccat ttctcaggga 960 catccacctc cagtgcccag accgccccgt gtggtggaca agctctctct gtgcattgct 1020 ccagggaaag cctgtagccc tggggccagt gagatcacag gtctgagcaa agctgagctg 1080 gaagtacagg ggcgtcaacg cttcgatgac aacctggcca tcctgctccg agccttcctg 1140 cagcgccagc cacagccttg ctgccttgtg gcacacaacg gtgaccgcta tgactttcct 1200 ctgctccaga cagagcttgc taggctgagc actcccagtc ccctagatgg taccttctgt 1260 gtggacagca tcgctgccct aaaggccttg gaacaagcta gcagcccctc agggaatggt 1320 tcgaggaaaa gctacagcct gggcagcatc tacacccgcc tgtactggca agcaccgaca 1380 gactcacata ctgctgaagg tgatgttcta accctgctca gcatctgtca gtggaagcca 1440 caggccctac tgcagtgggt ggacgaacat gcccggccct ttagcaccgt caagcccatg 1500 tacggcactc cggctaccac tggaacaaca gatctctccg gaggaggtgg ctcaggtggt 1560 ggaggatctg gaggaggtgg ctcaggtggt ggaggatctg gaggaggtgg gagtctcgag 1620 cccagaggtc ccacaatcaa gccctctcct ccatgcaaat gcccagcacc taacctcttg 1680 ggtggatcat ccgtcttcat cttccctcca aagatcagg atgtactcat gatctccctg 1740 agccccatgg tcacatgtgt ggtggtggat gtgagcgagg atgacccaga cgtccagatc 1800 agctggtttg tgacaacgt ggtacac acagctcaga cacaaccca ggaggaggat 1860 tacacagta ctctccgggt ggtcagtgcc ctcccatcc accaccagga ctggatgagt 1920 ggcaggagt tcaatgctc ggtcacaac aagacctcc cagcgtccat cgagagaacc 1980 atctcaaac ccagaggggcc agtaagagct ccacaggtat atgtcttgcc 2040 gagagatga ctaagaaga gttcagctctg acctgcatga tcacaggctt cttacctgcc 2100 gaaattgctg tggactggac cagcaatggg cgtacagagc aaactacaa gaacaccgca 2160 acagtcctgg actctgatgg ttcttactc atgtacagca agctcagagt aaaagagc 2220 acttgggaaa gaggaagtct ttcgcctgc tcagtggtcc acgaggctct gcacaatcac 2280 cttacgacta agagctctc tcggactccg ggtaaatgat aatcga 2328 <210> 134 <211> 1749 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 134 aagcttgcca ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtatgggccc tggagctcgc agacagggca ggattgtgca gggaaggcct 120 gagatgtgct tctgcccacc ccctacccca ctccctcccc ttcggatctt aacactgggc 180 actcacacac ccaccccatg ctcctctcca ggctcagcag caggtacgta cccaaccatg 240 ggctcgcagg ccctgccccc ggggcccatg cagaccctca tctttttcga catggaggcc 300 actggcttgc ccttctccca gcccaaggtc acggagctgt gcctgctggc tgtccacaga 360 tgtgccctgg agagcccccc cacctctcag gggccacctc ccacagttcc tccaccaccg 420 cgtgtggtag acaagctctc cctgtgtgtg gctccgggga aggcctgcag ccctgcagcc 480 agcgagatca caggtctgag cacagctgtg ctggcagcgc atgggcgtca atgttttgat 540 gacaacctgg ccaacctgct cctagccttc ctgcggcgcc agccacagcc ctggtgcctg 600 gtggcacaca atggtgaccg ctacgacttc cccctgctcc aagcagagct ggctatgctg 660 ggcctcacca gtgctctgga tggtgccttc tgtgtggata gcatcactgc gctgaaggcc 720 ctggagcgag caagcagccc ctcagaacac ggcccaagga agagctacag cctaggcagc 780 atctacactc gcctgtatgg gcagtcccct ccagactcgc acacggctga gggtgatgtc 840 ctggccctgc tcagcatctg tcagtggaga ccacaggccc tgctgcggtg ggtggatgct 900 cacgccaggc ctttcggcac catcaggccc atgtatgggg tcacagcctc tgctaggacc 960 aaagatctct ccggaggagg tggctcaggt ggtggaggat ctggaggagg tgggagtggt 1020 ggaggtggtt ctaccggtct cgagcccaaa tcttctgaca aaactcacac atgtccaccg 1080 tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 1140 gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 1200 gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1260 acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1320 ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1380 ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1440 tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 1500 gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1560 aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1620 aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1680 catgaggctc tgcacaacca ctacacgcag aagagcctct ctctgtctcc gggtaaatga 1740 taatctaga 1749 <210> 135 <211> 1758 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 135 aagcttgcca ccatggaaac cccagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtatgggccc tggagctcgc agacagggca ggattgtgca gggaaggcct 120 gagatgtgct tctgcccacc ccctacccca ctccctcccc ttcggatctt aacactgggc 180 actcacacac ccaccccatg ctcctctcca ggctcagcag caggtacgta cccaaccatg 240 ggctcgcagg ccctgccccc ggggcccatg cagaccctca tctttttcga catggaggcc 300 actggcttgc ccttctccca gcccaaggtc acggagctgt gcctgctggc tgtccacaga 360 tgtgccctgg agagcccccc cacctctcag gggccacctc ccacagttcc tccaccaccg 420 cgtgtggtag acaagctctc cctgtgtgtg gctccgggga aggcctgcag ccctgcagcc 480 agcgagatca caggtctgag cacagctgtg ctggcagcgc atgggcgtca atgttttgat 540 gacaacctgg ccaacctgct cctagccttc ctgcggcgcc agccacagcc ctggtgcctg 600 gtggcacaca atggtgaccg ctacgacttc cccctgctcc aagcagagct ggctatgctg 660 ggcctcacca gtgctctgga tggtgccttc tgtgtggata gcatcactgc gctgaaggcc 720 ctggagcgag caagcagccc ctcagaacac ggcccaagga agagctacag cctaggcagc 780 atctacactc gcctgtatgg gcagtcccct ccagactcgc acacggctga gggtgatgtc 840 ctggccctgc tcagcatctg tcagtggaga ccacaggccc tgctgcggtg ggtggatgct 900 cacgccaggc ctttcggcac catcaggccc atgtatgggg tcacagcctc tgctaggacc 960 aaagatctct ccggaggagg tggctcaggt ggtggaggat ctggaggagg tggctcaggt 1020 ggtggaggat ctggaggagg tgggagtctc gagcccaaat cttctgacaa aactcacaca 1080 tgtccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca 1140 aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 1200 gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1260 aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 1320 ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 1380 aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1440 ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg 1500 acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 1560 cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1620 ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1680 tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc tctgtctccg 1740 ggtaaatgat aatctaga 1758 <210> 136 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 136 Asp Leu Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Thr Gly Leu Glu 20 25 <210> 137 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 137 Asp Leu Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Gly Leu Glu 20 25 30 <210> 138 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 138 Asp Leu Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Leu Glu 20 25 30 <210> 139 <211> 262 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 139 Asp Ile Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr 1 5 10 15 Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser 20 25 30 Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr 35 40 45 Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp Thr 50 55 60 Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu 65 70 75 80 Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp Ser 85 90 95 Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Arg Asn Asp Thr Phe Asn 100 105 110 Arg Glu Pro Phe Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg 115 120 125 Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala 130 135 140 Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp 145 150 155 160 Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser 165 170 175 Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro 180 185 190 Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro 195 200 205 Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg 210 215 220 Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala 225 230 235 240 Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His Tyr Pro 245 250 255 Val Glu Val Met Leu Lys 260 <210> 140 <211> 262 <212> PRT <213> Homo sapiens <400> 140 Asp Ile Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr 1 5 10 15 Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser 20 25 30 Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr 35 40 45 Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp Thr 50 55 60 Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu 65 70 75 80 Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp Ser 85 90 95 Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Asn 100 105 110 Arg Glu Pro Ala Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg 115 120 125 Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala 130 135 140 Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp 145 150 155 160 Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser 165 170 175 Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro 180 185 190 Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro 195 200 205 Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg 210 215 220 Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala 225 230 235 240 Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His Tyr Pro 245 250 255 Val Glu Val Met Leu Lys 260 <210> 141 <211> 262 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 141 Asp Ile Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr 1 5 10 15 Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser 20 25 30 Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu Thr 35 40 45 Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro Asp Thr 50 55 60 Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu 65 70 75 80 Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser Ala Val Asp Ser 85 90 95 Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly Asn Asp Thr Phe Asn 100 105 110 Arg Glu Pro Phe Ile Val Arg Phe Phe Ser Arg Phe Thr Glu Val Arg 115 120 125 Glu Phe Ala Ile Val Pro Leu His Ala Ala Pro Gly Asp Ala Val Ala 130 135 140 Glu Ile Asp Ala Leu Tyr Asp Val Tyr Leu Asp Val Gln Glu Lys Trp 145 150 155 160 Gly Leu Glu Asp Val Met Leu Met Gly Asp Phe Asn Ala Gly Cys Ser 165 170 175 Tyr Val Arg Pro Ser Gln Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro 180 185 190 Thr Phe Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro 195 200 205 Thr His Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg 210 215 220 Gly Ala Val Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Ala 225 230 235 240 Tyr Gly Leu Ser Asp Gln Leu Ala Gln Ala Ile Ser Asp His Tyr Pro 245 250 255 Val Glu Val Met Leu Lys 260 <210> 142 <211> 265 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 142 Thr Gly Leu Lys Ile Ala Ala Phe Asn Ile Gln Thr Phe Gly Glu Thr 1 5 10 15 Lys Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser 20 25 30 Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser H...

Claims

1. A polypeptide consisting of a first nuclease domain, a second nuclease domain, and an Fc domain mutant, the first and second nuclease domains are linked in tandem to each other, the link between the first and second nuclease domains being direct or via a polypeptide linker, chemical conjugate, or genetic fusion, and the tandem array of nuclease domains is linked to either the C-terminus or the N-terminus of the Fc domain variant, the link between the nuclease domain and the Fc domain variant being direct, via a polypeptide linker, chemical conjugate, or genetic fusion; the first nuclease domain is an RNase and the second nuclease domain is a DNase, or the first nuclease domain is a DNase and the second nuclease domain is an RNase; the DNase is a mutant human DNase 1, The mutations in the Fc domain variant are one, two, three, or four amino acid substitutions that reduce binding to Fcγ receptors or complement proteins, or both, compared to the wild-type human IgG1 Fc domain; and The above polypeptide, wherein the Fc domain variant does not comprise a whole antibody.

2. The polypeptide of claim 1 , wherein the RNase is a wild-type or mutant RNase.

3. 3. The polypeptide of claim 1 or 2, wherein the RNase is human RNase or human pancreatic RNase 1.

4. The polypeptide of any one of claims 1 to 3, wherein the mutant human DNase 1 comprises an A114F mutation.

5. (a) the Fc domain variant comprises a hinge domain, a CH2 domain and a CH3 domain; and / or (b) the Fc domain variant comprises a modified hinge domain comprising a substitution of one or more of the three hinge cysteines, or comprises a modified hinge domain comprising a substitution of one or more of the three hinge cysteines with serines; and / or (c) the Fc domain variant comprises a modified CH2 domain comprising at least one substitution selected from P238S, P331S, N297S, or a combination thereof; A polypeptide according to any one of claims 1 to 4.

6. The polypeptide of any one of claims 1 to 5, wherein the Fc domain mutant comprises a modified hinge domain comprising a substitution at one or more of the three hinge cysteines.

7. The polypeptide of claim 6 , wherein the Fc domain mutant comprises a hinge domain modified by substitution of one or more serines with three hinge cysteines.

8. The Fc domain variant comprises a modified CH2 domain comprising at least one substitution selected from P238S, P331S, N297S, or a combination thereof; A polypeptide according to any one of claims 1 to 7.

9. The polypeptide of any one of claims 1 to 8, wherein the amino acid substitutions include one or more substitutions selected from P238S, P331S, K322S, and N297S.

10. (a) the RNase comprises the amino acid sequence set forth in SEQ ID NO: 149, with or without a leader sequence, or comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 149; and / or (b) the mutant human DNase 1 comprises an amino acid sequence set forth in SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO: 144, or comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO: 144; A polypeptide according to any one of claims 1 to 9.

11. The polypeptide according to any one of claims 1 to 10, wherein the nuclease domain and the Fc domain mutant are linked via a polypeptide linker that is a Gly-Ser linker.

12. A pharmaceutical composition comprising the polypeptide according to any one of claims 1 to 11 and a pharma- ceutically acceptable carrier.

13. A dimeric polypeptide comprising the polypeptide according to any one of claims 1 to 11.

14. The dimeric polypeptide of claim 13 which is a homodimer.

15. A composition comprising the dimeric polypeptide of claim 13 or 14 and a pharma- ceutically acceptable carrier.

16. A nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide according to any one of claims 1 to 11.

17. A nucleic acid molecule comprising a nucleic acid sequence encoding the dimeric polypeptide according to claim 13 or 14.

18. A recombinant expression vector comprising the nucleic acid molecule of claim 16 or 17.

19. A host cell transformed with the recombinant expression vector of claim 18.

20. Use of a polypeptide according to any one of claims 1 to 11 or a dimeric polypeptide according to claim 13 or 14 in the manufacture of a medicament for treating or preventing an autoimmune disease.

21. The use according to claim 20, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

22. The use according to claim 20, wherein the autoimmune disease is Sjogren's syndrome.

23. The use described in claim 20, wherein the autoimmune disease is lupus nephritis.

24. Use of a polypeptide described in any one of claims 1 to 11, a composition described in claim 12 or 15, or a dimeric polypeptide described in claim 13 or 14 in the manufacture of a medicament for treating SLE.

25. Use of a polypeptide according to any one of claims 1 to 11, a composition according to claim 12 or 15, or a dimeric polypeptide according to claim 13 or 14 in the manufacture of a medicament for treating lupus nephritis.

26. Use of a polypeptide according to any one of claims 1 to 11, a composition according to claim 12 or 15, or a dimeric polypeptide according to claim 13 or 14 in the manufacture of a medicament for treating Sjogren's syndrome.

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