Optimized binuclease fusion and method

Abstract

To provide optimized binuclease fusions and methods. [Solution] The present invention provides an optimized binuclease fusion protein having enhanced pharmacokinetic properties. The optimized binuclease fusion protein of the present invention comprises two or more nuclease domains, and the heterodimer is formed between a DNase operably coupled to an Fc domain and an RNase operably coupled to an Fc domain. The present invention also provides the use of such a binuclease fusion protein in methods for treating or preventing conditions associated with abnormal immune responses.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 357,756, filed Jul. 1, 2016. The content of the above application is incorporated herein by reference.

Background Art

[0002] Background Accumulation of (ribo)nucleoprotein particles from dead and dying cells is known to induce an inflammatory cascade in patients with systemic lupus erythematosus (SLE) by at least two mechanisms: (i) deposition or in situ formation of chromatin / anti-chromatin complexes causes nephritis, leading to loss of kidney function; and (ii) nucleic acids complexed with autoantibodies activate innate immunity via toll-like receptors (TLRs) 7, 8, and 9 and TLR-independent pathways. Release of nuclear proteins can function as a potent antigen for autoantibodies in SLE, providing amplification of B cell and DC activation via co-engagement of antigen receptors and TLRs. Thus, there is a need for means to remove nucleic acids bound to autoantibody antigens and / or reduce immune stimulation, immune amplification, and immune complex-mediated diseases in subjects that require removal of nucleic acids bound to autoantibody antigens, e.g., by digesting nucleic acids contained in circulating immune complexes, using long-acting nuclease molecules that attack circulating immune complexes.

Summary of the Invention

Means for Solving the Problems

[0004] In some embodiments, the optimized binuclease fusion protein is a tandem binuclease fusion protein comprising a first nuclease domain, a second nuclease domain, and an Fc region, wherein the first nuclease domain is DNase1 and the second nuclease domain is RNase1, and DNase1 is operably tandem to RNase1 from the N-terminus to the C-terminus, either by or without a linker, and RNase1 is operably tandem to the N-terminus or C-terminus of the Fc region. The tandem binuclease fusion protein exhibits enhanced pharmacokinetic activity compared to either the first or second nuclease domain alone. Such a tandem binuclease fusion protein exhibits altered, for example, an improved serum half-life, compared to either the first or second nuclease domain alone.

[0005] In some embodiments, the optimized binuclease fusion protein is a heterodimer binuclease fusion protein comprising a first nuclease domain, a second nuclease domain, and an Fc region, wherein the first nuclease domain is DNase1 and the second nuclease domain is RNase1, and DNase1 is operably linked to the N-terminus or C-terminus of the Fc region, either by or without a linker, and RNase1 is operably linked to the N-terminus or C-terminus of the Fc region, either by or without a linker, thereby forming a heterodimer. The heterodimer binuclease fusion protein exhibits enhanced pharmacokinetic activity compared to either the first or second nuclease domain alone. Such a heterodimer binuclease fusion protein exhibits altered, for example, an improved serum half-life, compared to either the first or second nuclease domain alone.

[0006] In some embodiments, the optimized double nuclease fusion protein is shown in Figure 1.

[0007] In some embodiments, the present invention provides an optimized double nuclease fusion protein comprising human DNase1, human RNase1 and mutant human IgG1 Fc, wherein the human DNase1 is operably linked from the N-terminus to the C-terminus of human RNase1 via a linker (e.g., a gly-ser linker), the human RNase1 is operably linked to the mutant human IgG1 Fc domain via a linker, and the mutant human IgG1 has a mutant hinge region (e.g., cysteine ​​substitution with serine, e.g., SCC) and one or more CH2 mutations (e.g., P238S, P331S, or both P238S and P331S, numbering according to the EU index) to reduce Fcγ receptor binding. In one embodiment, the optimized double nuclease fusion protein comprises human DNase1 operably linked to human RNase1 via a peptide linker (e.g., gly-ser linker) (N-terminus-DNase1-linker-RNase1-C-terminus), where human RNase1 is operably linked via the peptide linker (e.g., gly-ser linker) to a mutant hinge region SCC hinge and a mutant human IgG1 Fc domain having P238S and P331S mutations. In yet another embodiment, the Fc domain further includes mutations in the N-linked glycosylation site, e.g., substitution to N297 (numbering by Kabat).

[0008] In some embodiments, the optimized double nuclease fusion protein further comprises a first linker domain, the first nuclease domain being operably linked to a second nuclease domain via the first linker domain.

[0009] In some embodiments, the optimized double nuclease fusion protein further comprises a second linker domain, the second nuclease domain being operably linked to the Fc domain via the second linker domain.

[0010] In some embodiments, the RNase domain is wild-type RNase, e.g., wild-type human RNase 1. In other embodiments, the RNase domain is mutant RNase, e.g., aglycosylated, hypoglycosylated, or deglycosylated RNase 1, e.g., human RNase 1 N34S / N76S / N88S (SEQ ID NO: 28). In some embodiments, the RNase-containing optimized double nuclease fusion protein degrades circulating RNA and RNA in immune complexes, inhibits interferon-alpha production, or both. In yet another embodiment, the RNase activity is not less than approximately 10 times the activity of the control RNase molecule, e.g., less than 9 times, less than 8 times, less than 7 times, less than 6 times, less than 5 times, less than 4 times, less than 3 times, or less than 2 times. In yet another embodiment, the RNase activity is approximately equivalent to the activity of the control RNase molecule.

[0011] In some embodiments, the DNase domain is wild-type DNase, e.g., wild-type human DNase 1. In other embodiments, the DNase domain is a mutant DNase domain, e.g., mutant human DNase 1 A114F (SEQ ID NO: 21) or an aglycosylated, hypoglycosylated, or deglycosylated human DNase, e.g., mutant human DNase 1 N18S / N106S / A114F (SEQ ID NO: 24). In some embodiments, the DNase-containing optimized double nuclease fusion protein degrades circulating DNA and DNA in immune complexes, inhibits interferon-alpha production, or both. In yet other embodiments, the activity of the DNase is not less than about 10 times, e.g., less than 9 times, less than 8 times, less than 7 times, less than 6 times, less than 5 times, less than 4 times, less than 3 times, or less than 2 times, the activity of the control DNase molecule. In yet other embodiments, the activity of the DNase is approximately equivalent to the activity of the control DNase molecule.

[0012] In some embodiments, the optimized double nuclease fusion protein has a gly-ser linker that separates the first and second nuclease domains and / or the second nuclease domain from the Fc domain.

[0013] In some embodiments, the optimized double nuclease fusion protein has an increased serum half-life and / or activity compared to molecules that do not contain an Fc domain.

[0014] In some embodiments, the optimized double nuclease fusion protein may contain the mutant human DNase1 A114F domain described in SEQ ID NO: 21. In another embodiment, the optimized double nuclease fusion protein may contain the mutant human DNase1 N18S / N106S / A114F domain described in SEQ ID NO: 24. In some embodiments, the DNase domain is the mutant human DNase1 E13R / N74K / A114F / T205K (SEQ ID NO: 25). In other embodiments, the DNase domain is the mutant human DNase1 E13R / N74K / A114F / T205K / N18S / N106S (SEQ ID NO: 26).

[0015] In some embodiments, the DNase1 and RNase1 domains are aglycosylated, hypoglycosylated, or deglycosylated. In some embodiments, the DNase domain is a mutant DNase domain, e.g., mutant human DNase1, and an aglycosylated, hypoglycosylated, or deglycosylated DNase domain, e.g., aglycosylated, hypoglycosylated, or deglycosylated human DNase1. In one embodiment, human DNase1 includes a change (e.g., substitution) at one or more N-linked glycosylation sites, e.g., N18 and N106, and at least one further mutation selected from A114, E13, N74, T205, and combinations thereof. In another embodiment, human DNase1 includes a change (e.g., substitution) at N18, N106, or both N18 and N106, and further changes (e.g., substitution) at A114, E13, N74, T205, and combinations thereof. In yet another embodiment, human DNase1 includes alterations, e.g., substitutions, at N18, N106, A114, E13, N74, and T205, e.g., N18S / N106S / A114F / E13R / N74K / T205K (SEQ ID NO: 26). In yet another embodiment, the optimized double nuclease fusion protein with altered glycosylation includes the human wild-type RNase1 domain described in SEQ ID NO: 27. In yet another embodiment, the optimized double nuclease fusion protein with altered glycosylation includes the human mutant RNase1 N34S / N76S / N88S domain described in SEQ ID NO: 28.

[0016] In some embodiments, the present invention provides an optimized double nuclease fusion protein comprising a polypeptide having the amino acid sequences described in SEQ ID NOs: 1 to 17. In other embodiments, the optimized double nuclease fusion protein has an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequences described in SEQ ID NOs: 1 to 17.

[0017] In some embodiments, the optimized double nuclease fusion protein comprises a polypeptide comprising a first nuclease domain, a second nuclease domain, and an Fc domain, wherein the first nuclease domain is DNase1, the second nuclease domain is RNase1, and DNase1 is operably tandem-linked from the N-terminus to the C-terminus of RNase1, either by or without a linker, and RNase1 is operably linked to the Fc region, either by or without a linker. In some embodiments, RNase1 is operably linked to the N-terminus of the Fc domain without a linker. In some embodiments, RNase1 is operably linked to the C-terminus of the Fc domain without a linker. In some embodiments, DNase1 is operably linked to RNase1 via a linker. In some embodiments, the polypeptide comprises a tandem double nuclease fusion protein having the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the tandem double nuclease fusion protein is a homodimer containing any of the above polypeptides.

[0018] In some embodiments, the optimized double nuclease fusion protein is 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, and DNase1 is operably linked to the N-terminus or C-terminus of the first Fc domain, with or without a linker, and RNase1 is operably linked to the N-terminus or C-terminus of the second Fc domain, with or without a linker. In some embodiments of the above heterodimer, DNase1 is operably linked to the N-terminus of the first Fc domain without a linker, and RNase1 is operably linked to the N-terminus of the second Fc domain without a linker.

[0019] In some embodiments, DNase1 is operably linked to the N-terminus of the first Fc domain by a linker, and RNase1 is operably linked to the N-terminus of the second Fc domain by a linker. In some embodiments, DNase1 is operably linked to the N-terminus of the first Fc domain by a linker, and RNase1 is operably linked to the C-terminus of the second Fc domain without a linker. In some embodiments, DNase1 is operably linked to the N-terminus of the first Fc domain without a linker, and RNase1 is operably linked to the C-terminus of the second Fc domain without a linker. In some embodiments, DNase1 is operably linked to the N-terminus of the first Fc domain by a linker, and RNase1 is operably linked to the C-terminus of the second Fc domain by a linker. In some embodiments, DNase1 is operably linked to the C-terminus of a first Fc domain by a linker, and RNase1 is operably linked to the C-terminus of a second Fc domain by a linker. In some embodiments, DNase1 is operably linked to the C-terminus of a first Fc domain without a linker, and RNase1 is operably linked to the C-terminus of a second Fc domain without a linker. In some embodiments, DNase1 is operably linked to the C-terminus of a first Fc domain by a linker, and RNase1 is operably linked to the N-terminus of a second Fc domain without a linker. In some embodiments, DNase1 is operably linked to the C-terminus of a first Fc domain by a linker, and RNase1 is operably linked to the N-terminus of a second Fc domain by a linker.

[0020] In some embodiments, the optimized double nuclease fusion protein comprises (i) a first polypeptide containing the amino acid sequence described in SEQ ID NO: 3, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 3; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 4, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 4, or (ii) A first polypeptide containing the amino acid sequence described in SEQ ID NO: 7, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 7; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 8, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 8, or (iii) A first polypeptide containing the amino acid sequence described in SEQ ID NO: 9, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 9; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 10, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 10, or (iv) A first polypeptide containing the amino acid sequence described in SEQ ID NO: 11, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 11; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 12, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 12, or (v) A first polypeptide containing the amino acid sequence described in SEQ ID NO: 15, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 15; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 16, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 16. It is a heterodimer comprising a first and a second polypeptide sequence selected from the group consisting of.

[0021] Another aspect is 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, (i) The DNase1 is operably linked to the N-terminus of the first Fc domain either by a linker or without a linker, and the RNase1 is operably linked to the C-terminus of the first Fc domain either by a linker or without a linker, or (ii) The RNase1 is operably linked to the N-terminus of the first Fc domain either by a linker or without a linker, and the DNase1 is operably linked to the C-terminus of the first Fc domain either by a linker or without a linker.

[0022] In some embodiments of the above heterodimer, DNase1 is operably linked to the N-terminus of the first Fc domain without a linker, and RNase1 is operably linked to the C-terminus of the first Fc domain by a linker. In some embodiments, DNase1 is operably linked to the N-terminus of the first Fc domain by a linker, and RNase1 is operably linked to the C-terminus of the first Fc domain without a linker (with a without a linker). In some embodiments, RNase1 is operably linked to the N-terminus of the first Fc domain without a linker, and DNase1 is operably linked to the C-terminus of the first Fc domain by a linker. In some embodiments, RNase1 is operably linked to the N-terminus of the first Fc domain by a linker, and DNase1 is operably linked to the C-terminus of the first Fc domain by a linker.

[0023] In some embodiments, the heterodimer comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5; and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 6, or (ii) a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 14 and comprises first and second polypeptide sequences selected from the group consisting of

[0024] In some embodiments, both of the above heterodimers comprise one or more CH3 mutations in the Fc domain for preferentially forming the heterodimer. In some embodiments, the heterodimer comprises a first Fc domain comprising the CH3 mutations T350V, L351Y, F405A and Y407V and a second Fc domain comprising the CH3 mutations T350V, T366L, K392L, T394W (numbering follows the EU index).

[0025] Other aspects of the present disclosure relate to a composition comprising any of the above heterodimers and a pharmaceutically acceptable carrier. Also disclosed are nucleic acid molecules encoding the above heterodimers, recombinant expression vectors, and host cells transformed with the recombinant expression vectors, as well as methods of making the above heterodimers.

[0026] A method for producing a tandem-optimized double nuclease fusion protein as disclosed herein, comprising providing a host cell containing a nucleic acid sequence encoding the optimized double nuclease fusion protein, and maintaining the host cell under conditions in which the optimized double nuclease fusion protein is expressed, is also disclosed herein.

[0027] Methods for treating or preventing conditions related to abnormal immune responses are also disclosed herein by administering an effective amount of the optimized double nuclease fusion protein disclosed herein to patients who require treatment or prevention of conditions related to abnormal immune responses. In some embodiments, the conditions are autoimmune diseases. In some embodiments, the autoimmune diseases are insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyroidopathy, pernicious anemia, autoimmune atrophic gastritis, IgG4-related disease, Addison's disease, premature menopause, male infertility, juvenile diabetes mellitus, and Goodpasture disease. The group is selected from the following: leukemia syndrome, pemphigus vulgaris, bullous pemphigoid, sympathetic ophthalmitis, lens-induced uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-VE, idiopathic cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid LE, systemic lupus erythematosus (SLE), and connective tissue disease. In some embodiments, the autoimmune disease is SLE or Sjögren's syndrome.

[0028] Also disclosed herein are methods for treating SLE or Sjögren's syndrome, comprising administering to a subject a composition containing an optimized double nuclease fusion protein in an amount effective for degrading an immune complex containing RNA, DNA, or both RNA and DNA. In some embodiments, the composition comprises a pharmaceutically acceptable carrier and the optimized double nuclease fusion protein described herein. In other embodiments, the composition comprises an optimized double nuclease fusion protein having the amino acid sequence described in Sequence ID No. 1.

[0029] In another embodiment, the present invention relates to an optimized double nuclease fusion protein for use in the treatment of diseases characterized by impaired clearance or processing of apoptotic cells and cellular debris, such as SLE. In some embodiments, the optimized double nuclease fusion protein comprises the amino acid sequences described in SEQ ID NOs: 4 and 5.

[0030] In another embodiment, the present invention relates to the use of an optimized double nuclease fusion protein for producing a pharmaceutical for treating diseases characterized by impaired clearance or processing of apoptotic cells and cellular debris, such as SLE. In some embodiments, the optimized double nuclease fusion protein comprises the amino acid sequences described in SEQ ID NOs. 5 and 6.

[0031] In another embodiment, the present invention relates to the use of an optimized double nuclease fusion protein for producing a pharmaceutical for treating diseases characterized by impaired clearance or processing of apoptotic cells and cellular debris, such as SLE. In some embodiments, the optimized double nuclease fusion protein comprises the amino acid sequences described in SEQ ID NOs: 7 and 8.

[0032] In another embodiment, the present invention relates to the use of an optimized double nuclease fusion protein for producing a pharmaceutical for treating diseases characterized by impaired clearance or processing of apoptotic cells and cellular debris, such as SLE. In some embodiments, the optimized double nuclease fusion protein comprises the amino acid sequences described in SEQ ID NOs: 13 and 14.

[0033] In another embodiment, the present invention relates to the use of an optimized double nuclease fusion protein for producing a pharmaceutical for treating diseases characterized by impaired clearance or processing of apoptotic cells and cellular debris, such as SLE. In some embodiments, the optimized double nuclease fusion protein comprises the amino acid sequences described in SEQ ID NOs. 15 and 16.

[0034] These and other features, embodiments, and advantages of the present invention will be better understood with reference to the following description and accompanying drawings. In certain embodiments, for example, the following items are provided: (Item 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, the second nuclease domain is RNase1, the DNase1 is operably linked to the N-terminus or C-terminus of the first Fc domain, with or without a linker, and the RNase1 is operably linked to the N-terminus or C-terminus of the second Fc domain, with or without a linker. (Item 2) The heterodimer according to item 1, wherein the DNase1 is operably linked to the N-terminus of the first Fc domain without a linker, and the RNase1 is operably linked to the N-terminus of the second Fc domain without a linker. (Item 3) The heterodimer according to item 1, wherein the DNase 1 is operably linked to the N-terminus of the first Fc domain by a linker, and the RNase 1 is operably linked to the N-terminus of the second Fc domain by a linker. (Item 4) The heterodimer according to item 1, wherein the DNase1 is operably linked to the N-terminus of the first Fc domain by a linker, and the RNase1 is operably linked to the C-terminus of the second Fc domain without a linker. (Item 5) The heterodimer according to item 1, wherein the DNase 1 is operably linked to the N-terminus of the first Fc domain without a linker, and the RNase 1 is operably linked to the C-terminus of the second Fc domain without a linker. (Item 6) The heterodimer according to item 1, wherein the DNase 1 is operably linked to the N-terminus of the first Fc domain by a linker, and the RNase 1 is operably linked to the C-terminus of the second Fc domain by a linker. (Item 7) The heterodimer according to item 1, wherein the DNase 1 is operably linked to the C-terminus of the first Fc domain by a linker, and the RNase 1 is operably linked to the C-terminus of the second Fc domain by a linker. (Item 8) The heterodimer according to item 1, wherein the DNase 1 is operably linked to the C-terminus of the first Fc domain without a linker, and the RNase 1 is operably linked to the C-terminus of the second Fc domain without a linker. (Item 9) The heterodimer according to item 1, wherein the DNase1 is operably linked to the C-terminus of the first Fc domain by a linker, and the RNase1 is operably linked to the N-terminus of the second Fc domain without a linker. (Item 10) The heterodimer according to item 1, wherein the DNase 1 is operably linked to the C-terminus of the first Fc domain by a linker, and the RNase 1 is operably linked to the N-terminus of the second Fc domain by a linker. (Item 11) The heterodimer according to any one of items 1 to 10, wherein the RNase is wild-type human RNase 1 or a mutant RNase, such as aglycosylated, hypoglycosylated, or deglycosylated RNase 1, such as human RNase 1 N34S / N76S / N88S. (Item 12) The heterodimer described in item 11, wherein the RNase is wild-type human RNase 1. (Item 13) A heterodimer according to any one of items 1 to 12, wherein the DNase is wild-type human DNase1, mutant human DNase1 A114F, or aglycosylated, hypoglycosylated, or deglycosylated mutant human DNase1 N18S / N106S / A114F. (Item 14) A heterodimer according to any of the above items, wherein the first and second Fc domains include a hinge domain, a CH2 domain, and a CH3 domain. (Item 15) The heterodimer described in item 14, wherein the first and second Fc domains contain one or more substitutions of serine cysteine ​​residues in the three hinge region. (Item 16) The heterodimer described in item 15, wherein the first and second Fc domains contain mutations selected from the group consisting of SCC, SSS (residues 220, 226, and 229), G236R, L328R, L234A, and L235A (numbering follows the EU index). (Item 17) The heterodimer described in item 16, wherein the first and second Fc domains contain SCC mutations (residues 220, 226, and 229) (numbering follows the EU index). (Item 18) A heterodimer as described in any of the above items, wherein the first and second Fc domains contain the P238S and P331 mutations (numbering follows the EU index). (Item 19) A heterodimer according to any of the above items, wherein the first and second Fc domains contain one or more CH3 mutations for preferential heterodimer formation. (Item 20) The heterodimer described in item 19, wherein the first Fc domain contains CH3 mutants T350V, L351Y, F405A, and Y407V, and the second Fc domain contains CH3 mutants T350V, T366L, K392L, and T394W (numbering follows the EU index). (Item 21) The heterodimer according to any of the above items, wherein the linker domain is a polypeptide linker, for example, a gly-ser linker or an NLG linker (vdgasspvnvsspsvqdi). (Item 22) (i) A first polypeptide containing the amino acid sequence described in SEQ ID NO: 3, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 3; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 4, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 4, or (ii) A first polypeptide containing the amino acid sequence described in SEQ ID NO: 7, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 7; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 8, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 8, or (iii) A first polypeptide containing the amino acid sequence described in SEQ ID NO: 9, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 9; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 10, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 10, or (iv) A first polypeptide containing the amino acid sequence described in SEQ ID NO: 11, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 11; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 12, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 12, or (v) A first polypeptide containing the amino acid sequence described in SEQ ID NO: 15, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 15; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 16, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 16. A heterodimer comprising a first and a second polypeptide sequence selected from the group consisting of the following. (Item 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. (i) The DNase 1 is operably linked to the N-terminus of the first Fc domain by or without a linker, and the RNase 1 is operably linked to the C-terminus of the first Fc domain by or without a linker, or (i) A heterodimer in which RNase1 is operably linked to the N-terminus of the first Fc domain by or without a linker, and DNase1 is operably linked to the C-terminus of the first Fc domain by or without a linker. (Item 24) The heterodimer according to item 23, wherein the DNase1 is operably linked to the N-terminus of the first Fc domain without a linker, and the RNase1 is operably linked to the C-terminus of the first Fc domain by a linker. (Item 25) The heterodimer according to item 23, wherein the DNase1 is operably linked to the N-terminus of the first Fc domain by a linker, and the RNase1 is operably linked to the C-terminus of the first Fc domain by a linker without a linker. (Item 26) The heterodimer according to item 23, wherein the RNase1 is operably linked to the N-terminus of the first Fc domain without a linker, and the DNase1 is operably linked to the C-terminus of the first Fc domain by a linker. (Item 27) The heterodimer according to item 23, wherein the RNase1 is operably linked to the N-terminus of the first Fc domain by a linker, and the DNase1 is operably linked to the C-terminus of the first Fc domain by a linker. (Item 28) The heterodimer according to any one of items 23 to 27, wherein the RNase is wild-type human RNase 1 or a mutant RNase, such as aglycosylated, hypoglycosylated, or deglycosylated RNase 1, such as human RNase 1 N34S / N76S / N88S. (Item 29) The heterodimer described in item 28, wherein the RNase is wild-type human RNase 1. (Item 30) A heterodimer according to any one of items 23 to 29, wherein the DNase is wild-type human DNase1, mutant human DNase1 A114F, or aglycosylated, hypoglycosylated, or deglycosylated mutant human DNase1 N18S / N106S / A114F. (Item 31) A heterodimer according to any of the above items, wherein the first and second Fc domains include a hinge domain, a CH2 domain, and a CH3 domain. (Item 32) The heterodimer described in item 31, wherein the first and second Fc domains contain one or more substitutions of the three hinge region cysteine ​​residues by serine. (Item 33) The heterodimer described in item 32, wherein the first and second Fc domains contain mutations selected from the group consisting of SCC, SSS (residues 220, 226, and 229), G236R, L328R, L234A, and L235A (numbering follows the EU index). (Item 34) The heterodimer described in item 33, wherein the first and second Fc domains contain SCC mutations (residues 220, 226, and 229) (numbering follows the EU index). (Item 35) A heterodimer as described in any of the above items, wherein the first and second Fc domains contain the P238S and P331 mutations (numbering follows the EU index). (Item 36) A heterodimer according to any of the above items, wherein the first and second Fc domains contain one or more CH3 mutations for preferential heterodimer formation. (Item 37) The heterodimer described in item 36, wherein the first Fc domain contains CH3 mutants T350V, L351Y, F405A, and Y407V, and the second Fc domain contains CH3 mutants T350V, T366L, K392L, and T394W (numbering follows the EU index). (Item 38) The heterodimer according to any of the above items, wherein the linker domain is a polypeptide linker, for example, a gly-ser linker or an NLG linker (vdgasspvnvsspsvqdi). (Item 39) (i) A first polypeptide containing the amino acid sequence described in SEQ ID NO: 5, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 5; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 6, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 6, or (ii) A first polypeptide containing the amino acid sequence described in SEQ ID NO: 13, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 13; and a second polypeptide containing the amino acid sequence described in SEQ ID NO: 14, or a polypeptide containing an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 14. A heterodimer comprising a first and a second polypeptide sequence selected from the group consisting of the following. (Item 40) A composition comprising a heterodimer described in any of the above items and a pharmaceutically acceptable carrier. (Item 41) A nucleic acid molecule that encodes a heterodimer as described in any of the above items. (Item 42) A recombinant expression vector containing the nucleic acid molecule described in item 41. (Item 43) Host cells transformed with the recombinant expression vector described in item 42. (Item 44) A method for producing a heterodimer according to any one of items 1 to 39, comprising providing a host cell containing a nucleic acid sequence encoding the heterodimer; and maintaining the host cell under conditions in which the heterodimer is expressed. (Item 45) A method for treating or preventing a condition associated with an abnormal immune response, comprising administering an effective amount of any one of items 1 to 39 to a subject. (Item 46) The method described in item 45, wherein the aforementioned condition is an autoimmune disease. (Item 47) The method according to item 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, experimental uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes mellitus, Goodpasture syndrome, pemphigus vulgaris, bullous pemphigoid, sympathetic ophthalmitis, lens-induced uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-ve, idiopathic cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid LE, systemic lupus erythematosus (SLE), and connective tissue disease. (Item 48) The method described in item 47, wherein the autoimmune disease is SLE. (Item 49) The method described in item 47, wherein the aforementioned autoimmune disease is Sjögren's syndrome. (Item 50) A method for treating SLE, comprising administering to a subject an amount of a heterodimer effective for degrading an immune complex containing RNA, DNA, or both RNA and DNA, wherein the composition comprises a pharmaceutically acceptable carrier and a heterodimer as described in any one of items 1 to 39. (Item 51) A method for treating Sjögren's syndrome, comprising administering to a subject an amount of a heterodimer effective for degrading an immune complex containing RNA, DNA, or both RNA and DNA, wherein the composition comprises a pharmaceutically acceptable carrier and a heterodimer as described in any one of items 1 to 39. [Brief explanation of the drawing]

[0035] [Figure 1] Figure 1 is a depiction of an exemplary optimized double nuclease fusion protein.

[0036] [Figure 2]Figure 2 is a graph showing RNase activity measured by OD260.

[0037] [Figure 3] Figure 3 shows DNase activity measured by OD620 (left) and IC50 (right). [Modes for carrying out the invention]

[0038] Detailed explanation Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by the presence of high-titer autoantibodies against the body's own nucleoproteins. There is strong evidence that impaired clearance or processing of dead and dying cells in SLE leads to disease primarily through the accumulation of ribonucleoproteins and deoxyribonucleoproteins (abbreviated as nucleoproteins). Nucleoproteins cause damage through three mechanisms: i) activating the innate immune system to produce inflammatory cytokines; ii) acting as antigens to generate circulating immune complexes; and iii) acting as antigens to cause in situ complex formation at local sites such as the kidneys.

[0039] The present invention provides a method for treating diseases characterized by impaired clearance or processing of apoptotic cells and cellular debris, such as SLE and Sjögren's syndrome, by administering an effective amount of long-acting nuclease activity to degrade extracellular immune complexes containing RNA and DNA. Such treatment can inhibit the production of type I interferon (IFN), a prominent cytokine in SLE that is strongly correlated with disease activity and nephritis.

[0040] The present invention relates in part to providing such long-acting nucleases. In particular, the present invention relates to an optimized binuclease fusion protein, for example, a tandem binuclease fusion protein comprising a first nuclease domain, a second nuclease domain and an Fc region, wherein the first nuclease domain is DNase1, the second nuclease domain is RNase1, the DNase1 is operably linked in tandem from the N-terminus to the C-terminus of the RNase1, or with or without a linker, and the RNase1 is operably linked to the N-terminus or C-terminus of the Fc region.

[0041] In other embodiments, the present invention relates to an optimized binuclease fusion protein, for example, a heterodimer binuclease fusion protein comprising a first nuclease domain, a second nuclease domain, and an Fc region, wherein the first nuclease domain is DNase1, the second nuclease domain is RNase1, the DNase1 is operably linked to the N-terminus or C-terminus of the Fc region, with or without a linker, and the RNase1 is operably linked to the N-terminus or C-terminus of the Fc region, with or without a linker, thereby forming a heterodimer.

[0042] In some embodiments, optimized dual nuclease fusion proteins exhibit enhanced pharmacokinetic activity compared to either the first or second nuclease domain alone. Such optimized dual nuclease fusion proteins exhibit altered, for example, an improved serum half-life compared to either the first or second nuclease domain alone.

[0043] In some embodiments, the present invention provides an optimized double nuclease fusion protein comprising human DNase1, human RNase1 and mutant human IgG1 Fc, wherein the human DNase1 is operably linked from the N-terminus to the C-terminus of human RNase1 via a linker (e.g., a gly-ser linker), the human RNase1 is operably linked to the mutant human IgG1 Fc domain via a linker, and the mutant human IgG1 has a mutant hinge region (e.g., cysteine ​​substitution with serine, e.g., SCC) and one or more CH2 mutations (e.g., P238S, P331S, or both P238S and P331S, numbering according to the EU index) to reduce Fcγ receptor binding. In one embodiment, the optimized double nuclease fusion protein comprises human DNase1 operably linked to human RNase1 via a peptide linker (e.g., gly-ser linker) (N-terminus-DNase1-linker-RNase1-C-terminus), where human RNase1 is operably linked via the peptide linker (e.g., gly-ser linker) to a mutant hinge region SCC hinge and a mutant human IgG1 Fc domain having P238S and P331S mutations.

[0044] Therefore, in one embodiment, subjects having a disease characterized by impaired clearance or processing of apoptotic cells and cellular debris are treated by administering an optimized double nuclease fusion protein containing both DNase1 and RNase1 such that the optimized double nuclease fusion protein has increased bioavailability and / or serum half-life compared to the non-conjugate nuclease domain.

[0045] definition The claims and terms used herein are defined as follows, unless otherwise specified:

[0046] "Amino acids" refer to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as subsequently modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, namely hydrogen, a carboxyl group, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have a modified R group (e.g., norleucine) or a modified peptide skeleton, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimes refer to compounds that have a different structure from the general chemical structure of amino acids, but function similarly to naturally occurring amino acids.

[0047] Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may also be referred to by their commonly recognized single-letter codes.

[0048] "Amino acid substitution" refers to the replacement of at least one existing amino acid residue in a given amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different "replacement" amino acid residue. "Amino acid insertion" refers to the incorporation of at least one additional amino acid into a given amino acid sequence. Insertions typically consist of the insertion of one or two amino acid residues, but larger "peptide insertions," e.g., insertions of about 3 to 5, or even up to about 10, 15, or 20 amino acid residues, may occur. The inserted residues may be naturally occurring or may be non-naturally occurring as disclosed above. "Amino acid deletion" refers to the removal of at least one amino acid residue from a given amino acid sequence.

[0049] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and unnaturally occurring amino acid polymers.

[0050] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides, and polymers thereof in either single-stranded or double-stranded form. Unless otherwise specifically limited, the term encompasses nucleic acids containing known analogues of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized similarly to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res 1991;19:5081; Ohtsuka et al., JBC 1985;260:2605-8; Rossolini et al., Mol Cell Probes 1994;8:91-8). For arginine and leucine, modifications at the second base can also be conserved. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by gene.

[0051] The polynucleotides of the present invention may consist of any polyribonucleotide or polydeoxyribonucleotide (these may be unmodified RNA or DNA, or modified RNA or DNA). For example, polynucleotides may consist of single-stranded and double-stranded DNA, DNA which is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA which is a mixture of single-stranded and double-stranded regions, and hybrid molecules containing DNA and RNA (which may be single-stranded, more typically double-stranded, or a mixture of single-stranded and double-stranded regions). In addition, polynucleotides may consist of RNA or DNA, or triple-stranded regions containing both RNA and DNA. Polynucleotides may also contain one or more modified bases or DNA or RNA backbone modified for stability or for other reasons. Examples of “modified” bases include tritylated bases and special bases, such as inosine. Various modifications can be made to DNA and RNA; therefore, “polynucleotide” encompasses chemically, enzymatically, or metabolically modified forms.

[0052] As used herein, the terms “operably linked” or “operably coupled” refer to a juxtaposition of components that are in a relationship that enables them to function as intended.

[0053] As used herein, the terms "glycosylation" or "glycosylated" refer to the process or result of adding a sugar moiety to a molecule (e.g., an optimized double nuclease fusion protein).

[0054] As used herein, the term “altered glycosylation” refers to aglycosylated, deglycosylated, or deglycosylated molecule.

[0055] As used herein, the term “glycosylation site” refers to both a site that is potentially capable of accepting a carbohydrate moiety and a site within a protein to which a carbohydrate moiety is actually attached that contains any amino acid sequence capable of acting as an acceptor for oligosaccharides and / or carbohydrates.

[0056] As used herein, the terms “aglycosylation” or “aglycosylated” refer to the production of a non-glycosylated form of a molecule (e.g., an optimized double nuclease fusion protein) (e.g., by manipulating an optimized double nuclease fusion protein to lack an amino acid residue that functions as a glycosylation acceptor). Alternatively, an aglycosylated optimized double nuclease fusion protein may be produced, for example, by expressing an optimized double nuclease fusion protein in E. coli.

[0057] As used herein, the terms “desglycosylation” or “desglycosylated” refer to the process or result of enzymatic removal of a sugar portion from a molecule.

[0058] As used herein, the terms “low-glycosylated” or “low-glycosylated” refer to a molecule in which one or more carbohydrate structures that would normally be present when produced in mammalian cells are omitted, removed, modified, or shielded.

[0059] As used herein, the terms “Fc region” and “Fc domain” refer to a portion of a native immunoglobulin formed by each Fc domain (or Fc portion) of its two heavy chains, which does not have a variable region that binds to an antigen. In some embodiments, the Fc domain begins in the hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc domain includes at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc domain includes at least one of the hinge (e.g., upper, middle, and / or lower hinge regions) domains, a CH2 domain, a CH3 domain, a CH4 domain, or variants, portions, or fragments thereof. In other embodiments, the Fc domain includes a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In one embodiment, the Fc domain includes a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In another embodiment, the Fc domain includes a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In another embodiment, the Fc domain consists of a CH3 domain or a portion thereof. In another embodiment, the Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In another embodiment, the Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In another embodiment, the Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In one embodiment, the Fc domain lacks at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). In one embodiment, the Fc domain of the present invention includes at least a portion of the Fc molecule known in the art to be necessary for FcRn binding. In one embodiment, the Fc domain of the present invention includes at least a portion of the Fc molecule known in the art to be necessary for protein A binding. In one embodiment, the Fc domain of the present invention includes at least a portion of the Fc molecule known in the art to be necessary for protein G binding.In this specification, the term Fc domain generally refers to a polypeptide containing all or part of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides containing the entirety of the CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides containing, for example, only the hinge, CH2, and CH3 domains. The Fc domain may originate from any species and / or any subtype of immunoglobulin, including, but is not limited to, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. The term Fc domain encompasses both native Fc molecules and Fc variant molecules. Like Fc variants and native Fc, the term Fc domain includes molecules in monomeric or polymeric form, whether digested from a whole antibody or produced by other means.

[0060] As described herein, those skilled in the art will understand that any Fc domain can be modified so that its amino acid sequence differs from that of the native Fc domain of naturally occurring immunoglobulin molecules.

[0061] The Fc domain of the optimized double nuclease fusion protein of this disclosure may be derived from different immunoglobulin molecules. For example, the Fc domain of the optimized double nuclease fusion protein may include CH2 and / or CH3 domains derived from the IgG1 molecule, as well as a hinge region derived from the IgG3 molecule. In another example, the Fc domain may include a chimeric hinge region partially derived from the IgG1 molecule and partially derived from the IgG3 molecule. In yet another example, the Fc domain may include a chimeric hinge partially derived from the IgG1 molecule and partially derived from the IgG4 molecule. The wild-type human IgG1 Fc domain has the amino acid sequence described in Sequence ID No. 45.

[0062] As used herein, the term “serum half-life” refers to the time required for the in vivo serum concentration of optimized double nuclease fusion protein to decrease by 50%. A shorter serum half-life of the optimized double nuclease fusion protein will result in a shorter time required to exert a therapeutic effect.

[0063] As used herein, the term “optimized double nuclease fusion protein” refers to a polypeptide comprising at least two nuclease domains operably linked to an Fc domain or a variant or fragment thereof, with or without a linker, and the nucleic acid encoding such a polypeptide. In some embodiments, the optimized double nuclease fusion protein is a tandem double nuclease fusion protein, for example, in which one or more DNase1 domains and one or more RNase1 domains are tandem linked to either the N-terminus or C-terminus of one or more Fc domains. In some embodiments, the optimized double nuclease fusion protein is a heterodimer double nuclease fusion protein.

[0064] As used herein, the term “tandem double nuclease fusion protein” refers to a polypeptide comprising at least two nuclease domains linked in tandem (from N-terminus to C-terminus) and an Fc domain or a variant or fragment thereof, and the nucleic acid encoding such a polypeptide. For example, in one embodiment, a tandem double nuclease fusion protein is a polypeptide comprising at least one DNase1 domain and at least one RNase1 domain operably linked to at least one Fc domain. In another example, a tandem double nuclease fusion protein comprises, from N-terminus to C-terminus, a DNase1 domain, a first linker, an RNase1 domain, a second linker, and an Fc domain or a variant or fragment thereof.

[0065] As used herein, the term “heterodimerated double nuclease fusion protein” refers to a heterodimer comprising a first and second polypeptide each containing at least two nuclease domains, and two Fc domains or variants or fragments thereof, and the nucleic acid encoding such polypeptides. In some embodiments, the heterodimerated double nuclease fusion protein is a heterodimer comprising at least one DNase1 domain and at least one RNase1 domain operably linked to at least one Fc domain, wherein the DNase1 domain is operably linked to the N-terminus or C-terminus of the first Fc domain, with or without a linker, such that the DNase1 domain and the RNase1 domain are located at the opposite end (N-terminus or C-terminus) of either the same (first Fc domain) or a different Fc domain (second Fc domain), and the RNase1 domain is operably linked to the N-terminus or C-terminus of the same (first Fc domain) or a different Fc domain (second Fc domain), with or without a linker, such that the DNase1 domain and the RNase1 domain are located at the opposite end (N-terminus or C-terminus) of either the same (first Fc domain) or a different Fc domain (second Fc domain). In some embodiments, the heterodimer includes a DNase1 domain operably linked to the N-terminus or C-terminus of a first Fc domain, with or without a linker, and an RNase1 domain operably linked to the N-terminus or C-terminus of a second Fc domain, with or without a linker, such that the DNase1 and RNase1 domains are located in tandem at the same end (N-terminus or C-terminus) of the heterodimer. In some embodiments, the heterodimer includes a DNase1 domain operably linked to the N-terminus of a first Fc domain, with or without a linker, and an RNase1 domain operably linked to the C-terminus of a first Fc domain, with or without a linker. In some embodiments, RNase1 is operably linked to the N-terminus of a first Fc domain, with or without a linker, and DNase1 is operably linked to the C-terminus of a first Fc domain, with or without a linker.

[0066] As used herein, the term “variant” refers to a polypeptide derived from a wild-type nuclease or Fc domain that differs from the wild-type due to one or more alterations at one or more positions, i.e., substitution, insertion, and / or deletion. Substitution means the replacement of an amino acid occupying a position with a different amino acid. Deletion means the removal of an amino acid occupying a position. Insertion means the addition of one or more amino acids directly adjacent to an amino acid occupying a position, for example, 1 to 3 amino acids. Variant polypeptides always have less than 100% sequence identity or similarity to the wild-type polypeptide. In some embodiments, the variant polypeptide may have about 75% to less than 100% amino acid sequence identity or similarity to the wild-type polypeptide, or, for example, about 80% to less than 100%, about 85% to less than 100%, or about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) or about 95% to less than 100% amino acid sequence identity or similarity to the full length of the variant polypeptide.

[0067] In certain embodiments, the optimized double nuclease fusion protein utilizes one or more “linker domains,” such as polypeptide linkers. As used herein, the term “linker domain” refers to one or more amino acids that connect two or more peptide domains in a linear polypeptide sequence. As used herein, the term “polypeptide linker” refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) that connects two or more polypeptide domains in a linear amino acid sequence of a protein. For example, a polypeptide linker may be used to operably link the first and second nuclease domains to each other, or to link the first or second nuclease domain to an Fc domain. In some embodiments, such polypeptide linkers provide flexibility to the polypeptide molecule. In some embodiments, a polypeptide linker is used to link DNase1 to RNase1 and / or RNase1 to an Fc domain (e.g., genetically fuse). The optimized double nuclease fusion protein may contain one or more linker domains or peptide linkers. Various peptide linkers are known in this field.

[0068] As used herein, the term “gly-ser polypeptide linker” refers to a peptide comprising a glycine residue and a serine residue. An exemplary gly / ser polypeptide linker comprises the amino acid sequence (Gly4Ser)n (SEQ ID NO: 58). In some embodiments, n is 1 or greater, e.g., 2 or greater, 3 or greater, 4 or greater, 5 or greater, 6 or greater, 7 or greater, 8 or greater, 9 or greater, or 10 or greater (e.g., (Gly4Ser)10 (SEQ ID NO: 59)). Another exemplary gly / ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser)n (SEQ ID NO: 60). In some embodiments, n is 1 or greater, for example 2 or greater, 3 or greater, 4 or greater, 5 or greater, 6 or greater, 7 or greater, 8 or greater, 9 or greater, or 10 or greater (for example, Ser(Gly4Ser)10(SEQ ID NO: 61)).

[0069] As used herein, the terms “coupled,” “linked,” “fused,” and “fused” are interchangeable. These terms refer to the linking of two or more elements, components, or domains to one another by any means, including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art.

[0070] A polypeptide sequence or amino acid sequence "derived from" a specified polypeptide or protein refers to the origin of the polypeptide. Preferably, a polypeptide sequence or amino acid sequence derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or portion thereof (which consists of at least 10 to 20 amino acids, preferably at least 20 to 30 amino acids, more preferably at least 30 to 50 amino acids), or has an amino acid sequence in the sequence that can be otherwise identified by a person skilled in the art as having its origin. A polypeptide derived from another peptide may have one or more mutations compared to the starting polypeptide, for example, one or more amino acid residues that are substituted with another amino acid residue, or one or more amino acid residue insertions or deletions.

[0071] In one embodiment, there is a one-amino acid difference between the starting polypeptide sequence and the sequence derived therefrom. This sequence identity or similarity is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., the same residue) to the starting amino acid residue, after the sequences have been aligned and gaps introduced as necessary to achieve the maximum percentage of sequence identity.

[0072] In one embodiment, the polypeptides of the Disclosure consist of, are essentially, or include functionally active amino acid sequences and functionally active variants of the amino acid sequences listed in the sequence list or sequence listing disclosed herein. In embodiments, the polypeptides include amino acid sequences that are at least 80%, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequences listed in the sequence list or sequence listing disclosed herein. In some embodiments, the sequence includes a sequence that is at least 80%, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of amino acids described in the sequence list or sequence listing disclosed herein. In some embodiments, the polypeptide includes an amino acid sequence having at least 10 consecutive amino acids from the sequence list or sequence listing disclosed herein, for example, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 200, at least 300, at least 400 or at least 500 (or any integer within this range) of amino acid sequences.

[0073] In some embodiments, the optimized double nuclease fusion proteins of the Disclosure are encoded by nucleotide sequences. The nucleotide sequences of the Disclosure may be useful for many applications, including cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of hosts requiring it, antibody production for passive immunization, PCR, primer and probe generation, and siRNA design and production (see, for example, the Dharmacon siDesign website). In some embodiments, the nucleotide sequences of the Disclosure include, consist of, or are essentially composed of nucleotide sequences encoding the amino acid sequence of an optimized double nuclease fusion protein selected from a sequence listing or sequence list. In some embodiments, the nucleotide sequence includes a nucleotide sequence that is at least 80%, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence encoding the amino acid sequence of the sequence list or sequence listing disclosed herein. In some embodiments, the nucleotide sequence includes a sequence nucleotide sequence that is at least 80%, for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence nucleotide sequence that encodes the amino acid sequence described in the sequence list or sequence listing disclosed herein.In some embodiments, the nucleotide sequence includes a nucleotide sequence having at least 10, for example, at least 15, for example, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 200, at least 300, at least 400 or at least 500 (or any integer within this range) consecutive nucleotides of a nucleotide sequence encoding an amino acid sequence listed in the sequence list or sequence listing disclosed herein.

[0074] Furthermore, those skilled in the art will understand that optimized double nuclease fusion proteins can be modified so that their components (e.g., nuclease domain, linker domain, and Fc domain) have sequences different from the naturally occurring or native sequences from which they originate, while retaining the desired activity of the native sequences. For example, nucleotide or amino acid substitutions can be made that result in conserved substitutions or changes at "non-essential" amino acid residues. Isolated nucleic acid molecules encoding non-native variants can be created by introducing one or more nucleotide substitutions, additions, or deletions into the nucleotide sequence of an optimized double nuclease fusion protein, so that one or more amino acid substitutions, additions, or deletions are introduced into the encoding protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0075] Optimized double nuclease fusion proteins may include conserved amino acid substitutions in one or more amino acid residues, for example, in essential or non-essential amino acid residues. A “conserved amino acid substitution” is one in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the Art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues in the optimized double nuclease fusion protein are preferably replaced by other amino acid residues from the same side-chain family. In another embodiment, a sequence of amino acid chains may be replaced by structurally similar chains with different order and / or composition of members of the side-chain family. Alternatively, in another embodiment, mutations may be introduced randomly across all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants may be incorporated into the optimized double nuclease fusion protein and screened for their ability to bind to the desired target.

[0076] The term "improvement" refers to any therapeutically beneficial outcome in the treatment of a disease condition, such as an autoimmune disease condition (e.g., SLE, Sjögren's syndrome), including its prevention, reduction of severity or progression, remission, or cure.

[0077] The term "in situ" refers to a process that occurs in living cells that are growing independently of other organisms (for example, in tissue culture).

[0078] The term "in vivo" refers to a process that occurs in living organisms.

[0079] As used herein, the terms “mammal,” “subject,” or “patient” include, but are not limited to, both human and non-human animals, including humans, non-human primates, canids, felines, rodents, bovines, equids, and pigs.

[0080] In the context of two or more nucleic acid sequences or polypeptide sequences, the term “identity” percentage refers to two or more sequences or subsequences that have a specified percentage of nucleotide or amino acid residues that are identical when compared and aligned for maximum match using one of the following sequence comparison algorithms (e.g., BLASTP and BLASTN, or other algorithms available to those skilled in the art) or measured by visual inspection. Depending on the application, the “identity” percentage may exist across a region of the sequences being compared, e.g., a functional domain, or alternatively, across the entire length of the two sequences being compared.

[0081] For sequence comparison, typically, one sequence acts as a reference sequence for comparison with the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into a computer, and the coordinates of subsequences are specified as needed to specify the parameters of the sequence algorithm program. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence to the reference sequence based on the specified program parameters.

[0082] The optimal sequence alignment for comparison is, for example, by the local homology algorithm in Smith & Waterman, Adv Appl Math 1981;2:482, by the homology alignment algorithm in Needleman & Wunsch, J Mol Biol 1970;48:443, and by Pearson & Lipman, PNAS. Similarity search methods described in 1988;85:2444 can be performed by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.) or by visual inspection (see Ausubel et al. below for general information).

[0083] One example of a suitable algorithm for determining the percentage of sequence identity and sequence similarity is the BLAST algorithm, described by Altschul et al., J Mol Biol 1990;215:403-10. Software for performing BLAST analysis is available through the National Center for Biotechnology Information website.

[0084] The term "sufficient amount" means a quantity sufficient to produce the desired effect.

[0085] The term "therapeutic dose" refers to the amount that is effective in improving the symptoms of a disease. Since prevention can be considered a treatment, the therapeutic dose may also be called the "preventive dose."

[0086] The term "approximately" will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. If a term not obvious to those skilled in the art is used, considering the context in which it is used, "approximately" will mean within ±10% of a given value.

[0087] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless otherwise specifically indicated by the context.

[0088] Optimized dual nuclease fusion protein The optimized double nuclease fusion protein of this disclosure comprises an Fc domain or a variant or fragment thereof that alters the serum half-life of the nuclease molecule to which it is fused, compared to a nuclease molecule not fused to the Fc domain or a variant or fragment thereof.

[0089] In some embodiments, the compositions of the present disclosure include an optimized double nuclease fusion protein. In some embodiments, the optimized double nuclease fusion protein includes a nuclease domain operably coupled to an Fc domain or a variant or fragment thereof.

[0090] In some embodiments, the nuclease domain is operably coupled to the Fc domain or a variant or fragment thereof via a linker domain. In some embodiments, the linker domain is a linker peptide. In some embodiments, the linker domain is a linker nucleotide.

[0091] In some embodiments, the optimized double nuclease fusion protein includes a leader molecule, e.g., a leader peptide. In some embodiments, the leader molecule is a leader peptide located at the N-terminus of the nuclease domain. In some embodiments, the optimized double nuclease fusion protein of the present invention includes a leader peptide at the N-terminus of the molecule, and the leader peptide is subsequently cleaved from the optimized double nuclease fusion protein. Methods for generating nucleic acid sequences encoding a leader peptide fused to a recombinant protein are well known in the art. In some embodiments, any of the optimized double nuclease fusion proteins of the present invention may be expressed with or without the leader fused to their N-terminus. The protein sequences of the optimized double nuclease fusion proteins of this disclosure after cleavage of the fused leader peptide can be predicted and / or estimated by those skilled in the art.

[0092] In some embodiments, the leader is a VK3 leader peptide (VK3LP), which is fused to the N-terminus of an optimized double nuclease fusion protein. Such a leader sequence can enhance the levels of synthesis and secretion of the optimized double nuclease fusion protein in mammalian cells. In some embodiments, the leader is cleaved to produce an optimized double nuclease fusion protein. In some embodiments, the optimized double nuclease fusion protein of the present invention is expressed without the leader peptide fused to its N-terminus, and the resulting optimized double nuclease fusion protein has an N-terminal methionine.

[0093] In some embodiments, the optimized double nuclease fusion protein comprises two nuclease domains tadem operably coupled to each other, and further comprising two nuclease domains operably coupled to the N-terminus or C-terminus of the same or different Fc domain or variant or fragment thereof.

[0094] Figure 1 shows an exemplary configuration of the optimized double nuclease fusion protein, and the sequence listing provides sequences of exemplary optimized double nuclease fusion proteins in various configurations.

[0095] In some embodiments, the optimized double nuclease fusion protein is a multinuclease protein (e.g., both or two RNA or DNA nucleases, RNase and DNase, having different substrate specificities) fused to the same or different Fc domains or variants or fragments thereof that specifically bind to the extracellular immune complex.

[0096] In one embodiment, the nuclease domain is operably coupled to the N-terminus of the Fc domain or its variant or fragment (e.g., chemically conjugated (e.g., directly or via a polypeptide linker) or genetically fused). In another embodiment, the nuclease domain is operably coupled to the C-terminus of the Fc domain or its variant or fragment (e.g., chemically conjugated (e.g., directly or via a polypeptide linker) or genetically fused). In yet another embodiment, the nuclease domain is operably coupled via the amino acid side chain of the Fc domain or its variant or fragment (e.g., chemically conjugated (e.g., directly or via a polypeptide linker) or genetically fused).

[0097] In certain embodiments, the optimized double nuclease fusion protein of this disclosure comprises two or more nuclease domains and at least one Fc domain or a variant or fragment thereof. For example, a nuclease domain may be operably coupled to both the N-terminus and C-terminus of the same or different Fc domain or variant or fragment by an optional linker between the nuclease domain and the Fc domain, its variant or fragment. In some embodiments, the nuclease domains are identical (e.g., RNase and RNase, or DNase1 and DNase1). In other embodiments, the nuclease domains are different (e.g., DNase and RNase).

[0098] In some embodiments, two or more nuclease domains are operably coupled in series with each other (e.g., via a polypeptide linker), and tandem-aligned nuclease domains are operably coupled to either the N-terminus or C-terminus of the same or different Fc domain or its variant or fragment (e.g., chemically conjugated (e.g., directly or via a polypeptide linker) or genetically fused). In other embodiments, tandem-aligned nuclease domains are operably coupled to both the N-terminus and C-terminus of the same Fc domain or its variant or fragment. In some embodiments, nuclease domains are operably tandem (e.g., N-DNase-RNase-C or N-RNase-DNase-C) to the N-terminus or C-terminus of the same or different Fc domain, with or without a linker. In some embodiments, the tandem double nuclease fusion protein forms a homodimer or a heterodimer.

[0099] In other embodiments, one or more nuclease domains may be inserted between two Fc domains or variants or fragments thereof. For example, one or more nuclease domains may form all or part of the polypeptide linker of the optimized double nuclease fusion protein of the Disclosure.

[0100] In some embodiments, the optimized double nuclease fusion protein comprises at least two nuclease domains (e.g., RNase and DNase), at least one linker domain, and at least one Fc domain or a variant or fragment thereof.

[0101] In some embodiments, the optimized double nuclease fusion protein of the present disclosure comprises an Fc domain or a variant or fragment thereof, as described above, thereby increasing the serum half-life and bioavailability of the optimized double nuclease fusion protein.

[0102] In some embodiments, the optimized double nuclease fusion protein comprises one or more polypeptides, for example, a polypeptide having an amino acid sequence shown in any of SEQ ID NOs: 1 to 17.

[0103] Those skilled in the art will understand that other configurations of nuclease domains and Fc domains are possible by including optional linkers between nuclease domains and / or between nuclease domains and Fc domains. They will also understand that the orientation of the domains can be altered, as long as the nuclease domains are active in the particular configuration being tested.

[0104] In certain embodiments, the optimized double nuclease fusion protein of the Disclosure has at least one nuclease domain specific to a target molecule that mediates a biological effect. In other embodiments, binding of the optimized double nuclease fusion protein of the Disclosure to a target molecule (e.g., DNA or RNA) results in a reduction or elimination of the target molecule, for example, from cells, tissues, or circulation.

[0105] In other embodiments, the optimized double nuclease fusion proteins of the present disclosure may be assembled with each other or other polypeptides to form a binding protein having two or more polypeptides ("multimer"), where at least one polypeptide of the multimer is the optimized double nuclease fusion protein of the present invention. Exemplary multimer forms include dimers, trimers, tetramers, and hexamer change-binding proteins. In one embodiment, the polypeptides of the multimer are the same (i.e., homomeric change-binding proteins, e.g., homodimer, homotetramer). In another embodiment, the polypeptides of the multimer are different (e.g., heteromeric).

[0106] In some embodiments, the optimized double nuclease fusion protein has a serum half-life that is increased by at least about 1.5 times, for example, at least 3 times, at least 5 times, at least 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1000 times, or 1000 times or more, compared to the corresponding nuclease molecule that is not fused to the Fc domain or its variant or fragment. In other embodiments, the optimized double nuclease fusion protein has a serum half-life that is reduced by at least about 1.5 times, for example, at least 3 times, at least 5 times, at least 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or 500 times or less, compared to the corresponding nuclease molecule that is not fused to the Fc domain or its variant or fragment. Routine methods accepted in the art may be used to determine the serum half-life of the optimized double nuclease fusion protein of this disclosure.

[0107] In some embodiments, the activity of the RNase in the optimized double nuclease fusion protein is not less than approximately 10 times, e.g., less than 9 times, less than 8 times, less than 7 times, less than 6 times, less than 5 times, less than 4 times, less than 3 times, or less than 2 times, compared to the activity of the control RNase molecule. In some embodiments, the activity of the RNase in the optimized double nuclease fusion protein is approximately equivalent to the activity of the control RNase molecule.

[0108] In some embodiments, the activity of DNase in the optimized double nuclease fusion protein is not less than approximately 10 times, e.g., less than 9 times, less than 8 times, less than 7 times, less than 6 times, less than 5 times, less than 4 times, less than 3 times, or less than 2 times, compared to the activity of the control DNase molecule. In some embodiments, the activity of DNase in the optimized double nuclease fusion protein is approximately equivalent to the activity of the control DNase molecule.

[0109] In some embodiments, the optimized double nuclease fusion protein may be active against extracellular immune complexes containing, for example, DNA and / or RNA deposited in soluble or insoluble forms.

[0110] In some embodiments, the activity of the optimized double nuclease fusion protein is detectable in vitro and / or in vivo. In some embodiments, the optimized double nuclease fusion protein binds to cells, malignant cells, or cancer cells and interferes with their biological activity.

[0111] In another embodiment, a multifunctional RNase or DNase molecule attached to an scFv is provided, which has binding specificity to another enzyme or antibody, for example, a multifunctional RNase or DNase molecule that targets RNA or DNA or a second nuclease domain with the same or different specificity as the first domain.

[0112] In some embodiments, the linker domain includes (gly4ser)3,4, or5 variants that alter the linker length by advancing it by 5 amino acids. In other embodiments, the linker domain is approximately 18 amino acids long and includes an N-linked glycosylation site that may be sensitive to protease cleavage in vivo. In some embodiments, the N-linked glycosylation site may protect the optimized double nuclease fusion protein from cleavage in the linker domain. In some embodiments, the N-linked glycosylation site may assist in the separation of folding of independent functional domains isolated by the linker domain.

[0113] In some embodiments, the linker domain is the NLG linker (VDGASSPVNVSSPSVQDI) (SEQ ID NO: 41).

[0114] In some embodiments, the optimized double nuclease fusion protein comprises substantially all or at least an enzymatically active fragment of DNase. In some embodiments, the DNase is type I secretory DNase, preferably human DNase, e.g., mature human pancreatic DNase 1 (UniProtKB entry P24855, SEQ ID NO: 20). In some embodiments, the naturally occurring variant allele A114F (SEQ ID NO: 21), which exhibits reduced actin sensitivity, is included in the DNase 1 optimized double nuclease fusion protein (see Pan et al., JBC 1998;273:18374-81; Zhen et al., BBRC 1997;231:499-504; Rodriguez et al., Genomics 1997;42:507-13). In other embodiments, the naturally occurring variant allele G105R (SEQ ID NO: 22), which exhibits higher DNase activity compared to wild-type DNase1, is included in the DNase1 optimized double nuclease fusion protein (see Yasuda et al., Int J Biochem Cell Biol 2010;42:1216-25). In some embodiments, this mutation is introduced into the optimized double nuclease fusion protein to produce a more stable derivative of human DNase1. In some embodiments, the DNase is either human wild-type DNase1 or human DNase1 A114F (i.e., human DNase1 N18S / N106S / A114F, SEQ ID NO: 24) mutated to remove all potential N-linked glycosylation sites, i.e., the asparagine residues at positions 18 and 106 of the DNase1 domain described in SEQ ID NO: 20 (which correspond to the asparagine residues at positions 40 and 128, respectively, of full-length pancreatic DNase1 with a native leader (SEQ ID NO: 23)).

[0115] In some embodiments, the DNase is human DNase 1 containing one or more basic (i.e., positively charged) amino acid substitutions to increase DNase functionality and chromatin cleavage. In some embodiments, basic amino acids are introduced to the DNA-binding surface of human DNase 1 to enhance binding to charged phosphate on the DNA substrate (see U.S. Patent No. 7,407,785; U.S. Patent No. 6,391,607). This overactive DNase 1 may be referred to as a "chromatin cutter."

[0116] In some embodiments, one, two, three, four, five, or six basic amino acid substitutions are introduced into DNase1. For example, one or more of the following residues are mutated to enhance DNA binding: Gln9, Glu13, Thr14, His44, Asn74, Asn110, Thr205. In some embodiments, one or more of the above amino acids are substituted with basic amino acids, such as arginine, lysine, and / or histidine. For example, mutant human DNase may contain one or more of the following substitutions: Q9R, E13R, T14K, H44K, N74K, N110R, T205K. In some embodiments, mutant human DNase1 also contains the A114F substitution that reduces actin sensitivity (see U.S. Patent No. 6,348,343). In one embodiment, the mutant human DNase1 includes the following substitutions: E13R, N74K, A114F, and T205K.

[0117] In some embodiments, mutant human DNase1 further includes mutations to remove potential glycosylation sites, e.g., asparagine residues at positions 18 and 106 of the DNase1 domain described in SEQ ID NO: 20 (which correspond to asparagine residues at positions 40 and 128, respectively, of full-length pancreatic DNase1 with a native leader). In one embodiment, mutant human DNase1 includes the following substitution: E13R / N74K / A114F / T205K / N18S / N106S.

[0118] In some embodiments, DNase is DNase1-like (DNaseL) enzymes 1-3 (UniProtKB entry Q13609; SEQ ID NO: 46). In some embodiments, DNase is 3-prime repair exonuclease 1 (TREX1; UniProtKB entry Q9NSU2; SEQ ID NO: 47). In some embodiments, DNase is DNase2. In some embodiments, DNase2 is DNAse2α (i.e., DNase2; UnitProtKB entry O00115; SEQ ID NO: 48) or DNase2β (i.e., DNase2-like acid DNase; UnitProtKB entry Q8WZ79; SEQ ID NO: 49). In some embodiments, the N-linked glycosylation site of DNase1L3, TREX1, DNase2α, or DNase2β is mutated to remove a potential N-linked glycosylation site. In some embodiments, a DNase-linker-Fc domain containing 20 or 25aa linker domains is constructed.

[0119] In some embodiments, the optimized double nuclease fusion protein comprises RNase1 of the RNaseA family, preferably human pancreatic RNase1 (UniProtKB entry P07998; SEQ ID NO: 27). In some embodiments, human RNase1 is mutated to remove potential N-linked glycosylation sites, i.e., asparagine residues at positions 34, 76, and 88 of the RNase1 domain described in SEQ ID NO: 27 (this corresponds to asparagine residues at positions 62, 104, and 116, respectively, of full-length pancreatic RNase1 with a native leader (SEQ ID NO: 29) (human RNase1 N34S / N76S / N88S, SEQ ID NO: 28). In some embodiments, an RNase1-linker-Fc containing 20 or 25aa linker domains is constructed.

[0120] In some embodiments, the optimized double nuclease fusion protein comprises DNase-linker-RNase-Fc, with the RNase1 domain located on the COOH side of Fc. In other embodiments, the optimized double nuclease fusion protein comprises DNase-linker-RNase-Fc, with the RNase1 domain located on the NH2 side of Fc. In some embodiments, the optimized double nuclease fusion protein comprises DNase-Fc and RNase-Fc; DNase1-Fc-linker-RNase and Fc domains; DNase1-Fc and Fc-linker-RNase; Fc-linker-DNase1 and Fc-linker-RNase; RNase-Fc-linker-DNase and Fc domains; Fc-linker-DNase and RNase-Fc; and RNase-Fc-linker-DNase.

[0121] In some embodiments, the fusion conjugation between the enzyme domain of the optimized double nuclease fusion protein and the other domain is optimized.

[0122] In some embodiments, the target of the RNase enzyme activity of the optimized double nuclease fusion protein is primarily extracellular, consisting of RNA contained in immune complexes, for example, with anti-RNP autoantibodies, and RNA expressed on the surface of apoptotic cells. In some embodiments, the optimized double nuclease fusion protein is active in the acidic environment of endocytic vesicles. In some embodiments, the optimized double nuclease fusion protein, including an Fc domain or a variant or fragment thereof, is adapted to be active in both extracellular and endocytic environments. In some embodiments, this allows the optimized double nuclease fusion protein, including a wild-type Fc domain or a variant or fragment thereof, to halt TLR7 signaling by pre-phagocytosed immune complexes or by RNA that activates TLR7 after viral infection. In some embodiments, the wild-type RNase of the optimized double nuclease fusion protein is not resistant to inhibition by RNase cytoplasmic inhibitors. In some embodiments, the wild-type RNase of the optimized double nuclease fusion protein is not active in the cytoplasm of cells.

[0123] In some embodiments, the optimized double nuclease fusion protein contains both DNase and RNase. In some embodiments, these optimized double nuclease fusion proteins digest or degrade immune complexes containing RNA, DNA, or a combination of both RNA and DNA, and are active extracellularly, thus improving the treatment of SLE.

[0124] FC Domain In some embodiments, polypeptides comprising one or more nuclease domains are operably coupled to Fc domains that function as scaffolds and as means for increasing the serum half-life of the polypeptide. In some embodiments, one or more nuclease domains and / or Fc domains are aglycosylated, deglycosylated, or hypoglycosylated.

[0125] Suitable Fc domains are well known in the art and are not limited to Fc and Fc variants, such as those disclosed in International Publication No. 2011 / 053982, International Publication No. 02 / 060955, International Publication No. 02 / 096948, International Publication No. 05 / 047327, International Publication No. 05 / 018572 and U.S. Patent Application Publication No. 2007 / 0111281 (the contents of which are incorporated herein by reference). It is within the capabilities of those skilled in the art to use routine methods (e.g., cloning, conjugation) to introduce Fc domains (with or without altered glycosylation) into optimized double nuclease fusion proteins disclosed herein.

[0126] In some embodiments, the Fc domain is, for example, wild-type human IgG1 Fc as shown in SEQ ID NO: 45.

[0127] In some embodiments, the Fc domain is altered or modified by mutations resulting in, for example, amino acid addition, deletion, or substitution. As used herein, the term “Fc domain variant” refers to an Fc domain having at least one amino acid modification, e.g., an amino acid substitution, compared to the wild-type Fc from which the Fc domain is derived. For example, if the Fc domain is derived from a human IgG1 antibody, the variant includes at least one amino acid mutation (e.g., substitution) compared to the wild-type amino acid at the corresponding position in the human IgG1 Fc region. The amino acid substitution in the Fc variant may be located at a position within the Fc domain where the residue is considered to correspond to a position number given to it in the Fc region of the antibody (numbering follows the EU index).

[0128] In one embodiment, the Fc variant includes one or more amino acid substitutions at amino acid positions located in or part of the hinge region. In another embodiment, the Fc variant includes one or more amino acid substitutions at amino acid positions located in or part of the CH2 domain. In another embodiment, the Fc variant includes one or more amino acid substitutions at amino acid positions located in or part of the CH3 domain. In another embodiment, the Fc variant includes one or more amino acid substitutions at amino acid positions located in or part of the CH4 domain.

[0129] In some embodiments, the Fc region has a mutation at N83 (i.e., N297 according to Kabat numbering), resulting in an aglycosylated Fc region (e.g., Fc N83S; SEQ ID NO: 50). In some embodiments, the Fc domain contains a mutation in one or more of the three hinged cysteine ​​regions (residues 220, 226, and 229, numbering according to the EU index). In some embodiments, one or more of the three hinged cysteines in the Fc domain may be mutated to SCC (SEQ ID NO: 51) or SSS (SEQ ID NO: 52), where "S" represents an amino acid substitution of cysteine ​​by serine. Therefore, "SCC" indicates that only the first cysteine ​​of the three hinge region cysteines is substituted with serine (residues 220, 226, and 229, numbering follows the EU index), while "SSS" indicates that all three cysteines in the hinge region are substituted with serine (residues 220, 226, and 229, numbering follows the EU index).

[0130] In some embodiments, the Fc domain is a mutant human IgG1 Fc domain. In some embodiments, the mutant Fc domain includes one or more mutations in the hinge domain, the CH2 domain, and / or the CH3 domain.

[0131] CH2 substitution In some embodiments, the mutant Fc domain contains the P238S mutation. In some embodiments, the mutant Fc domain contains the P331S mutation. In some embodiments, the mutant Fc domain contains both the P238S and P331S mutations. In some embodiments, the mutant Fc domain contains P238S and / or P331S, and may contain mutations in one or more of the three hinge cysteine ​​residues (residues 220, 226, and 229) (numbering follows the EU index). In some embodiments, the mutant Fc domain contains P238S and / or P331S, and / or one or more mutations in the three hinge cysteine ​​residues (residues 220, 226, and 229) (numbering follows the EU index). In some embodiments, the mutant Fc domain includes mutations in P238S and / or P331S, and / or SCC in hinge cysteine, or mutations in SSS in three hinge cysteine. In some embodiments, the mutant Fc domain includes mutations in P238S and P331S, and at least one of the three hinge cysteine. In some embodiments, the mutant Fc domain includes P238S and P331S and SCC. In some embodiments, the mutant Fc domain includes P238S and P331S and SSS. In some embodiments, the mutant Fc domain includes P238S and SCC or SSS. In some embodiments, the mutant Fc domain includes P331S and SCC or SSS. (All numbering follows the EU index).

[0132] In some embodiments, the mutant Fc domain includes a mutation at an N-linked glycosylation site such as N297, for example, a substitution of asparagine with another amino acid such as serine, e.g., N297S. In some embodiments, the mutant Fc domain includes a mutation at an N-linked glycosylation site such as N297, for example, a substitution of asparagine with another amino acid such as serine, e.g., N297S, and a mutation in one or more of the three hingecysteines. In some embodiments, the mutant Fc domain includes a mutation at an N-linked glycosylation site such as N297, for example, a substitution of asparagine with another amino acid such as serine, e.g., N297S, and a mutation to SCC in one of the three hingecysteines, or a mutation to SSS in all three hingecysteines. In some embodiments, the mutant Fc domain includes mutations in N-linked glycosylation sites such as N297, e.g., substitution of asparagine with another amino acid such as serine, e.g., N297, and one or more mutations in the CH2 domain that reduce FcγR binding and / or complement activation, e.g., mutations in P238 or P331 or both, e.g., P238S or P331S or both P238S and P331S. In some embodiments, such mutant Fc domains may further include mutations in the hinge region, e.g., SCC or SSS. (All numbering follows the EU index). In some embodiments, the mutant Fc domains are as shown in the sequence listing or sequence list herein.

[0133] CH3 substitution Heterodimers can preferentially be formed by mutations in the CH3 domain of the Fc domain on the heterodimer double nuclease fusion proteins disclosed herein. Initially, the heavy chain was manipulated for heterodimerization using the “knob-into-hole” strategy (Rigway B et al., Protein Eng., 9(1996) pp.617-621). The term “knob-into-hole” refers to a technique that induces two polypeptides to pair with each other in vitro or in vivo by introducing a convex portion (knob) into one polypeptide and a concave portion (hole) into the other polypeptide at their interaction surface. See, for example, International Publication No. 96 / 027011, International Publication No. 98 / 050431, U.S. Patent No. 5,731,168, U.S. Patent Application Publication No. 2007 / 0178552, International Publication No. 2009089004, and U.S. Patent Application Publication No. 20090182127. In particular, combinations of mutations in the CH3 domain, such as S354C, T366W in the “knob” heavy chain and Y349C, T366S, L368A, Y407V in the “hole” heavy chain, may be used to preferentially form heterodimers. In some embodiments, the heterodimeric double nuclease fusion proteins disclosed herein include a first CH3 domain having the knob mutation T366W and a second CH3 domain having the hole mutations T366S, L368A, and Y407V. (Numbering follows the EU index).

[0134] In some embodiments, the CH3 mutation is derived from Zymeworks (U.S. Patent Application Publication No. 2012 / 0149876, which is incorporated herein by reference) and Von As described by Kreudenstein, TS et al., mABs, 5 (2013), pp. 646-654), the following mutations are included: T350V, L351Y, F405A, and Y407V (first CH3 domain); and T350V, T366L, K392L, and T394W (second CH3 domain). In some embodiments, the heterodimeric double nuclease fusion proteins disclosed herein include a first CH3 domain having the T350V, L351Y, F405A, and Y407V mutations and a second CH3 domain having the T350V, T366L, K392L, and T394W mutations. (Numbering follows EU index).

[0135] In some embodiments, the CH3 mutations are those described by Moore, GL et al. (mABs, 3(2011), pp. 546-557), including the following mutations: S364H and F405A (first CH3 domain); and Y349T and T394F (second CH3 domain). In some embodiments, the heterodimeric double nuclease fusion proteins disclosed herein include a first CH3 domain having the S364H and F405A mutations and a second CH3 domain having the Y349T and T394F mutations. (Numbering follows EU index).

[0136] In some embodiments, the CH3 mutations are those described by Gunasekaran, K et al. (J. Biol. Chem., 285 (2010), pp. 19637-19646), including the following mutations: K409D and K392D (first CH3 domain); and D399K and E365K (second CH3 domain). In some embodiments, the heterodimeric double nuclease fusion proteins disclosed herein include a first CH3 domain having the K409D and K392D mutations and a second CH3 domain having the D399K and E365K mutations. (Numbering follows EU index).

[0137] The optimized double nuclease fusion proteins of this disclosure may use Fc variants known to confer effector function and / or alteration of FcR binding, which are recognized in the art.For example, International Publication No. 88 / 07089, International Publication No. 96 / 14339, International Publication No. 98 / 05787, International Publication No. 98 / 23289, International Publication No. 99 / 51642, International Publication No. 99 / 58572, International Publication No. 00 / 09560, International Publication No. 00 / 32767, International Publication No. 00 / 42072, International Publication No. 02 / 44215, International Publication No. 02 / 060919, International Publication No. 03 / 074569, International Publication No. 04 / 016750, International Publication No. 04 / 029207, International Publication No. 04 / 035752, International Publication No. 0 International Publication No. 4 / 063351, International Publication No. 04 / 074455, International Publication No. 04 / 099249, International Publication No. 05 / 040217, International Publication No. 04 / 044859, International Publication No. 05 / 070963, International Publication No. 05 / 077981, International Publication No. 05 / 092925, International Publication No. 05 / 123780, International Publication No. 06 / 019447, International Publication No. 06 / 047350 and International Publication No. 06 / 085967; U.S. Patent Application Publication No. 2007 / 0231329, U.S. Patent Application Publication No. 2007 / 0231329, U.S. Patent Application Publication U.S. Patent Application Publication No. 2007 / 0237765, U.S. Patent Application Publication No. 2007 / 0237766, U.S. Patent Application Publication No. 2007 / 0237767, U.S. Patent Application Publication No. 2007 / 0243188, U.S. Patent Application Publication No. 20070248603, U.S. Patent Application Publication No. 20070286859, U.S. Patent Application Publication No. 20080057056; or U.S. Patent No. 5,648,260; U.S. Patent No. 5,739,277; U.S. Patent No. 5,834,250; U.S. Patent No. 5,869,046; U.S. Patent No. 6,096,871; U.S. Patent No. 6 Changes (e.g., substitutions) at one or more amino acid positions disclosed in U.S. Patent No. 121,022; U.S. Patent No. 6,194,551; U.S. Patent No. 6,242,195; U.S. Patent No. 6,277,375; U.S. Patent No. 6,528,624; U.S. Patent No. 6,538,124; U.S. Patent No. 6,737,056; U.S. Patent No. 6,821,505; U.S. Patent No. 6,998,253; U.S. Patent No. 7,083,784; and U.S. Patent No. 7,317,091 (each of which is incorporated herein by reference).In one embodiment, a specific modification (e.g., a specific substitution of one or more amino acids disclosed in the Art) may be made at one or more of the disclosed amino acid positions. In another embodiment, a different modification (e.g., a different substitution of one or more amino acid positions disclosed in the Art) may be made at one or more of the disclosed amino acid positions.

[0138] Other amino acid mutations in the Fc domain are thought to reduce binding to the Fcγ receptor and Fcγ receptor subtypes. The assignment of amino acid residue numbers to the Fc domain follows Kabat's definition. For example, see Sequences of Proteins. See also: of Immunological Interest (Table of Contents, Introduction, and Constant Region Sequences sections), 5th edition, Bethesda, MD:NIH vol.1:647-723 (1991); Kabat et al., "Introduction," Sequences of Proteins of Immunological Interest, US Dept of Health and Human Services, NIH, 5th edition, Bethesda, MD vol.1:xiii-xcvi (1991); Chothia & Lesk, J.Mol.Biol.196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989) (these are incorporated herein by reference for all purposes).

[0139] For example, as described in U.S. Patent No. 6,737,056 issued on 18 May 2004 (which is incorporated herein by reference in its entirety), the locations of Fc areas 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294 Mutations in 295, 296, 298, 301, 303, 305, 307, 312, 315, 322, 324, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 356, 360, 373, 376, 378, 379, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439 can alter binding. This patent reported that changing Pro331 to Ser in IgG3 resulted in a 6-fold lower affinity compared to non-mutant IgG3, indicating the involvement of Pro331 in FcγRI binding. In addition, U.S. Patent No. 5,624,821, issued on 29 April 1997 (which is incorporated herein by reference in its entirety), discloses that amino acid modifications at positions 234, 235, 236 and 237, 297, 318, 320 and 322 potentially alter receptor binding affinity. (Numbering follows EU index).

[0140] Further variations intended for use include, for example, those described in U.S. Patent Application Publication No. 2006 / 0235208, published on 19 October 2006 (which is incorporated herein by reference in its entirety). This publication includes Fc variants that exhibit reduced binding to the Fcγ receptor, reduced antibody-dependent cell-mediated cytotoxicity, or reduced complement-dependent cytotoxicity, specifically 232G, 234G, 234H, 235D, 235G, 235H, 236I, 236N, 236P, 236R, 237K, 237L, 237N, 237P, 238K, 239R, 265G, 267R, 269R, 270H, 297S, 299A, 299I, 299V, 325A, 325L, 327R, 328R, 329K, 330I, 330L, 330N, 330P, 3 Fc variants including 30R and 331L (numbering follows the EU index), which include at least one amino acid modification in the Fc region, as well as double mutants 236R / 237K, 236R / 325L, 236R / 328R, 237K / 325L, 237K / 328R, 325L / 328R, 235G / 236R, 267R / 269R, 234G / 235G, 236R / 237K / 325L, 236R / 325L / 328R, 235G / 236R / 237K, and 237K / 325L / 328R are described. Other variants intended for use as described in this publication include 227G, 234D, 234E, 234G, 234I, 234Y, 235D, 235I, 235S, 236S, 239D, 246H, 255Y, 258H, 260H, 2641, 267D, 267E, 268D, 268E, 272H, 272I, 272R, 281D, 282G, 283H, 284E, 293R, 295E, 304T, 324G, 324I, 327D, 327A, 3 28A, 328D, 328E, 328F, 328I, 328M, 328N, 328Q, 328T, 328V, 328Y, 330I, 330L, 330Y, 332D, 332E, 335D, insertion of G between positions 235 and 236, insertion of A between positions 235 and 236, insertion of S between positions 235 and 236, insertion of T between positions 235 and 236, insertion of N between positions 235 and 236, insertion of D between positions 235 and 236,Insertion of V between positions 235 and 236, insertion of L between positions 235 and 236, insertion of G between positions 235 and 236, insertion of A between positions 235 and 236, insertion of S between positions 235 and 236, insertion of T between positions 235 and 236, insertion of N between positions 235 and 236, insertion of D between positions 235 and 236, insertion of V between positions 235 and 236, insertion of L between positions 235 and 236, position 29 Examples include the insertion of G between 7 and 298, the insertion of A between positions 297 and 298, the insertion of S between positions 297 and 298, the insertion of D between positions 297 and 298, the insertion of G between positions 326 and 327, the insertion of A between positions 326 and 327, the insertion of T between positions 326 and 327, the insertion of D between positions 326 and 327, and the insertion of E between positions 326 and 327 (numbering follows the EU index). In addition, the variants described in U.S. Patent Application Publication No. 2006 / 0235208 include 227G / 332E, 234D / 332E, 234E / 332E, 234Y / 332E, 234I / 332E, 234G / 332E, 235I / 332E, 235S / 332E, 235D / 332E, 235E / 332E, and 236S / 3 32E, 236A / 332E, 236S / 332D, 236A / 332D, 239D / 268E, 246H / 332E, 255Y / 332E, 258H / 332E, 260H / 332E, 264I / 332E, 267E / 332E, 267D / 332E, 268D / 332D, 268E / 332D, 268E / 332E, 268D / 332E, 268E / 330Y, 268D / 330Y, 272R / 332E, 272H / 332E, 283H / 332E, 284E / 332E, 293R / 332 E, 295E / 332E, 304T / 332E, 324I / 332E, 324G / 332E, 324I / 332D, 324G / 332D, 327D / 332E, 32 8A / 332E, 328T / 332E, 328V / 332E, 328I / 332E, 328F / 332E, 328Y / 332E, 328M / 332E, 328D / 3 32E, 328E / 332E, 328N / 332E, 328Q / 332E, 328A / 332D, 328T / 332D, 328V / 332D, 328I / 332D,328F / 332D, 328Y / 332D, 328M / 332D, 328D / 332D, 328E / 332D, 328N / 332D, 328Q / 332D, 330L / 332E, 330Y / 332E, 330I / 332E, 332D 239 D / 332E / 327D、239D / 332E / 284E、239D / 268E / 330Y、239D / 332E / 268E / 330Y、239D / 332E / 327A、239D / 332E / 268E / 327A、239D / 33 2E / 330Y / 327A, 332E / 330Y / 268E / 327A, 239D / 332E / 268E / 330Y / 327A, Insert G>297-298 / 332E, Insert A>297-298 / 332E, Insert S>297-298 / 3 32E, Insertion D>297-298 / 332E, Insertion G>326-327 / 332E, Insertion A>326-327 / 332E, Insertion T>326-327 / 332E, Insertion D>326-327 / 332E, Insertion E>326-327 / 332E, Insertion G>235-236 / 332E, Insertion A>235-236 / 332E, Insertion S>235-236 / 332E, Insertion T>235-236 / 332E, Insertion N>235-236 / 332E, Insertion D>235-236 / 332E, Insertions V>235-236 / 332E, L>235-236 / 332E, G>235-236 / 332D, A>235-236 / 332D, S>235-236 / 332D, T>235-236 / 332D, N>235-236 / 332D, D>235-236 / 332D, V>235-236 / 332D, and L>235-236 / 332D (numbering follows EU index) are examples of those intended for use. For example, U.S. Patent Application Publication No. 2003 / 0108548, published on 12 June 2003 (which is incorporated herein by reference in its entirety), describes the variant L234A / L235A. In embodiments, the modifications described may be included individually or in combination. (Numbering follows the EU index).

[0141] Linker Domain In some embodiments, the optimized double nuclease fusion protein includes a linker domain. In some embodiments, the optimized double nuclease fusion protein includes multiple linker domains. In some embodiments, the linker domain is a polypeptide linker. In certain embodiments, it is desirable to use a polypeptide linker to fuse Fc or a variant or fragment thereof to one or more nuclease domains to form the optimized double nuclease fusion protein.

[0142] 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) that comprises an amino acid sequence (which may or may not be naturally occurring) linked by a linear amino acid sequence to a sequence (which may or may not be naturally occurring) that is not originally linked (which may or may not be naturally occurring) (e.g., an Fc sequence). For example, a polypeptide linker may be a modified form of a naturally occurring polypeptide (e.g., including mutations, e.g., additions, substitutions, or deletions) or a non-naturally occurring polypeptide comprising a first amino acid sequence (which may or may not be naturally occurring). The polypeptide linker of the present invention can be used, for example, to ensure that Fc or its variant or fragment is juxtaposed to ensure proper folding and formation of a functional Fc or its variant or fragment. Preferably, a polypeptide linker compatible with the present invention will be relatively non-immunogenic and will not inhibit any non-covalent association between monomeric subunits of the binding protein.

[0143] In certain embodiments, the optimized double nuclease fusion protein uses the NLG linker described in SEQ ID NO: 41.

[0144] In certain embodiments, the optimized double nuclease fusion protein of this disclosure uses a polypeptide linker to in-frame link any two or more domains in a single polypeptide chain. In one embodiment, the two or more domains may be independently selected from any of the Fc domains or their variants or fragments or nuclease domains discussed herein. For example, in certain embodiments, the polypeptide linker may be used to fusion identical Fc fragments, thereby forming a homodimeric Fc region. In other embodiments, the polypeptide linker may be used to fusion different Fc fragments, thereby forming a heterodimeric Fc region. In other embodiments, the polypeptide linker of the present invention may be used to genetically fuse the C-terminus of a first Fc fragment to the N-terminus of a second Fc fragment to form a complete Fc domain.

[0145] In one embodiment, the polypeptide linker includes a portion of the Fc domain or a variant or fragment thereof. For example, in one embodiment, the polypeptide linker may include an Fc fragment (e.g., a C domain or an N domain) or a different portion of the Fc domain or a variant thereof.

[0146] 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 the amino acid sequence of formula (Gly4Ser)n (SEQ ID NO: 62) (wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5)). A preferred gly / ser linker is (Gly4Ser)4 (SEQ ID NO: 63). Another preferred gly / ser linker is (Gly4Ser)3 (SEQ ID NO: 30). Another preferred gly / ser linker is (Gly4Ser)5 (SEQ ID NO: 64). In certain embodiments, a gly-ser linker may be inserted between two other sequences of the polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In other embodiments, the gly-ser linker is attached to one or both ends of another sequence of the polypeptide linker (e.g., any of the polypeptide linker sequences described herein). In yet another embodiment, two or more gly-ser linkers are incorporated in series with the polypeptide linker.

[0147] In other embodiments, the polypeptide linker of the present invention includes a biologically relevant peptide sequence or a portion of its sequence. For example, the biologically relevant peptide sequence may be, but is not limited to, a sequence derived from a rejection-inhibiting peptide or an anti-inflammatory peptide. The rejection-inhibiting peptide or anti-inflammatory peptide may be selected from the group consisting of cytokine-inhibiting peptides, cell adhesion-inhibiting peptides, thrombin-inhibiting peptides, and platelet-inhibiting peptides. In preferred embodiments, the polypeptide linker includes a peptide sequence selected from the group consisting of IL-1 inhibitory or antagonist peptide sequences, erythropoietin (EPO) mimetic peptide sequences, thrombopoietin (TPO) mimetic peptide sequences, G-CSF mimetic peptide sequences, TNF antagonist peptide sequences, integrin-binding peptide sequences, selectin antagonist peptide sequences, antipathogen peptide sequences, vasoactive intestinal peptide (VIP) mimetic peptide sequences, calmodulin antagonist peptide sequences, mast cell antagonists, SH3 antagonist peptide sequences, urokinase receptor (UKR) antagonist peptide sequences, somatostatin or cortistatin mimetic peptide sequences, and macrophage and / or T cell inhibitory peptide sequences. Exemplary peptide sequences (any 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).

[0148] Other linkers suitable for use in optimized double nuclease fusion proteins are known in the art, for example, the serine-rich linker disclosed in U.S. Patent No. 5,525,491, the helix-forming peptide linker disclosed in Arai et al., Protein Eng 2001;14:529-32 (e.g., A(EAAAK)nA(n=2~5) (SEQ ID NO: 65)), and the stable linker disclosed in Chen et al., Mol Pharm 2011;8:457-65, namely the dipeptide linker LE, the thrombin-sensitive disulfide cyclopeptide linker, and the α-helix-forming linker LEA(EAAAK)4ALEA(EAAAK)4ALE (SEQ ID NO: 53).

[0149] Other exemplary linkers include the GS linker (i.e., (GS)n) (SEQ ID NO: 66), the GGSG (SEQ ID NO: 70) linker (i.e., (GGSG)n (SEQ ID NO: 67)), the GSAT linker (SEQ ID NO: 44), the SEG linker, and the GGS linker (i.e., (GGSGGS)n (SEQ ID NO: 68)) (wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5)). Other suitable linkers for use in optimized double nuclease fusion proteins can be found using publicly available databases, such as the Linker Database (ibi.vu.nl / programs / linkerdbwww). The Linker Database is a database of interdomain linkers of multifunctional enzymes that are candidate linkers in novel fusion proteins (see, e.g., George et al., Protein Engineering 2002;15:871-9).

[0150] It will be understood that variant forms 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, so that one or more amino acid substitutions, additions, or deletions are introduced into the polypeptide linker. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0151] The polypeptide linkers of this disclosure are at least one amino acid long and may be of varying lengths. In one embodiment, the polypeptide linkers of the present invention are about 1 to about 50 amino acid long. As used in this context, the term "about" indicates + / - 2 amino acid residues. Since the linker length must be a positive integer, a length of about 1 amino acid to about 50 amino acids means a length of 1 amino acid to 48 to 52 amino acids. In another embodiment, the polypeptide linkers of this disclosure are about 10 to 20 amino acid long. In yet another embodiment, the polypeptide linkers of this disclosure are about 15 to about 50 amino acid long.

[0152] In another embodiment, the polypeptide linker of the Disclosure is about 20 to about 45 amino acid lengths. In another embodiment, the polypeptide linker of the Disclosure is about 15 to about 25 amino acid lengths. In another embodiment, the polypeptide linker of the Disclosure is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61 or more amino acid lengths.

[0153] Polypeptide linkers can be introduced into polypeptide sequences using techniques known in the art. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transform host cells for stable production of the polypeptides produced.

[0154] Exemplary optimized double nuclease fusion protein The optimized double nuclease fusion protein of the present invention is modular and can be configured to incorporate various individual domains. For example, in one embodiment, the optimized double nuclease fusion protein may include the mutant human DNase1 A114F domain described in (SEQ ID NO: 21). In another embodiment, the optimized double nuclease fusion protein may include the mutant human DNase1 N18S / N106S / A114F domain described in SEQ ID NO: 24. In yet another embodiment, the optimized double nuclease fusion protein may include the human wild-type RNase1 domain described in SEQ ID NO: 27. In yet another embodiment, the optimized double nuclease fusion protein may include the human mutant RNase1 N34S / N76S / N88S domain described in SEQ ID NO: 28. In yet another embodiment, the optimized double nuclease fusion protein may include the (Gly4Ser)3 linker domain described in SEQ ID NO: 30. In yet another embodiment, the optimized double nuclease fusion protein may include the NLG linker described in SEQ ID NO: 41. In another embodiment, the optimized double nuclease fusion protein may include a VK3LP reader (SEQ ID NO: 54). Those skilled in the art will understand that these individual domains can be operably coupled to each other in any order to form an enzymatically active optimized double nuclease fusion protein. For example, as detailed in the following specific examples, RNase1 can be operably coupled to an Fc domain. In another example, RNase1 can be operably coupled to an Fc domain via a (Gly4Ser)3 (SEQ ID NO: 30) linker domain. In yet another example, DNase1 A114F can be operably coupled to an Fc domain. In yet another example, DNase1 A114F can be operably coupled to an Fc domain via a (Gly4Ser)3 (SEQ ID NO: 30) linker domain. A variety of other configurations are possible using the non-limiting exemplary configurations disclosed herein, in Figure 1 and in the sequence listing.

[0155] In some embodiments, the optimized double nuclease fusion protein comprises a wild-type human RNase1 domain operably coupled to a mutant Fc domain or fragment thereof containing an SCC hinge and CH2 mutants P238S and P331S, and a mutant human DNase1 domain operably coupled to human RNase1, thereby forming a tandem homodimer. In some embodiments, DNase1 is linked to RNase1 via a peptide linker, such as the NLG linker disclosed herein. In some embodiments, RNase1 is operably linked to the N-terminus of the Fc domain, either by or without a linker. In some embodiments, the optimized double nuclease fusion protein comprises a polypeptide having the amino acid sequence described in SEQ ID NO: 1. In some embodiments, RNase1 is operably linked to the C-terminus of the Fc domain, either by or without a linker. In some embodiments, the optimized double nuclease fusion protein comprises a polypeptide having the amino acid sequence described in SEQ ID NO: 2. In some embodiments, the optimized double nuclease fusion protein is a homodimer or a heterodimer.

[0156] In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising, by or without a linker, an SCC hinge and a mutant human DNase1 domain operably coupled to a first mutant Fc domain or a variant or fragment thereof having CH2 mutations P238S, P331S and CH3 mutations T350V, L351Y, F405A, and Y407V, and by or without a linker, an SCC hinge and a wild-type human RNase1 domain operably coupled to a second mutant Fc domain or fragment thereof having CH2 mutations P238S and P331S and CH3 mutations T350V, T366L, K392L, and T394W. In some embodiments, both DNase1 and RNase1 are ligated to the N-terminus of their respective Fc domains. In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising a polypeptide containing the amino acid sequence described in SEQ ID NO: 3 and a polypeptide containing the amino acid sequence described in SEQ ID NO: 4.

[0157] In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising a mutant human DNase1 domain and a wild-type human RNase1 domain, both operably coupled by a linker or not to an SCC hinge and a first mutant Fc domain or fragment thereof containing CH2 mutations P238S and P331S and CH3 mutations T350V, T366L, K392L and T394W, and a second mutant Fc domain or fragment thereof having mutations T350V, T366L, K392L and T394W. In some embodiments, DNase1 and RNase1 are ligated to the N-terminus and C-terminus of the first and second Fc domains, respectively. In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising a polypeptide containing the sequence described in SEQ ID NO: 5 and a polypeptide containing the amino acid sequence described in SEQ ID NO: 6.

[0158] In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising an SCC hinge and a mutant human DNase1 domain operably coupled by a linker or not to a mutant Fc domain or fragment thereof containing CH2 mutants P238S, P331S and CH3 mutants T350V, L351Y, F405A, and Y407V, and a wild-type human RNase1 domain operably coupled by a linker or not to an SCC hinge and a mutant Fc domain or fragment thereof containing CH2 mutants P238S and P331S and CH3 mutants T350V, T366L, K392L, and T394W. In some embodiments, DNase1 is ligated to the N-terminus of the Fc domain and RNase1 is ligated to the C-terminus of the Fc domain. In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising a polypeptide containing the sequence described in SEQ ID NO: 7 and a polypeptide containing the amino acid sequence described in SEQ ID NO: 8.

[0159] In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising an SCC hinge and a mutant human DNase1 domain operably coupled by a linker or not to a mutant Fc domain or fragment thereof containing CH2 mutants P238S, P331S and CH3 mutants T350V, L351Y, F405A, and Y407V, and a wild-type human RNase1 domain operably coupled by a linker or not to an SCC hinge and a mutant Fc domain or fragment thereof containing CH2 mutants P238S and P331S and CH3 mutants T350V, T366L, K392L, and T394W. In some embodiments, both DNase1 and RNase1 are ligated to the C-terminus of their respective Fc domains. In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising a polypeptide containing the sequence described in SEQ ID NO: 9 and a polypeptide containing the amino acid sequence described in SEQ ID NO: 10. In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising a polypeptide containing the sequence described in SEQ ID NO: 11 and a polypeptide containing the amino acid sequence described in SEQ ID NO: 12.

[0160] In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising a mutant human DNase1 domain and a wild-type human RNase1 domain, both operably coupled by a linker or not to an SCC hinge and a mutant Fc domain or fragment thereof containing CH2 mutants P238S, P331S and CH3 mutants T350V, L351Y, F405A, and Y407V, and a mutant Fc domain or fragment thereof containing CH2 mutants P238S, P331S and CH3 mutants T350V, T366L, K392L, and T394W. In some embodiments, DNase1 and RNase1 are ligated to the C-terminus and N-terminus, respectively, of the Fc domain. In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising a polypeptide containing the sequence described in SEQ ID NO: 13 and a polypeptide containing the amino acid sequence described in SEQ ID NO: 14.

[0161] In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising an SCC hinge and a mutant human DNase1 domain operably coupled by a linker or not to a mutant Fc domain or fragment thereof containing CH2 mutants P238S, P331S and CH3 mutants T350V, L351Y, F405A, and Y407V, and a wild-type human RNase1 domain operably coupled by a linker or not to an SCC hinge and a mutant Fc domain or fragment thereof containing CH2 mutants P238S and P331S and CH3 mutants T350V, T366L, K392L, and T394W. In some embodiments, DNase1 is ligated to the C-terminus of the Fc domain and RNase1 is ligated to the N-terminus of the Fc domain. In some embodiments, the optimized double nuclease fusion protein is a heterodimer comprising a polypeptide containing the sequence described in SEQ ID NO: 15 and a polypeptide containing the amino acid sequence described in SEQ ID NO: 16.

[0162] In some embodiments, the optimized double nuclease fusion protein comprises a polypeptide having an amino acid sequence that is at least 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at least 99.5% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 17. In some embodiments, the polypeptide comprises the amino acid sequence described in any one of SEQ ID NOs: 1 to 17.

[0163] In some embodiments, the optimized double nuclease fusion protein has a leader sequence.

[0164] Those skilled in the art will understand that the leader and linker sequences are of arbitrary selection and not limited to those described in the embodiments above. For example, the RNase and / or DNase domain may be directly fused to the N-terminus and / or C-terminus of Fc or its variant or fragment; the leader domain may be any of those known in the art that are useful for their intended purpose, for example, to increase protein expression and / or secretion (e.g., Gaussia luciferase signal peptide (MGVKVLFALICIAVAEA; SEQ ID NO: 31)); the linker may be any linker known in the art, for example, (Gly4Ser)n (SEQ ID NO: 58), NLG (VDGASSPVNVSSPSVQDI; SEQ ID NO: 41), LE, the thrombin-sensitive disulfide cyclopeptide linker, LEA(EAAAK)4ALEA(EAAAK)4 (SEQ ID NO: 32), or an in vitro cleavable disulfide linker. It will also be understood that making the corresponding changes to the amino acid sequence of an optimized double nuclease fusion protein using routine cloning and recombination methods is within the capabilities of those skilled in the art. It will also be understood that asparagine residues in the nuclease domain (i.e., N34, N76, and N88 in RNase1, and N18 and N106 in DNase1) can be substituted with non-serine amino acids (e.g., glutamine), provided that the amino acids do not function as acceptors for N-linked glycosylation.

[0165] Method for producing optimized double nuclease fusion proteins The optimized double nuclease fusion proteins of this disclosure can be primarily produced in transformed or transfected host cells using recombinant DNA technology. To do so, recombinant DNA molecules encoding the peptide are prepared. Methods for preparing such DNA molecules are well known in the art. For example, the peptide-encoding sequence can be cleaved from DNA using appropriate restriction enzymes. Alternatively, the DNA molecule can be synthesized using chemical synthesis techniques such as the phosphoramidate method. A combination of these techniques may also be used.

[0166] The present invention also includes vectors capable of expressing peptides in a suitable host. The vector comprises a DNA molecule encoding a peptide operably coupled to an expression regulatory sequence. Methods for influencing this operable coupling, either before or after inserting the DNA molecule into the vector, are well known. Expression regulatory sequences include promoters, activators, enhancers, operators, ribosomal nuclease domains, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the regulation of transcription or translation.

[0167] The resulting vector, which has a DNA molecule on it, is used to transform or transfect a suitable host. This transformation or transfection can be carried out using methods well known in the art.

[0168] Many available and well-known host cells can be used in carrying out 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 or transfection ratio, ease of peptide recovery, expression characteristics, biosafety, and cost. These factors must be balanced, with the understanding that not all hosts are equally effective for the expression of a particular DNA sequence. These general guidelines indicate that useful microbial hosts include bacterial (e.g., Escherichia coli) cells in culture, yeast (e.g., Saccharomyces) and other fungi, insects, plants, mammalian (including human) cells, or other hosts known in the art. In a preferred embodiment, the optimized double nuclease fusion protein is produced in CHO cells.

[0169] Next, the transformed or transfected host is cultured and purified. The host cells can be cultured under conventional fermentation or culture conditions so that the desired compound is expressed. Such fermentation and culture conditions are well known in the art. Finally, the peptide is purified from the culture by methods well known in the art.

[0170] The compounds can also be prepared by synthetic methods. For example, solid-phase synthesis techniques can be used. Suitable techniques are well known in the art, as seen in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85:2149; Davis et al., Biochem Intl 1985; 10:394-414; Stewart and Young (1969), Solid Examples include those described in Phase Peptide Synthesis; U.S. Patent No. 3,941,763; Finn et al., (1976), The Proteins (3rd ed.) 2:105-253; and Erickson et al., (1976), The Proteins (3rd ed.) 2:257-527. Solid-phase synthesis is the most cost-effective method for producing small peptides and is therefore a preferred technique for producing individual peptides. Compounds containing derivatized peptides or non-peptide groups can be synthesized by well-known organic chemical techniques.

[0171] Other methods for molecular expression / synthesis are generally known to those skilled in the art.

[0172] Optimized double nuclease fusion protein with altered glycosylation Glycosylation (e.g., O-linked or N-linked glycosylation) can affect the serum half-life of the optimized binuclease fusion protein of this disclosure by minimizing their removal from circulation by, for example, mannose and asialoglycoprotein receptors and other lectin-like receptors. Therefore, in some embodiments, the optimized binuclease fusion protein of this disclosure may have a limited serum half-life. The fusion protein is prepared in an aglycosylated, deglycosylated, or hypoglycosylated form. Preferably, the N-linked glycosylation is altered, and the optimized double nuclease fusion protein is aglycosylated.

[0173] In some embodiments, all asparagine residues in an optimized double nuclease fusion protein conforming to the Asn-X-Ser / Thr (where X can be any other naturally occurring amino acid besides Pro) consensus are mutated to residues that do not function as N-linked glycosylation acceptors (e.g., serine, glutamine), thereby eliminating glycosylation of the optimized double nuclease fusion protein when synthesized in cells that glycosylate the protein.

[0174] In some embodiments, the optimized double nuclease fusion protein lacking an N-linked glycosylation site is produced in mammalian cells. In one embodiment, the mammalian cell is a CHO cell. Therefore, in certain embodiments, the aglycosylation-optimized double nuclease fusion protein is produced in a CHO cell.

[0175] In other embodiments, the reduction or absence of N-glycosylation is achieved, for example, by producing an optimized double nuclease fusion protein in a host (e.g., bacteria such as E. coli), mammalian cells manipulated to lack one or more enzymes important for glycosylation, or mammalian cells treated with a drug that prevents glycosylation, such as tunicamycin (an inhibitor of Dol-PP-GlcNAc formation).

[0176] In some embodiments, the optimized double nuclease fusion protein is produced in lower eukaryotes that have been engineered to produce glycoproteins with complex N-glycans rather than high-mannose sugars (see, for example, U.S. Patent Application Publication 2007 / 0105127).

[0177] In some embodiments, glycosylation-optimized binuclease fusion proteins (e.g., those produced in mammalian cells such as CHO cells) are chemically or enzymatically treated to remove one or more carbohydrate residues (e.g., one or more mannose, fucose, and / or N-acetylglucosamine residues), or to modify or shield one or more carbohydrate residues. Such modification or shielding may reduce the binding of the optimized binuclease fusion protein to mannose receptors and / or asialoglycoprotein receptors and / or other lectin-like receptors. Chemical deglycosylation can be achieved, for example, by treating the optimized double nuclease fusion protein with trifluoromethanesulfonic acid (TFMS), as disclosed in Sojar et al., JBC 1989;264:2552-9 and Sojar et al., Methods Enzymol 1987;138:341-50, or by treating it with hydrogen fluoride, as disclosed in Sojar et al. (1987, op. cit.). Enzymatic removal of N-linked carbohydrates from the optimized double nuclease fusion protein can be achieved, for example, by treating the optimized double nuclease fusion protein with protein N-glycosidase (PNGase) A or F, as disclosed in Thotakura et al. (Methods Enzymol 1987;138:350-9). Other commercially available deglycosylating enzymes recognized in the art and suitable for use include endo-α-N-acetyl-galactosaminidase, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3, and endoglycosidase H. In some embodiments, one or more of these enzymes may be used to deglycosylate the optimized double nuclease fusion protein of this disclosure. Alternative methods for deglycosylation are disclosed, for example, in U.S. Patent No. 8,198,063.

[0178] In some embodiments, the optimized double nuclease fusion protein is partially deglycosylated. Partial deglycosylation can be achieved by treating the optimized double nuclease fusion protein with an endoglycosidase (e.g., endoglycosidase H) (which cleaves N-linked high-mannose carbohydrates but not complex carbohydrates, leaving a single GlcNAc residue linked to asparagine). The optimized double nuclease fusion protein treated with endoglycosidase H will lack high-mannose carbohydrates, and consequently, its interaction with the hepatic mannose receptor will be reduced. This receptor recognizes terminal GlcNAc, but the potential for productive interaction with a single GlcNAc on the protein surface is not as great as in the case of the intact high-mannose structure.

[0179] In other embodiments, to reduce the clearance of the optimized double nuclease fusion protein from blood, the glycosylation of the optimized double nuclease fusion protein is modified by, for example, oxidation, reduction, dehydration, substitution, esterification, alkylation, sialylation, carbon-carbon bond cleavage, etc. In some embodiments, to modify the carbohydrate structure, the optimized double nuclease fusion protein is treated with periodate and sodium borohydride. Periodate treatment oxidizes adjacent diols, cleaves carbon-carbon bonds, and replaces hydroxyl groups with aldehyde groups; borohydride reduces aldehydes to hydroxyls. Many sugar residues contain vicinal diols and are therefore cleaved by this treatment. The extension of serum half-lives by periodates and sodium borohydride is exemplified by sequential treatment of the lysosomal enzyme β-glucuronidase with these agents (see, e.g., Houba et al., (1996) Bioconjug Chem 1996:7:606-11; Stahl et al., PNAS 1976;73:4045-9; Achord et al., Pediat.Res 1977;11:816-22; Achord et al., Cell 1978;15:269-78). Methods for treatment with periodates and sodium borohydride are disclosed in Hickman et al., BBRC 1974;57:55-61. A method for treating lysine with periodate and cyanoboron hydride to increase its serum half-life and tissue distribution is disclosed by Thorpe et al. in Eur J Biochem 1985;147:197-206.

[0180] In one embodiment, the carbohydrate structure of the optimized double nuclease fusion protein may be shielded by the addition of one or more further parts (e.g., carbohydrate groups, phosphate groups, alkyl groups, etc.) that interfere with structural recognition by mannose, asialoglycoprotein receptors, or other lectin-like receptors.

[0181] In some embodiments, one or more potential glycosylation sites are removed by mutations in the nucleic acid encoding the optimized double nuclease fusion protein, thereby reducing the glycosylation (deglycosylation) of the optimized double nuclease fusion protein when synthesized in cells that glycosylate the protein, such as mammalian cells, such as CHO cells. In some embodiments, for example, if a deglycosylated optimized double nuclease fusion protein exhibits increased activity or contributes to an increased serum half-life, it may be desirable to selectively deglycosylate the nuclease domain of the optimized double nuclease fusion protein by mutating a potential N-linked glycosylation site within it. In other embodiments, for example, if such modification improves the serum half-life of the optimized double nuclease fusion protein, it may be desirable to deglycosylate a portion of the optimized double nuclease fusion protein such that regions other than the nuclease domain lack N-glycosylation. Alternatively, by modifying other amino acids near the glycosylation acceptor, the recognition motif of the glycosylation enzyme can be disrupted without necessarily altering the amino acids that are normally glycosylated.

[0182] In some embodiments, glycosylation of the optimized double nuclease fusion protein can be altered by introducing glycosylation sites. For example, the amino acid sequence of the optimized double nuclease fusion protein can be modified to introduce a consensus sequence for N-linked glycosylation of Asp-X-Ser / Thr (where X is any amino acid other than proline). Further N-linked glycosylation sites can be added at any location in the amino acid sequence of the optimized double nuclease fusion protein. Preferably, the glycosylation sites are introduced at a location in the amino acid sequence that does not substantially reduce the nuclease (e.g., RNase and / or DNase) activity of the optimized double nuclease fusion protein.

[0183] The addition of an O-linked glycosylation site has been reported to alter the serum half-life of proteins, such as growth hormone, follicle-stimulating hormone, IGFBP-6, factor IX, and many others (as disclosed in, e.g., Okada et al., Endocr Rev 2011;32:2-342; Weenen et al., J Clin Endocrinol Metab 2004;89:5204-12; Marinaro et al., European Journal of Endocrinology 2000;142:512-6; U.S. Patent Application Publication No. 2011 / 0154516). Therefore, in some embodiments, the O-linked glycosylation (on serine / threonine residues) of the optimized double nuclease fusion protein is altered. Methods for altering O-linked glycosylation are routine in the art, for example, by β-elimination (e.g., Huang et al., Rapid Communications in Mass Spectrometry 2002;16:1199-204;Conrad,Curr Protoc Mol Biol 2001;Chapter 17:Unit17.15A;Fukuda,Curr See Protoc Mol Biol 2001; Chapter 17; Unit 17.15B; Zachara et al., Curr Protoc Mol Biol 2011; Unit 17.6;; this can be achieved by using a commercially available kit (e.g., GlycoProfile® Beta-Elimination Kit, Sigma); or by subjecting the optimized double nuclease fusion protein to a series of exoglycosidases, e.g., β1-4 galactosidase and β-N-acetylglucosaminidase, until only Gal β1-3GalNAc and / or GlcNAc β1-3GalNAc remains, followed by treatment with, for example, endo-α-N-acetylgalactosaminidase (i.e., O-glycosidase). Such enzymes are commercially available, for example, from New England Biolabs. In further embodiments, as disclosed, for example, in Okada et al. (cited above), Weenen et al. (cited above), U.S. Patent Application Publication No. 2008 / 0274958; and U.S. Patent Application Publication No. 2011 / 0171218, the optimized double nuclease fusion protein is modified to introduce O-linked glycosylation in the optimized double nuclease fusion protein. In some embodiments, one or more O-linked glycosylation consensus sites, such as CXXGGT / SC (SEQ ID NO: 33) (van den Steen et al., In Critical Reviews in Biochemistry and Molecular Biology, Michael Cox, ed., 1998;33:151-208), NST-E / DA (SEQ ID NO: 34), NITQS (SEQ ID NO: 35), QSTQS (SEQ ID NO: 36), D / E-FT-R / KV (SEQ ID NO: 37), CE / D-SN (SEQ ID NO: 38), and GGSC-K / R (SEQ ID NO: 39), are introduced into the optimized double nuclease fusion protein. Further O-linked glycosylation sites may be added at any location in the amino acid sequence of the optimized double nuclease fusion protein.Preferably, the glycosylation site is introduced at an amino acid sequence position that does not substantially reduce the nuclease (e.g., RNase and / or DNase) activity of the optimized double nuclease fusion protein. Alternatively, the O-linked sugar moiety is introduced by chemically modifying the amino acids of the optimized double nuclease fusion protein, as described, for example, in International Publication No. 87 / 05330 and Aplin et al., CRC Crit Rev Biochem 1981;259-306).

[0184] In some embodiments, both N-linked and O-linked glycosylation sites are preferably introduced at amino acid sequence positions that do not substantially reduce the nuclease (e.g., RNase and / or DNase) activity of the optimized double nuclease fusion protein.

[0185] It is well within the capabilities of those skilled in the art to introduce, reduce, or eliminate glycosylation (e.g., N-linked or O-linked glycosylation) in an optimized double nuclease fusion protein, and to determine, using routine methods in the art, whether such modification of the glycosylation state increases or decreases the nuclease activity or serum half-life of the optimized double nuclease fusion protein.

[0186] In some embodiments, the optimized double nuclease fusion protein may contain altered sugar forms (e.g., non-fucosylated glycans or fucose-free glycans).

[0187] In some embodiments, an optimized binuclease fusion protein having altered glycosylation has a serum half-life that is at least about 1.5 times, for example, at least 3 times, at least 5 times, at least 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 600 times, at least about 700 times, at least about 800 times, at least about 900 times, at least about 1000 times, or 1000 times or more increased compared to a corresponding glycosylated optimized binuclease fusion protein (e.g., an optimized binuclease fusion protein in which the potential N-linked glycosylation site is not mutated). Routine methods accepted in the art may be used to determine the serum half-life of an optimized binuclease fusion protein having altered glycosylation status.

[0188] In some embodiments, an optimized binuclease fusion protein having altered glycosylation (e.g., an aglycosylated, deglycosylated, or hypoglycosylated optimized binuclease fusion protein) retains at least 50%, for example, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the activity of the corresponding glycosylated optimized binuclease fusion protein (e.g., an optimized binuclease fusion protein in which the potential N-linked glycosylation site is not mutated).

[0189] In some embodiments, altering the glycosylation state of an optimized binuclease fusion protein can increase nuclease activity by directly increasing enzyme activity or by increasing bioavailability (e.g., serum half-life). Thus, in some embodiments, the nuclease activity of an optimized binuclease fusion protein with altered glycosylation is increased by at least 1.3 times, for example, at least 1.5 times, at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 6.5 times, at least 7 times, at least 7.5 times, at least 8 times, at least 8.5 times, at least 9 times, at least 9.5 times or 10 times or more compared to the corresponding glycosylated optimized binuclease fusion protein (e.g., an optimized binuclease fusion protein with no mutation in the potential N-linked glycosylation site).

[0190] Those skilled in the art can easily determine the glycosylation state of an optimized double nuclease fusion protein using methods accepted in the art. In preferred embodiments, the glycosylation state is determined by mass spectrometry. In other embodiments, the interaction with concanavalin A (ConA) may be evaluated to determine whether the optimized double nuclease fusion protein is hypoglycosylated. Hypoglycosylated optimized double nuclease fusion proteins are expected to show reduced binding to ConA-Sepharose compared to the corresponding glycosylated optimized double nuclease fusion proteins. SDS-PAGE analysis may also be used to compare the mobility of hypoglycosylated proteins and the corresponding glycosylated proteins. Hypoglycosylated proteins are expected to have greater mobility on SDS-PAGE compared to glycosylated proteins. Other suitable methods accepted in the art for analyzing protein glycosylation state are disclosed, for example, Roth et al., International Journal of Carbohydrate Chemistry 2012;1-10.

[0191] The pharmacokinetics of optimized double nuclease fusion proteins with different glycosylation states, such as their serum half-lives, can be assayed using routine methods, for example, by introducing the optimized double nuclease fusion protein intravenously into mice, collecting blood samples at predetermined time points, and assaying and comparing the levels and / or enzymatic activity of the optimized double nuclease fusion protein in the samples.

[0192] Pharmaceutical composition In certain embodiments, the optimized binuclease fusion protein is administered alone. In certain embodiments, the optimized binuclease fusion protein is administered before the administration of at least one other therapeutic agent. In certain embodiments, the optimized binuclease fusion protein is administered concurrently with the administration of at least one other therapeutic agent. In certain embodiments, the optimized binuclease fusion protein is administered after the administration of at least one other therapeutic agent. In other embodiments, the optimized binuclease fusion protein is administered before the administration of at least one other therapeutic agent. As will be recognized by those skilled in the art, in some embodiments, the optimized binuclease fusion protein is combined with other drugs / compounds. In some embodiments, the optimized binuclease fusion protein and other drugs are administered concurrently. In some embodiments, the optimized binuclease fusion protein and other drugs are not administered concurrently, and the optimized binuclease fusion protein is administered before or after the administration of the drugs. In some embodiments, the subject receives both the optimized binuclease fusion protein and other drugs during the same prophylactic, injury-onset and / or treatment period.

[0193] The pharmaceutical compositions of the present invention may be administered in combination therapy, i.e., in combination with other agents. In certain embodiments, the combination therapy comprises an optimized double nuclease fusion protein in combination with at least one other agent. The agents include, but are not limited to, chemical compositions, antibodies, antigen-binding domains, and combinations and conjugates thereof, which are synthetically prepared in vitro. In certain embodiments, the agent may act as an agonist, antagonist, allosteric modulator, or toxin.

[0194] In certain embodiments, the present invention provides a pharmaceutical composition comprising an optimized binuclease fusion protein together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0195] In certain embodiments, the present invention provides a pharmaceutical composition comprising an optimized binuclease fusion protein and at least one therapeutically effective amount of a further therapeutic agent, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0196] In certain embodiments, the acceptable formulation materials are preferably non-toxic to the recipient at the dosage and concentration used. In some embodiments, the formulation materials are for subcutaneous and / or intravenous administration. In certain embodiments, the pharmaceutical composition may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, aroma, sterility, stability, rate of dissolution or release, adsorption, or permeability of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antibacterial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffering agents (such as borates, bicarbonates, tris-HCl, citrates, phosphates, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as gelatin); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; and salt-forming counterions (sodium Examples include: 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 acid, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyroxapol, etc.); stability enhancers (such as sucrose or sorbitol); tensile strength enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents; excipients; and / or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18th Edition, ARGennaro, 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.

[0197] In certain embodiments, the optimized double nuclease fusion protein and / or therapeutic molecule is ligated to a half-life extension vehicle known in the art. Such vehicles include, but are not limited to, polyethylene glycol, glycogen (e.g., glycosylation of the optimized double nuclease fusion protein), 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 published International Publication No. 99 / 25044.

[0198] In certain embodiments, the optimal pharmaceutical composition can be determined by those skilled in the art, for example, depending on the intended route of administration, delivery method, and desired dose. See, for example, Remington's Pharmaceutical Sciences, cited above. In certain embodiments, such compositions may affect the physical state, stability, in vivo release rate, and in vivo efflux rate of the antibody of the present invention.

[0199] In certain embodiments, the primary vehicle or carrier in the pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable vehicle or carrier may be water for injection, physiological saline, or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. In some embodiments, the physiological saline includes isotonic phosphate-buffered saline. In certain embodiments, the pharmaceutical composition includes Tris buffer at approximately pH 7.0–8.5, or acetate buffer at approximately pH 4.0–5.5 (which may further include sorbitol or a suitable substitute thereof). In certain embodiments, a composition comprising an optimized double nuclease fusion protein with or without at least one further therapeutic agent may be prepared for storage by mixing a selected composition of desired purity with an optional formulation agent in the form of a lyophilized cake or aqueous solution (Remington's Pharmaceutical Sciences, cited above). Furthermore, in certain embodiments, compositions comprising an optimized binuclease fusion protein, either with or without at least one further therapeutic agent, may be formulated as lyophilized products using a suitable excipient such as sucrose.

[0200] In certain embodiments, the pharmaceutical composition may be selected for parenteral delivery. In certain embodiments, the composition may be selected for inhalation or for delivery via the gastrointestinal tract (e.g., orally). The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0201] In certain embodiments, the formulation components are present at concentrations acceptable to the administration site. In certain embodiments, buffers are used to maintain the composition at or slightly below physiological pH, typically within a pH range of about 5 to about 8.

[0202] In certain embodiments, when parenteral administration is intended, the therapeutic composition may be in the form of a parenterally acceptable pyrogen-free aqueous solution containing the desired optimized double nuclease fusion protein with or without further therapeutic agents in a pharmaceutically acceptable vehicle. In certain embodiments, the vehicle for parenteral injection is sterile distilled water formulated as a properly preserved sterile isotonic solution with or without further therapeutic agents containing the optimized double nuclease fusion protein. In certain embodiments, the preparation may involve formulating the desired molecule with an agent that can provide controlled or sustained release of the product, such as injection microspheres, biodegradable particles, polymer compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes, which can then be delivered via depot injection. In certain embodiments, hyaluronic acid may also be used, which may have the effect of promoting duration in circulation. In certain embodiments, an implantable drug delivery device may be used to introduce the desired molecule.

[0203] In certain embodiments, the pharmaceutical composition may be formulated for inhalation. In certain embodiments, the optimized double nuclease fusion protein may be formulated as a dry powder for inhalation, with or without at least one further therapeutic agent. In certain embodiments, an inhalation solution containing the optimized double nuclease fusion protein may be formulated with a spray for aerosol delivery, with or without at least one further therapeutic agent. In certain embodiments, the solution may be sprayed. Lung administration is further described in International Patent Application No. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0204] In certain embodiments, the formulation is intended to be administered orally. In certain embodiments, the optimized binuclease fusion protein administered in this manner may be formulated with or without a carrier commonly used in the formulation of solid dosage forms such as tablets and capsules, with or without a carrier carrier, with or without carrier, with or without carrier, with or without carrier, carrier, carrier, carrier, carrier.

[0205] In certain embodiments, the pharmaceutical composition may contain an effective amount of optimized binuclease fusion protein in a mixture with non-toxic excipients suitable for the manufacture of tablets, with or without at least one further therapeutic agent. In certain embodiments, the solution may be prepared in unit dose form by dissolving the tablets in sterile water or another suitable vehicle. In certain embodiments, suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or 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.

[0206] Further pharmaceutical compositions comprising formulations containing an optimized binuclease fusion protein in a sustained-release or controlled-release formulation, either with or without at least one further therapeutic agent, will be apparent to those skilled in the art. In certain embodiments, various other sustained-release or controlled-release means, e.g., liposome carriers, biodegradable microparticles or porous beads, and techniques for formulating depot injections are also known to those skilled in the art. See, for example, International Patent Application PCT / US93 / 00829, which describes the controlled release of porous polymer microparticles for the delivery of pharmaceutical compositions. In certain embodiments, the sustained-release preparation may include, for example, a semipermeable polymer matrix in the form of a molded article, e.g., a film or microcapsule. The sustained-release matrix may include polyester, hydrogel, polylactide (U.S. Patent No. 3,773,919 and European Patent Application Publication No. 058,481), copolymer of L-glutamic acid and γ-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., cited above), or poly-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. 133,988). In certain embodiments, the sustained-release composition may also include liposomes that can be prepared by any of several methods known in the art. See, for example, Eppstein et al., PNAS, 82:3688-3692 (1985); European Patent Application Publication No. 036,676; European Patent Application Publication No. 088,046 and European Patent Application Publication No. 143,949.

[0207] Pharmaceutical compositions intended for in vivo administration are typically sterile. In certain embodiments, this can be achieved by filtration through a sterile filtration membrane. In certain embodiments, if the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. In certain embodiments, compositions for parenteral administration can be stored in lyophilized form or in solution. In certain embodiments, parenteral compositions are generally placed in containers with a sterile access port, such as intravenous solution bags or vials with a stopper that can be penetrated by a subcutaneous injection needle.

[0208] In certain embodiments, once a pharmaceutical composition has been formulated, it may be stored in a suitable sterile vial as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. In certain embodiments, such formulations may be stored either in an immediate-use form or in a form that is reconstituted before administration (e.g., lyophilized).

[0209] In certain embodiments, a kit for producing single-dose units is provided. In certain embodiments, the kit may include both a first container containing a dry protein and a second container containing an aqueous formulation. In certain embodiments, the kit includes single and multi-chamber prefilled syringes (e.g., liquid syringes and rio-syringes).

[0210] In certain embodiments, the effective amount of a pharmaceutical composition containing an optimized binuclease fusion protein to be used therapeutically, with or without at least one further therapeutic agent, will depend, for example, on the therapeutic situation and purpose. Those skilled in the art will therefore recognize that, according to certain embodiments, the appropriate dose level for treatment will vary, in part, depending on the indication, route of administration, and the size (body weight, body surface area, or organ size) and / or condition (age and overall health) of the patient in which the optimized binuclease fusion protein is used with or without at least one further therapeutic agent when the molecule is delivered. In certain embodiments, the clinician may set the dose and modify the route of administration to obtain the optimal therapeutic effect. In certain embodiments, a typical dose may range from about 0.1 μg / kg to or exceeding about 100 mg / kg, depending on the factors described above. In certain embodiments, the dosage may range from 0.1 μg / kg to a maximum of approximately 100 mg / kg; or from 1 μg / kg to a maximum of approximately 100 mg / kg; or from 5 μg / kg to a maximum of approximately 100 mg / kg.

[0211] In certain embodiments, the administration frequency will take into account the pharmacokinetic parameters of the optimized binuclease fusion protein and / or any further therapeutic agents in the formulation used. In certain embodiments, the clinician will administer the composition until a dose is reached that achieves the desired effect. In certain embodiments, the composition may therefore be administered as a single dose, or as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implantable device or catheter. Further fine-tuning of the appropriate dose is routinely performed by those skilled in the art and is within the scope of work routinely carried out by those skilled in the art. In certain embodiments, the appropriate dose may be determined by using appropriate dose-response data.

[0212] In certain embodiments, the route of administration of the pharmaceutical composition is by oral injection via known methods, such as intravenous, intraperitoneal, intracerebral (intraparum), intraventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal, or intrafocal routes; by a continuous-release system; or by an implantable device. In certain embodiments, the composition may be administered by bolus injection, by continuous infusion, or by an implantable device.

[0213] In certain embodiments, the composition may be administered topically via an implantation of a membrane, sponge, or other suitable material on which the desired molecule is adsorbed or encapsulated. In certain embodiments, if an implantation device is used, the device may be implanted in any suitable tissue or organ, and the delivery of the desired molecule may be by diffusion, sustained-release bolus, or continuous administration.

[0214] In certain embodiments, it may be desirable to use a pharmaceutical composition comprising an optimized binuclease fusion protein ex vivo, either with or without at least one further therapeutic agent. In such cases, cells, tissues, and / or organs removed from a patient are exposed to a pharmaceutical composition comprising an optimized binuclease fusion protein, either with or without at least one further therapeutic agent, and subsequently the cells, tissues, and / or organs are re-implanted in the patient's body.

[0215] In certain embodiments, optimized binuclease fusion proteins and / or any further therapeutic agents may be delivered by embedding specific cells genetically engineered to express and secrete polypeptides using methods such as those described herein. In certain embodiments, such cells may be animal or human cells and may be autologous, xenogeneic, or xenogenic. In certain embodiments, the cells may be immortalized. In certain embodiments, cells may be encapsulated to avoid invasion of surrounding tissues in order to reduce the opportunity for an immunological response. In certain embodiments, the encapsulation material is typically a biocompatible semipermeable polymer encapsulant or membrane that allows for the release of protein products but prevents cell destruction by the patient's immune system or other harmful factors from surrounding tissues.

[0216] In vitro assay Various in vitro assays known in the art can be used to evaluate the efficacy of the optimized double nuclease fusion protein of the present invention.

[0217] For example, cultured human PBMCs derived from normal or lupus patient PBMCs are isolated, cultured, and treated with various stimuli (e.g., TLR ligands, co-stimulatory antibodies, immune complexes, and normal or autoimmune serum) in or out of the presence of the optimized double nuclease fusion protein. Cytokine production by stimulated cells can be measured using commercially available reagents for various cytokines (e.g., IL-6, IL-8, IL-10, IL-4, IFN-γ, and TNF-α), e.g., antibody pairing kits from Biolegend (San Diego, CA). Culture supernatants are collected at various time points (e.g., 24 hours, 48 ​​hours, or later) as needed for the assay to determine the effect of the optimized double nuclease fusion protein on cytokine production. IFN-α production can be measured using, for example, PBL. Measurements are performed using anti-human IFN-α antibodies and standard curve reagents available from interferon source (Piscataway, NJ). Similar assays are performed using human lymphocyte subpopulations (isolated monocytes, B cells, pDCs, T cells, etc.) purified using a commercially available magnetic bead-based isolation kit available from Miltenyi Biotech (Auburn, CA).

[0218] Multicolor flow cytometry may be used to evaluate the effect of optimized double nuclease fusion proteins on immune cell activation by measuring the expression of lymphocyte-activating receptors, such as CD5, CD23, CD69, CD80, CD86, and CD25, in PBMCs or isolated cell subpopulations at various time points after stimulation, using routine methods accepted in the art.

[0219] The efficacy of optimized double nuclease fusion proteins can also be tested by incubating SLE patient serum with normal human pDCs to activate IFN output, as described, for example, Ahlin et al., Lupus 2012:21:586-95; Mathsson et al., Clin Expt Immunol 2007;147:513-20; and Chiang et al., J Immunol 2011;186:1279-1288. While not bound by theory, circulating nucleic acid-containing immune complexes in SLE patient serum facilitate nucleic acid antigen entry into pDC endosomes via Fc receptor-mediated endocytosis, followed by the binding of nucleic acids to endosomal TLR7, 8, and 9 and their activation. To evaluate the effects of optimized double nuclease fusion proteins, SLE patient serum or plasma is pretreated with optimized double nuclease fusion proteins and subsequently added to cultures of pDC cells isolated from healthy volunteers. Next, the levels of IFN-α produced are determined at multiple time points. By degrading nucleic acids containing immune complexes, effective optimized double nuclease fusion proteins are expected to reduce the amount of IFN-α produced.

[0220] The efficacy of the optimized binuclease fusion protein is demonstrated by comparing the results of assays from cells treated with the optimized binuclease fusion protein disclosed herein with the results of assays from cells treated with a control formulation. After treatment, the levels of the various markers (e.g., cytokines, cell surface receptors, proliferation) are generally improved in the group treated with the effective optimized binuclease fusion protein compared to the marker levels present before treatment or compared to the levels measured in the control group.

[0221] Treatment method The optimized dual nuclease fusion proteins of this disclosure are particularly effective in treating autoimmune disorders or abnormal immune responses. In this regard, it will be recognized that the optimized dual nuclease fusion proteins of this disclosure can be used to control, suppress, modulate, treat or eliminate undesirable immune responses to both external and autoantigens.

[0222] In another embodiment, the optimized double nuclease fusion protein is adapted to prevent (preventively) or treat (therapeutically) a disease or disorder in a mammal, such as an autoimmune disease, by administering a therapeutically effective or sufficient amount of the optimized double nuclease fusion protein to a mammal in need, and the disease is prevented or treated. Any suitable route of administration to achieve the desired effect is contemplated by the present invention (e.g., intravenously, intramuscularly, subcutaneously). Treatment of the disease condition may result in a reduction of symptoms associated with the condition (this may be long-term, short-term, or even a temporarily beneficial effect).

[0223] Numerous disease conditions are suitable for treatment with the optimized double nuclease fusion protein of this disclosure. For example, in some embodiments, the disease or disorder may be an autoimmune disease or cancer. In some such embodiments, autoimmune diseases include insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes mellitus, Goodpasture syndrome, pemphigus vulgaris, bullous pemphigoid, sympathetic ophthalmitis, lens-induced uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis Hbs-VE, idiopathic cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid LE, SLE, or connective tissue disease.

[0224] In certain embodiments, the optimized double nuclease fusion protein is used to prevent or treat SLE or Sjögren's syndrome. The efficacy of the optimized double nuclease fusion protein is demonstrated by comparing IFN-α levels, IFN-α response gene levels, autoantibody titers, renal function and disease status, and / or circulating immune complex levels in mammals treated with the optimized double nuclease fusion protein disclosed herein with those in mammals treated with a control formulation.

[0225] For example, human subjects requiring treatment are selected or identified (e.g., patients meeting the American College of Rheumatology criteria for SLE, or patients meeting the American-European Consensus Sjogren's Classification Criteria). Subjects may, for example, require relief of the cause or symptoms of SLE or Sjogren's syndrome. Subject identification may be performed in a clinical setting or elsewhere, for example, at the subject's home, by the subject using a self-test kit.

[0226] At time 0 hours, an appropriate initial dose of optimized binuclease fusion protein is administered to the subject. The optimized binuclease fusion protein is formulated as described herein. At intervals following the initial dose, e.g., 7, 14, and 21 days, the subject's condition is assessed by measuring, for example, IFN-α levels, IFN-α response gene levels, autoantibody titers, renal function and disease state, and / or circulating immune complex levels. Other relevant criteria may also be measured. The number and intensity of administrations are adjusted according to the subject's needs. After treatment, the subject's IFN-α levels, IFN-α response gene levels, autoantibody titers, renal function and disease state, and / or circulating immune complex levels are decreased and / or increased compared to levels present before treatment or compared to levels measured in an untreated, similarly affected / control subject.

[0227] In another example, a rodent subject requiring treatment is selected or identified (see, e.g., Example 7). Subject identification may be performed in a laboratory setting or elsewhere. At time 0 hours, an appropriate initial dose of the optimized binuclease fusion protein is administered to the subject. The optimized binuclease fusion protein is formulated as described herein. At intervals following the initial dose, e.g., 7, 14, and 21 days, the subject's condition is assessed by measuring, for example, IFN-α levels, IFN-α response gene levels, autoantibody titers, renal function and disease status, and / or circulating immune complex levels. Other relevant criteria may also be measured. The number and intensity of doses are adjusted according to the subject's needs.

[0228] Following treatment, the subject's IFN-α levels, IFN-α response gene levels, autoantibody titers, renal function and disease status, and / or circulating immune complex levels decrease and / or increase compared to levels present before treatment or compared to levels measured in untreated, similarly affected / control subjects.

[0229] Another aspect of the present invention involves using one or more optimized double nuclease fusion proteins to use gene therapy methods for treating or preventing disorders, diseases, and conditions. The gene therapy methods relate to introducing optimized double nuclease fusion protein nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal requiring them to achieve the expression of one or more polypeptides of the present disclosure. The methods may include introducing one or more polynucleotides encoding the optimized double nuclease fusion protein of the present disclosure, operably coupled to a promoter and any other genetic elements necessary for polypeptide expression by the target tissue.

[0230] In gene therapy applications, optimized double nuclease fusion protein genes are introduced into cells to achieve in vivo synthesis of therapeutically effective gene products. "Gene therapy" includes both conventional gene therapy, where a sustained effect is achieved with a single treatment, and the administration of gene therapy agents involving one or repeated doses of therapeutically effective DNA or mRNA. Oligonucleotides can be modified to enhance their uptake, for example, by substituting their charged phosphodiester groups with uncharged groups. [Examples]

[0231] The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended in any way to limit the scope of the present invention. With regard to the figures used (e.g., quantities, temperatures, etc.), efforts have been made to ensure accuracy, but naturally, some experimental error and deviation should be allowed.

[0232] Unless otherwise specified, the implementation of this invention will utilize conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology within the scope of the art. Such techniques are well described in the literature. For example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); ALLehninger, 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 Sundberg, Advanced Organic Chemistry 3. rd See Ed. (Plenum Press) Vols A and B (1992).

[0233] Example 1 Generation of an optimized double nuclease fusion protein encoding an expression vector Various embodiments of the optimized double nuclease fusion protein of this disclosure are shown in Figure 1, and the amino acid sequences of each are shown in the sequence listing. As an exemplary optimized double nuclease fusion protein, a double nuclease fusion protein having the configuration shown in Figure 1 was constructed. Specifically, starting from the amino acid sequence of the optimized double nuclease fusion protein, the polynucleotide encoding the optimized double nuclease fusion protein was directly synthesized using codon optimization by Genescript (Genescript, Piscatawy, NJ) to enable optimal expression in mammalian cells. The optimization process involved, for example, avoiding regions with very high (>80%) or very low (<30%) GC content where possible, and avoiding cis-acting sequence motifs, such as internal TATA boxes, chi sites and ribosome entry sites, AT-rich or GC-rich sequence stretches, RNA instability motifs, repetitive sequences and RNA secondary structures, as well as potential splice donor and acceptor sites in higher eukaryotes. The DNA encoding the optimized double nuclease fusion protein was cloned into a pcDNA3.1+ mammalian expression vector. We generated an optimized double nuclease fusion protein with the following configuration.

[0234] The tandem homodimer RSLV-145 (SEQ ID NO: 1) has a DNase-linker-RNase-Fc structure, in which the wild-type human RNase1 domain (SEQ ID NO: 27) is operably coupled to the N-terminus of the mutant Fc region, which includes an SCC hinge and CH2 mutations P238S and P331S (SEQ ID NO: 55), without the use of a linker, and the mutant human DNase1 domain (SEQ ID NO: 25) is operably coupled to the N-terminus of the RNase1 domain via an NLG linker (SEQ ID NO: 41).

[0235] To preferentially form heterodimers, each Fc domain in the following constructs contained complementary CH3 mutations: T350V, L351Y, F405A, and Y407V; as well as T350V, T366L, K392L, and T394W (numbering follows the EU index).

[0236] The tandem heterodimer RSLV-147 has a DNase-Fc (SEQ ID NO: 3) and an RNase-Fc (SEQ ID NO: 4) domain. The mutant human DNase1 domain (SEQ ID NO: 25) is operably coupled to the N-terminus of a first mutant Fc region containing an SCC hinge, CH2 mutations P238S and P331S, and a CH3 mutation. The wild-type human RNase1 domain (SEQ ID NO: 27) is operably coupled to the N-terminus of a second mutant Fc region containing an SCC hinge, CH2 mutations P238S and P331S, and a CH3 mutation.

[0237] The heterodimer RSLV-148 has the following structure: DNase-first Fc domain-linker-RNase (SEQ ID NO: 5) and second mutant Fc domain (containing CH2 mutations P238S, P331S and a CH3 mutation) (SEQ ID NO: 6) (N-terminal cleavage type containing the first cysteine ​​at the CCC hinge). The mutant human DNase1 domain (SEQ ID NO: 25) is operably coupled to the SCC hinge and the N-terminus of the first mutant Fc region containing CH2 mutations P238S, P331S and a CH3 mutation, while the wild-type human RNase1 domain (SEQ ID NO: 27) is operably coupled to the C-terminus of the first Fc region via an NLG linker.

[0238] The heterodimer RSLV-149 has a DNase-Fc (SEQ ID NO: 7) and Fc-linker-RNase (SEQ ID NO: 8) structure. The mutant human DNase1 domain (SEQ ID NO: 25) is operably coupled via an SCC hinge to the N-terminus of the first mutant Fc region, which contains CH2 mutations P238S, P331S, and a CH3 mutation. The wild-type human RNase1 domain (SEQ ID NO: 27) is operably coupled via an NLG linker to the C-terminus of the second mutant Fc region (SEQ ID NO: 6), which contains CH2 mutations P238S, P331S, and a CH3 mutation.

[0239] The heterodimer RSLV-152 has the following structure: RNase-first mutant Fc-linker-DNase (SEQ ID NO: 13) and second mutant Fc domain (containing CH2 mutations P238S, P331S and a CH3 mutation) (SEQ ID NO: 14). The wild-type human RNase1 domain (SEQ ID NO: 27) is operably coupled to the N-terminus of the first mutant Fc region containing the CH2 mutations P238S, P331S and the CH3 mutation via an SCC hinge, and the mutant human DNase1 domain (SEQ ID NO: 25) is operably coupled to the C-terminus of the first mutant Fc region via an NLG linker (NLG liner).

[0240] The heterodimer RSLV-153 has an Fc-linker-DNase (SEQ ID NO: 15) and RNase-Fc (SEQ ID NO: 16) configuration. The mutant human DNase1 domain (SEQ ID NO: 25) is operably coupled via an NLG linker to the C-terminus of the first mutant Fc region, which includes CH2 mutations P238S, P331S and a CH3 mutation. The wild-type human RNase1 domain (SEQ ID NO: 27) is operably coupled via an SCC hinge to the N-terminus of the second mutant Fc region, which includes CH2 mutations P238S, P331S and a CH3 mutation.

[0241] Constructs RLSV-327 (a dual nuclease containing RNase1 and DNase1 linked to human serum albumin; RNase-linker-HSA-linker-DNase E13R / N74K / A114F / T205K) and RSLV-132 (RNase-Fc) (containing DNase and RNase moieties) were used as controls.

[0242] Example 2 Transient expression of stable mammalian cell lines expressing optimized dual nuclease fusion proteins For transient expression, the expression vector of Example 1 containing the optimized dual nuclease fusion protein insert was transiently transfected into Chinese hamster ovary (CHO) cells, such as CHO-S cells (e.g., FreeStyle™ CHO-S cells, Invitrogen), using the transfection protocol recommended by the manufacturer and FreeStyle™ MAX reagent. CHO-S cells were maintained in FreeStyle™ CHO expression medium containing 2 mM L-glutamine and penicillin-streptomycin.

[0243] Generate a stable CHO-S cell line expressing an optimized dual nuclease fusion protein using routine methods known in the art. For example, infect CHO-S cells with a virus (e.g., retrovirus, lentivirus) containing the nucleic acid sequence of the optimized dual nuclease fusion protein and a nucleic acid sequence encoding a marker (e.g., GFP, a surface marker selectable by magnetic beads) selected using, for example, flow cytometry or magnetic bead separation (e.g., MACSelect™ system). Alternatively, transfect CHO-S cells with a vector containing the nucleic acid sequence of the optimized dual nuclease fusion protein and a selectable marker using any transfection method known in the art, such as electroporation (Lonza) or the above FreeStyle™ MAX reagent, and subsequently select using, for example, flow cytometry. The selectable marker can be incorporated into the same vector as the one encoding the optimized dual nuclease fusion protein or a separate vector.

[0244] Capture the molecule using a column packed with Protein A Sepharose beads, then wash with a column wash buffer (e.g., 90 mM Tris, 150 mM NaCl, 0.05% sodium azide), and elute the molecule from the column using an appropriate elution buffer (e.g., 0.1 M citrate buffer, pH 3.0) to purify the optimized dual nuclease fusion protein from the culture supernatant. Further concentrate the eluted material by buffer exchange by continuous spin in PBS using a Centricon concentrator and then filter through a 0.2 μm filter device. Determine the concentration of the optimized dual nuclease fusion protein using standard spectrophotometric methods (e.g., Bradford, BCA, Lowry, Biuret assays).

[0245] Example 3 Nuclease Activity of the Purified Optimized Dual Nuclease Fusion Protein The RNase activity of optimized double nuclease fusion proteins present in mouse serum was analyzed. Proteins were added to 50 mM Hepes and 100 mM NaCl (pH 7.3) at doses of 12.5–100 ng to 2.5 mg / ml of poly-IC(Sigma) and incubated at 37°C for 50 minutes. TCA was added to a final concentration of 5%, and the mixture was left on ice. The samples were filtered to remove precipitates and OD (Oral Dispersion). 260 The filtrate was collected for reading. The results are shown in Figure 2. Both RSLV-132 and RSLV-145 (both containing two RNase moieties per molar equivalent of construct) were active, with RSLV-145 being more active than RSLV-132. Other constructs containing only one RNase moiety on a molar basis (RSLV-147, RSLV-152, RSLV-153, and RSLV-327) were similar and consistent with RSLV-132. Surprisingly, RSLV-148 and RSLV-149 had higher activity than RSLV-132 and the other single RNase constructs. Each of these constructs contains an RNase moiety attached to the C-terminus of the Fc domain.

[0246] The DNase1 activity of an optimized double nuclease fusion protein containing the DNase1 domain was measured using ODN-2006-G5 (InvivoGen, tlrl-2006g5) (a DNA oligonucleotide agonist of TLR9). Protein doses ranging from 0.24 ng / ml to 500 ng / ml were incubated with ODN-2006-G5 at 37°C for 1 hour in DMEM containing 25 mM Hepes and 10% FBS. The reaction mixture was applied overnight at 37°C to hTLR9 HEKBlue cells engineered to secrete alkaline phosphatase (SEAP) in response to the TLR9 agonist. The culture medium was harvested and assayed for SEAP using a colorimetric substrate, and then ODN-2006-G5 was used. 620The readings were obtained using GraphPadPrism® version 6.0e software to calculate the IC50 values. The results are shown in Figure 3. All six constructs (RSLV-145, RSLV-147, RSLV-148, RSLV-149, RSLV-152, and RSLV-153) exhibited robust DNase activity, being at least 5000 times more active than recombinant huDNase1. RSLV-145 appeared to be approximately twice as active as the other heterodimer constructs and RSLV-327, which is likely due to the presence of two DNase domains in RSLV-145 compared to one in the other constructs. RSLV-152 was consistently the least active construct.

[0247] Example 4 Efficacy of optimized dual nuclease fusion protein in vitro Effect of optimized double nuclease fusion protein on cytokine expression Human PBMCs were isolated and cultured from normal and lupus patients. The cells were treated with various stimulating TLR ligands, co-stimulatory antibodies, immune complexes, and normal or autoimmune serum, with and without the optimized double nuclease fusion protein of Example 2. Culture supernatants were collected at various time points (e.g., 6 hours, 12 hours, 24 hours, 48 ​​hours, etc.), and the levels of a group of cytokines, including human IL-6, IL-8, IL-10, IL-4, IFN-γ, IFN-α, and TNF-α, were measured using a commercially available ELISA kit, e.g., from Thermo Fisher Scientific, Inc. An effective optimized double nuclease fusion protein was expected to reduce the levels of cytokines produced by stimulated PBMCs compared to a control.

[0248] Effect of optimized double nuclease fusion protein on lymphocyte-activating receptor expression Human PBMCs were isolated and cultured from normal and lupus patients. The cells were treated with various stimulating TLR ligands, co-stimulatory antibodies, immune complexes, and normal or autoimmune serum, with and without the optimized double nuclease fusion protein of Example 2. The cells were then subjected to multicolor flow cytometry to measure the expression of lymphocyte-activating receptors CD5, CD23, CD69, CD80, CD86, and CD25 at various time points after stimulation (e.g., 6 hours, 12 hours, 24 hours, 48 ​​hours, etc.) using routine methods accepted in the art. Suitable antibodies for these receptors are commercially available, for example, from BD / PharMingen. An effective optimized double nuclease fusion protein is expected to reduce the expression of lymphocyte-activating receptors in stimulated PBMCs compared to controls.

[0249] Effect of optimized dual nuclease fusion protein on plasmacytoid dendritic cell (pDC) interferon output pDCs derived from healthy volunteers are isolated using methods accepted in the art or commercially available kits, such as the EasySep® Human EpCAM Positive Selection Kit (StemCell Technologies, Inc.). For example, in a 96-well flat-bottom plate, 5 × 10¹⁶ pDCs are isolated in 0.1 ml of appropriate medium (e.g., complete RPMI medium containing 10% FBS, 2 mM glutamine, 55 μM β-mercaptoethanol, 1 mM sodium pyruvate, 100 U / ml penicillin, and 100 μg / ml streptomycin). 4 ~2.5×10 5Isolated pDCs are cultured at densities ranging from cells / well. The cultured pDCs are activated by adding serum or plasma from individual SLE patients diluted in a 1:5 ratio with culture medium, and 0.1 ml of these samples are added to the cell-containing wells (final patient serum concentration is 10%). The cultures are incubated at 37°C for 40 hours, after which the conditioned medium is collected and evaluated for IFNα content using a commercially available ELISA kit. Serum samples obtained from healthy volunteers are used as controls. To evaluate the effect of optimized double nuclease fusion protein, serum or plasma from SLE patients is pretreated with optimized double nuclease fusion protein (1-10 μg / ml) for 30 minutes and added to the pDC cultures. Effective optimized double nuclease fusion protein is expected to reduce the amount of IFNα produced as a result of degrading nucleic acid-containing ICs.

[0250] While the present invention has been described in particular with reference to preferred embodiments and various alternative embodiments, those skilled in the art will understand that various modifications in form and detail can be made without departing from the spirit and scope of the invention.

[0251] All references, published patents, and patent applications cited herein are incorporated herein by reference in their entirety for any purpose. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] Table 1-7 Table 1-8 Table 1-9 Table 1-10

Claims

1. A homodimer comprising a first nuclease domain, a second nuclease domain, and an Fc domain, wherein the first nuclease domain is human DNase1, the second nuclease domain is human RNase1, the human DNase1 is operably linked in tandem from the N-terminus to the C-terminus of the human RNase1, with or without a linker, the human RNase1 is operably linked with or without a linker to the N-terminus of the Fc domain, the homodimer comprises a polypeptide having an amino acid sequence that is at least 90% identical to the sequence described in SEQ ID NO: 1, and the homodimer retains the activity of a homodimer having the sequence described in SEQ ID NO:

1.

2. A homodimer comprising a first nuclease domain, a second nuclease domain, and an Fc domain, wherein the first nuclease domain is human DNase 1, the second nuclease domain is human RNase 1, the human RNase 1 is operably linked to the C-terminus of the Fc domain by or without a linker, the human DNase 1 is operably linked to the C-terminus of the human RNase 1 by a linker, the homodimer comprises a polypeptide having an amino acid sequence identical to at least 90% of the sequence described in SEQ ID NO: 2, and the homodimer retains the activity of a homodimer having an amino acid sequence described in SEQ ID NO:

2.

3. The homodimer according to claim 1 or claim 2, wherein the DNase comprises modifications of E13R, N74K, A114F, and T205K.

4. A composition comprising the homodimer described in any one of Claims 1 to 3.

5. The composition according to claim 4, comprising a pharmaceutically acceptable carrier.

6. A nucleic acid molecule comprising a nucleotide sequence encoding the homodimer described in any one of claims 1 to 3.

7. A composition for forming a homodimer according to any one of claims 1 to 3, comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide, wherein the polypeptide, together with other polypeptides encoded by the nucleic acid molecule, forms a homodimer according to any one of claims 1 to 3.

8. A recombinant expression vector comprising the nucleic acid molecule described in Claim 6.

9. The composition according to claim 7, wherein the nucleic acid molecule is present as a recombinant expression vector.

10. Host cells transformed with the recombinant expression vector described in Claim 8.

11. The composition according to claim 9, wherein host cells are transformed with the recombinant expression vector.

12. A method for producing a homodimer according to any one of claims 1 to 3, comprising the steps of: providing a host cell containing a nucleic acid molecule encoding the polypeptide of the homodimer; and maintaining the host cell under conditions in which the polypeptide is expressed.

13. A method for producing a homodimer, comprising the step of maintaining the cells described in claim 10 under conditions that allow expression of the polypeptide of the homodimer.

14. The method according to claim 12 or 13, wherein the polypeptide is expressed and thereby the homodimer is formed.

15. The method according to claim 14, further comprising the step of obtaining the homodimer.

16. A composition for treating or preventing a condition related to an abnormal immune response, comprising the homodimer described in any one of claims 1 to 3.

17. The composition according to claim 4 or 5 for treating or preventing a condition related to an abnormal immune response.

18. The composition according to claim 16 or 17, wherein the condition is an autoimmune disease.

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

20. The composition according to claim 18, wherein the autoimmune disease is SLE.

21. The composition according to claim 18, wherein the autoimmune disease is Sjögren's syndrome.

22. A composition for treating SLE, comprising a pharmaceutically acceptable carrier and a homodimer according to any one of claims 1 to 3, wherein the amount of the homodimer is effective in degrading an immune complex containing RNA, DNA, or both RNA and DNA.

23. A composition for treating Sjögren's syndrome, comprising a pharmaceutically acceptable carrier and a homodimer according to any one of claims 1 to 3, wherein the amount of the homodimer is effective in degrading an immune complex containing RNA, DNA, or both RNA and DNA.

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