Optimized binuclease fusions and methods

Abstract

The invention provides for optimized binuclease fusion proteins with increased pharmacokinetic properties. The optimized binuclease fusion proteins of the invention two or more nuclease domains (e.g., RNase and DNase domain) operably coupled to an Fc domain. The invention also provides methods of treating or preventing a condition associated with an abnormal immune response.

We claim: 
     
         1 . A heterodimer comprising a first nuclease domain, a second nuclease domain, a first Fc domain and a second Fc domain, wherein the first nuclease domain is DNase1 and the second nuclease domain is RNase1, wherein the DNase1 is operably linked with or without a linker to the N- or C-terminus of the first Fc domain, and the RNase1 is operably linked with or without a linker to the N- or C-terminus of the second Fc domain. 
     
     
         2 . The heterodimer of  claim 1 , wherein the DNase 1 is operably linked without a linker to the N-terminus of the first Fc domain and the RNase1 is operably linked without a linker to the N-terminus of the second Fc domain. 
     
     
         3 . The heterodimer of  claim 1 , wherein the DNase 1 is operably linked with a linker to the N-terminus of the first Fc domain and the RNase1 is operably linked with a linker to the N-terminus of the second Fc domain. 
     
     
         4 . The heterodimer of  claim 1 , wherein the DNase 1 is operably linked with a linker to the N-terminus of the first Fc domain and the RNase1 is operably linked without a linker to the C-terminus of the second Fc domain. 
     
     
         5 . The heterodimer of  claim 1 , wherein the DNase 1 is operably linked without a linker to the N-terminus of the first Fc domain and the RNase1 is operably linked without a linker to the C-terminus of the second Fc domain. 
     
     
         6 . The heterodimer of  claim 1 , wherein the DNase 1 is operably linked with a linker to the N-terminus of the first Fc domain and the RNase1 is operably linked with a linker to the C-terminus of the second Fc domain. 
     
     
         7 . The heterodimer of  claim 1 , wherein the DNase 1 is operably linked with a linker to the C-terminus of the first Fc domain and the RNase1 is operably linked with a linker to the C-terminus of the second Fc domain. 
     
     
         8 . The heterodimer of  claim 1 , wherein the DNase 1 is operably linked without a linker to the C-terminus of the first Fc domain and the RNase1 is operably linked without a linker to the C-terminus of the second Fc domain. 
     
     
         9 . The heterodimer of  claim 1 , wherein the DNase 1 is operably linked with a linker to the C-terminus of the first Fc domain and the RNase1 is operably linked without a linker to the N-terminus of the second Fc domain. 
     
     
         10 . The heterodimer of  claim 1 , wherein the DNase 1 is operably linked with a linker to the C-terminus of the first Fc domain and the RNase1 is operably linked with a linker to the N-terminus of the second Fc domain. 
     
     
         11 . The heterodimer of any one of  claims 1 - 10 , wherein the RNase is a wild type human RNase1, or a mutant RNase, such as an aglycosylated, underglycosylated, or deglycosylated RNase 1, such as human RNase1 N34S/N76S/N88S. 
     
     
         12 . The heterodimer of  claim 11 , wherein the RNase is wild type human RNase1. 
     
     
         13 . The heterodimer of any one of  claims 1 - 12 , wherein the DNase is a wild type human DNase1, or a mutant human DNase1 A114F, or an aglycosylated, underglycosylated, or deglycosylated mutant human DNase 1 N18S/N106S/A114F. 
     
     
         14 . The heterodimer of any of the preceding claims wherein the first and second Fc domains comprise a hinge domain, a CH2 domain and a CH3 domain. 
     
     
         15 . The heterodimer of  claim 14 , wherein the first and second Fc domains comprise a substitution of one or more of three hinge region cysteine residues with serine. 
     
     
         16 . The heterodimer of  claim 15 , wherein the first and second Fc domains comprise a mutation selected from the group consisting of SCC, SSS (residues 220, 226, and 229), G236R, L328R, L234A, and L235A, numbering according to the EU index. 
     
     
         17 . The heterodimer of  claim 16 , wherein the first and second Fc domains comprise an SCC mutation (residues 220, 226, and 229), numbering according to the EU index. 
     
     
         18 . The heterodimer of any of the preceding claims, wherein the first and second Fc domains comprise a P238S and a P331 mutation, numbering according to the EU index. 
     
     
         19 . The heterodimer of any of the preceding claims, wherein the first and second Fc domains comprise one or more CH3 mutations to preferentially form heterodimers. 
     
     
         20 . The heterodimer of  claim 19 , wherein the first Fc domain comprises CH3 mutations T350V, L351Y, F405A, and Y407V, and the second Fc domain comprises CH3 mutations T350V, T366L, K392L, T394W, numbering according to the EU index. 
     
     
         21 . The heterodimer of any of the preceding claims, wherein the linker domain is a polypeptide linker, such as a gly-ser linker or an NLG linker (vdgasspvnvsspsvqdi). 
     
     
         22 . A heterodimer comprising a first and second polypeptide sequence selected from the group consisting of:
 (i) a first polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 3, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3; and a second polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 4, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4, or   (ii) a first polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 7, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:7; and a second polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 8, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:8, or   (iii) a first polypeptide comprising an amino acid sequence set forth in SEQ ID NO:9, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:9; and a second polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 10, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:10, or   (iv) a first polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 11, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 11; and a second polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 12, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:12, or   (v) a first polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 15, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 15; and a second polypeptide comprising an amino acid sequence set forth in SEQ ID NO:16, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:16.   
     
     
         23 . A heterodimer comprising a first nuclease domain, a second nuclease domain and a first Fc domain and a second Fc domain, wherein the first nuclease domain is DNase1 and the second nuclease domain is RNase1, wherein
 (i) the DNase1 is operably linked with or without a linker to the N-terminus of the first Fc domain, and the RNase1 is operably linked with or without a linker to the C-terminus of the first Fc domain, or   (i) the RNase1 is operably linked with or without a linker to the N-terminus of the first Fc domain, and the DNase1 is operably linked with or without a linker to the C-terminus of the first Fc domain.   
     
     
         24 . The heterodimer of  claim 23 , wherein the DNase 1 is operably linked without a linker to the N-terminus of the first Fc domain and the RNase1 is operably linked with a linker to the C-terminus of the first Fc domain. 
     
     
         25 . The heterodimer of  claim 23 , wherein the DNase 1 is operably linked with a linker to the N-terminus of the first Fc domain and the RNase1 is operably linked with a without a linker to the C-terminus of the first Fc domain. 
     
     
         26 . The heterodimer of  claim 23 , wherein the RNase 1 is operably linked without a linker to the N-terminus of the first Fc domain and the DNase 1 is operably linked with a linker to the C-terminus of the first Fc domain. 
     
     
         27 . The heterodimer of  claim 23 , wherein the RNase 1 is operably linked with a linker to the N-terminus of the first Fc domain and the DNase 1 is operably linked with a linker to the C-terminus of the first Fc domain. 
     
     
         28 . The heterodimer of any one of  claims 23 - 27 , wherein the RNase is a wild type human RNase1, or a mutant RNase, such as an aglycosylated, underglycosylated, or deglycosylated RNase 1, such as human RNase1 N34S/N76S/N88S. 
     
     
         29 . The heterodimer of  claim 28 , wherein the RNase is wild type human RNase1. 
     
     
         30 . The heterodimer of any one of  claims 23 - 29 , wherein the DNase is a wild type human DNase1, or a mutant human DNase1 A114F, or an aglycosylated, underglycosylated, or deglycosylated mutant human DNase1 N18S/N106S/A114F. 
     
     
         31 . The heterodimer of any of the preceding claims wherein the first and second Fc domains comprise a hinge domain, a CH2 domain and a CH3 domain. 
     
     
         32 . The heterodimer of  claim 31 , wherein the first and second Fc domains comprise a substitution of one or more of three hinge region cysteine residues with serine. 
     
     
         33 . The heterodimer of  claim 32 , wherein the first and second Fc domains comprise a mutation selected from the group consisting of SCC, SSS (residues 220, 226, and 229), G236R, L328R, L234A, and L235A, numbering according to the EU index. 
     
     
         34 . The heterodimer of  claim 33 , wherein the first and second Fc domains comprise an SCC mutation (residues 220, 226, and 229), numbering according to the EU index. 
     
     
         35 . The heterodimer of any of the preceding claims, wherein the first and second Fc domains comprise a P238S and a P331 mutation, numbering according to the EU index. 
     
     
         36 . The heterodimer of any of the preceding claims, wherein the first and second Fc domains comprise one or more CH3 mutations to preferentially form heterodimers. 
     
     
         37 . The heterodimer of  claim 36 , wherein the first Fc domain comprises CH3 mutations T350V, L351Y, F405A, and Y407V, and the second Fc domain comprises CH3 mutations T350V, T366L, K392L, T394W, numbering according to the EU index. 
     
     
         38 . The heterodimer of any of the preceding claims, wherein the linker domain is a polypeptide linker, such as a gly-ser linker or an NLG linker (vdgasspvnvsspsvqdi). 
     
     
         39 . A heterodimer comprising a first and second polypeptide sequence selected from the group consisting of:
 (i) a first polypeptide comprising an amino acid sequence set forth in SEQ ID NO:5, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:5; and a second polypeptide comprising an amino acid sequence set forth in SEQ ID NO:6, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:6, or   (ii) a first polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 13, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; and a second polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 14, or a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:14.   
     
     
         40 . A composition comprising the heterodimer of any of the preceding claims and a pharmaceutically acceptable carrier. 
     
     
         41 . A nucleic acid molecule encoding the heterodimer of any of the preceding claims. 
     
     
         42 . A recombinant expression vector comprising a nucleic acid molecule according to  claim 41 . 
     
     
         43 . A host cell transformed with the recombinant expression vector according to  claim 42 . 
     
     
         44 . A method of making the heterodimer of any one of  claims 1 - 39 , comprising: providing a host cell comprising a nucleic acid sequence that encodes the heterodimer; and maintaining the host cell under conditions in which the heterodimer is expressed. 
     
     
         45 . A method for treating or preventing a condition associated with an abnormal immune response, comprising administering to a subject an effective amount of a heterodimer of any one of  claims 1 - 39 . 
     
     
         46 . The method of  claim 45 , wherein the condition is an autoimmune disease. 
     
     
         47 . The method of  claim 46 , wherein the autoimmune disease is selected from the group consisting of insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, an experimental form of uveoretinitis, Hashimoto's thyroiditis, primary myxoedema, thyrotoxicosis, pernicious anaemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, phacogenic uveitis, autoimmune haemolytic anaemia, idiopathic leucopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, cryptogenic cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid LE, systemic lupus erythematosus (SLE), and connective tissue disease. 
     
     
         48 . The method of  claim 47 , wherein the autoimmune disease is SLE. 
     
     
         49 . The method of  claim 47 , wherein the autoimmune disease is Sjogren's syndrome 
     
     
         50 . A method of treating SLE comprising administering to a subject an amount of a heterodimer effective to degrade immune complexes containing RNA, DNA or both RNA and DNA, wherein the composition comprises a pharmaceutically acceptable carrier and a heterodimer of any one of  claims 1 - 39 . 
     
     
         51 . A method of treating Sjogren's syndrome comprising administering to a subject an amount of a heterodimer effective to degrade immune complexes containing RNA, DNA or both RNA and DNA, wherein the composition comprises a pharmaceutically acceptable carrier and a heterodimer of any one of  claims 1 - 39 .