Optimized dinuclease fusions and methods

Abstract

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

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201780053637.9, whose application date is June 30, 2017 and invention name is “Optimized dinuclease fusion and method”.

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 357,756, filed on July 1, 2016. The contents of the above application are incorporated herein by reference. Background Art

[0004] It is known that the accumulation of (ribo)nucleoprotein particles from dead and dying cells induces an inflammatory cascade in patients with systemic lupus erythematosus (SLE) by at least two mechanisms: (i) deposition or in situ formation of chromatin / antichromatin complexes causes nephritis and leads to loss of renal function; and (ii) nucleic acids complexed with autoantibodies activate innate immunity through Toll-like receptors (TLRs) 7, 8 and 9 and non-TLR-dependent pathways. The release of nucleoproteins can serve as potent antigens for autoantibodies in SLE, thereby providing expansion of B cells and activation of DCs through co-engagement of antigen receptors and TLRs. Therefore, there is a need for methods of removing nucleic acids bound to autoantibody antigens and / or attenuating immune stimulation, immune amplification and immune complex-mediated diseases in subjects in need, for example, using long-acting nuclease molecules that attack circulating immune complexes by digesting the nucleic acids contained therein. Summary of the invention

[0005] The present invention relates in part to the dinuclease (binuclease) fusion protein of optimization, it is the dinuclease fusion protein or heterodimer dinuclease fusion protein of the series connection that can be combined with multiple substrates with high nuclease activity.In some respects, the dinuclease fusion protein of series connection comprises one or more DNase1 and one or more RNase1 domains that are operably connected in series with one or more Fc domains.In some respects, the heterodimer dinuclease fusion protein comprises a single DNase1 domain and a single RNase1 domain that are operably connected with one or more Fc domains, so that DNase1 and RNase1 domains are located at the N- or C- end of the Fc domain.In some respects, the dinuclease fusion protein of optimization has eased the problem of expressing bifunctional nuclease-Fc chain and has alleviated the potential steric hindrance of one or more nuclease domains.In some respects, the heterodimer dinuclease fusion protein comprises one or more mutations in the Fc domain to maximize the formation of heterodimers.

[0006] In some embodiments, the dinuclease fusion protein of optimization is the dinuclease fusion protein of series connection, it comprises the first nuclease domain, the second nuclease domain and Fc district, wherein the first nuclease domain is DNase1 and the second nuclease domain is RNase1, wherein DNase1 is operably connected to RNase1 in series from N-terminal to C-terminal with or without joint, and RNase1 is operably connected to N-or C-terminal of Fc district.The dinuclease fusion protein of series connection shows enhanced pharmacokinetic activity relative to the first or second nuclease domain alone.This series connection dinuclease fusion protein shows the serum half-life (for example improved) of change relative to the first or second nuclease domain alone.

[0007] In some respects, the dinuclease fusion protein optimized is a heterodimer dinuclease fusion protein, which comprises 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, wherein DNase1 is operably connected to the N- or C-terminal of the Fc region with or without a joint, and RNase1 is operably connected to the N- or C-terminal of the Fc region with or without a joint, thereby forming a heterodimer. Heterodimer dinuclease fusion protein shows enhanced pharmacokinetic activity relative to the first or second independent nuclease domain. This heterodimer dinuclease fusion protein shows a serum half-life (e.g., improved) of change relative to the first or second independent nuclease domain.

[0008] In some aspects, the optimized dinuclease fusion protein is Figure 1 Those presented in .

[0009] In some aspects, the present invention provides an optimized dinuclease fusion protein comprising human DNase1, human RNase1, and mutant human IgG1 Fc, wherein human DNase1 is operably linked to human RNase1 from N-terminus to C-terminus via a linker (e.g., a gly-ser linker), and wherein human RNase1 is operably linked to a mutant human IgG1 Fc domain via a linker, wherein the mutant human IgG1 has a mutant hinge region (e.g., a cysteine ​​substitution, such as a substitution with serine, such as SCC)) and one or more CH2 mutations to reduce Fcγ receptor binding (e.g., P238S, P331S, or both P238S and P331S, numbered according to the EU index). In one embodiment, the optimized dinuclease fusion protein comprises a human DNase1 (N-terminal-DNase1-linker-RNase1-C-terminal) operably linked to human RNase1 via a peptide linker (e.g., a gly-ser linker), and human RNase1 is operably linked to a mutant human IgG1 Fc domain having a mutant hinge region, an SCC hinge, and P238S and P331S mutations via a peptide linker (e.g., a gly-ser linker). In yet another embodiment, the Fc domain further comprises a mutation at an N-linked glycosylation site, such as a substitution at N297 (according to Kabat numbering).

[0010] In some embodiments, the optimized di-nuclease fusion protein further comprises a first linker domain, and the first nuclease domain is operably coupled to the second nuclease domain via the first linker domain.

[0011] In some embodiments, the optimized di-nuclease fusion protein further comprises a second linker domain, and the second nuclease domain is operably coupled to the Fc domain via the second linker domain.

[0012] In some embodiments, the RNase domain is a wild-type RNase, such as wild-type human RNase1. In other embodiments, the RNase domain is a mutant RNase, such as non-glycosylated, hypoglycosylated or deglycosylated RNase1, such as human RNase1N34S / N76S / N88S (SEQ ID NO: 28). In some embodiments, the optimized two-nuclease fusion protein containing RNase degrades RNA in circulating RNA and immune complexes, or inhibits interferon-α production, or both. In other embodiments, the activity of RNase is not less than about 10 times the activity of the control RNase molecule, such as 9 times lower, 8 times lower, 7 times lower, 6 times lower, 5 times lower, 4 times lower, 3 times lower or 2 times lower. In other embodiments, the activity of RNase is approximately equal to the activity of the control RNase molecule.

[0013] In some embodiments, the DNase domain is a wild-type DNase, such as wild-type human DNase1. In other embodiments, the DNase domain is a mutant DNase domain, such as mutant human DNase1 A114F (SEQ ID NO: 21) or non-glycosylated, low-glycosylated or deglycosylated human DNase, such as mutant human DNase1N18S / N106S / A114F (SEQ ID NO: 24). In some embodiments, the optimized two-nuclease fusion protein containing DNase degrades circulating DNA and DNA in immune complexes, or inhibits the production of interferon-α, or both. In yet other embodiments, the activity of DNase is not less than about 10 times the activity of the control DNase molecule, such as 9 times lower, 8 times lower, 7 times lower, 6 times lower, 5 times lower, 4 times lower, 3 times lower or 2 times lower. In other embodiments, the activity of DNase is approximately equal to the activity of the control DNase molecule.

[0014] In some embodiments, the optimized di-nuclease fusion protein has a gly-ser linker separating the first and second nuclease domains and / or the second nuclease domain from the Fc domain.

[0015] In some embodiments, the optimized di-nuclease fusion protein has increased serum half-life and / or activity relative to a molecule without an Fc domain.

[0016] In some aspects, the optimized dinuclease fusion protein may include the mutant human DNase1 A114F domain shown in SEQ ID NO: 21. In another embodiment, the optimized dinuclease fusion protein may include the mutant human DNase1 N18S / N106S / A114F domain shown in SEQ ID NO: 24. In some embodiments, the DNase domain is mutant human DNase1 E13R / N74K / A114F / T205K (SEQ ID NO: 25). In other embodiments, the DNase domain is mutant human DNase1 E13R / N74K / A114F / T205K / N18S / N106S (SEQ ID NO: 26).

[0017] In some embodiments, DNase1 and RNase1 domains are non-glycosylated, low glycosylated or deglycosylated. In some embodiments, the DNase domain is a mutant DNase domain, such as a mutant human DNase1 and a non-glycosylated, low glycosylated or deglycosylated DNase domain, such as a non-glycosylated, low glycosylated or deglycosylated human DNase1. In one embodiment, human DNase1 includes changes (e.g., displacements) at one or more N-linked glycosylation sites (e.g., N18 and N106), and at least one other mutation selected from A114, E13, N74, T205 and combinations thereof. In another embodiment, human DNase1 includes changes (e.g., displacements) at N18, N106 or both N18 and N106 and other changes (e.g., displacements) at A114, E13, N74, T205 and combinations thereof. In yet another embodiment, human DNase1 includes changes at N18, N106, A114, E13, N74 and T205, such as substitutions, for example N18S / N106S / A114F / E13R / N74K / T205K (SEQ ID NO: 26). In another embodiment, the optimized dinuclease fusion protein with altered glycosylation includes the human wild-type RNase1 domain shown in SEQ ID NO: 27. In another embodiment, the optimized dinuclease fusion protein with altered glycosylation includes the human mutant RNase1 N34S / N76S / N88S domain as shown in SEQ ID NO: 28.

[0018] In some aspects, the present invention provides an optimized dinuclease fusion protein comprising a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1-17. In other aspects, the optimized dinuclease fusion protein has an amino acid sequence that is at least 90% identical or at least 95% identical to the amino acid sequence as shown in SEQ ID NO: 1-17.

[0019] In some aspects, the optimized two-nuclease fusion protein comprises a polypeptide containing a first nuclease domain, a second nuclease domain and an Fc domain, wherein the first nuclease domain is DNase1, and the second nuclease domain is RNase1, wherein DNase1 is operably connected to RNase1 in series from N-terminus to C-terminus with or without a joint, and RNase1 is operably connected to the Fc region with or without a joint. In some aspects, RNase 1 is operably connected to the N-terminus of the Fc domain without a joint. In some aspects, RNase 1 is operably connected to the C-terminus of the Fc domain without a joint. In some aspects, DNase1 is operably connected to RNase1 through a joint. In some aspects, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, or a tandem fusion protein comprising an amino acid sequence at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. In some aspects, the tandem two-nuclease fusion protein is a homodimer comprising any of the aforementioned polypeptides.

[0020] In some respects, the optimized two-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, wherein DNase1 is operably connected to the N- or C-terminal of the first Fc domain with or without a joint, and RNase1 is operably connected to the N- or C-terminal of the second Fc domain with or without a joint. In some aspects of the aforementioned heterodimer, DNase1 is operably connected to the N-terminal of the first Fc domain without a joint, and RNase1 is operably connected to the N-terminal of the second Fc domain without a joint.

[0021] In some aspects, DNase 1 is operably connected to the N-terminal of the first Fc domain when there is a joint, and RNase1 is operably connected to the N-terminal of the second Fc domain when there is a joint. In some aspects, DNase1 is operably connected to the N-terminal of the first Fc domain when there is a joint, and RNase1 is operably connected to the C-terminal of the second Fc domain when there is no joint. In some aspects, DNase1 is operably connected to the N-terminal of the first Fc domain when there is no joint, and RNase1 is operably connected to the C-terminal of the second Fc domain when there is no joint. In some aspects, DNase 1 is operably connected to the N-terminal of the first Fc domain when there is a joint, and RNase1 is operably connected to the C-terminal of the second Fc domain when there is a joint. In some aspects, DNase 1 is operably connected to the C-terminal of the first Fc domain when there is a joint, and RNase1 is operably connected to the C-terminal of the second Fc domain when there is a joint. In some aspects, DNase1 is operably connected to the C-terminal of the first Fc domain without a joint, and RNase1 is operably connected to the C-terminal of the second Fc domain without a joint. In some aspects, DNase 1 is operably connected to the C-terminal of the first Fc domain with a joint, and RNase1 is operably connected to the N-terminal of the second Fc domain without a joint. In some aspects, DNase 1 is operably connected to the C-terminal of the first Fc domain with a joint, and RNase1 is operably connected to the N-terminal of the second Fc domain with a joint.

[0022] In some aspects, the optimized di-nuclease fusion protein is a heterodimer comprising a first and a second polypeptide sequence selected from the group consisting of:

[0023] (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:3; and a second polypeptide comprising the amino acid sequence of SEQ ID NO:4, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:4, or

[0024] (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO:7, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:7; and a second polypeptide comprising the amino acid sequence of SEQ ID NO:8, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:8, or

[0025] (iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO:9, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:9; and a second polypeptide comprising the amino acid sequence of SEQ ID NO:10, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:10, or

[0026] (iv) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 11, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 12, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, or

[0027] (v) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 15, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 16, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16.

[0028] Other aspects relate to heterodimers comprising a first nuclease domain, a second nuclease domain, and a first Fc domain and a second Fc domain, wherein the first nuclease domain is DNase1, and the second nuclease domain is RNase1, wherein

[0029] (i) DNase1 is operably linked to the N-terminus of the first Fc domain with or without a linker, and RNase1 is operably linked to the C-terminus of the first Fc domain with or without a linker, or

[0030] (ii) RNase1 is operably linked to the N-terminus of the first Fc domain with or without a linker, and DNase1 is operably linked to the C-terminus of the first Fc domain with or without a linker.

[0031] In some aspects of the aforementioned heterodimer, DNase 1 is operably connected to the N-terminal of the first Fc domain in the absence of a joint, and RNase1 is operably connected to the C-terminal of the first Fc domain in the presence of a joint. In some aspects, DNase 1 is operably connected to the N-terminal of the first Fc domain in the presence of a joint, and RNase1 is operably connected to the C-terminal of the first Fc domain in the absence of a joint. In some aspects, RNase 1 is operably connected to the N-terminal of the first Fc domain in the absence of a joint, and DNase 1 is operably connected to the C-terminal of the first Fc domain in the presence of a joint. In some aspects, RNase 1 is operably connected to the N-terminal of the first Fc domain in the presence of a joint, and DNase 1 is operably connected to the C-terminal of the first Fc domain in the presence of a joint.

[0032] In some aspects, the heterodimer comprises a first and a second polypeptide sequence selected from the group consisting of:

[0033] (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:5, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:5; and a second polypeptide comprising the amino acid sequence of SEQ ID NO:6, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:6, or

[0034] (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 14.

[0035] In some aspects, any of the aforementioned heterodimers include one or more CH3 mutations in the Fc domain to preferentially form heterodimers. In some aspects, the heterodimer includes a first Fc domain containing CH3 mutations T350V, L351Y, F405A and Y407V numbered according to the EU index, and a second Fc domain containing CH3 mutations T350V, T366L, K392L, T394W.

[0036] Other aspects of the present disclosure relate to compositions comprising any of the aforementioned heterodimers and a pharmaceutically acceptable carrier. Also disclosed are nucleic acid molecules encoding the aforementioned heterodimers, recombinant expression vectors, and host cells transformed with the recombinant expression vectors, as well as methods for preparing the aforementioned heterodimers.

[0037] Also disclosed herein is a method for preparing the tandem optimized dinuclease fusion protein disclosed herein, comprising providing a host cell comprising a nucleic acid sequence encoding the optimized dinuclease fusion protein; and maintaining the host cell under conditions where the optimized dinuclease fusion protein is expressed.

[0038] Also disclosed herein are methods of treating or preventing a disorder associated with an abnormal immune response by administering an effective amount of the optimized dinuclease fusion protein disclosed herein to a patient in need thereof. In some embodiments, the disorder is an autoimmune disease. In some embodiments, the autoimmune disease is selected from insulin-dependent diabetes, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental uveitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, IgG4-related disease, Addison's disease, premature menopause, male infertility, juvenile diabetes, Goodpasture syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, crystalline uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis HBs-ve, cryptogenic cirrhosis, ulcerative colitis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid LE, systemic lupus erythematosus (SLE) and connective tissue disease. In some embodiments, the autoimmune disease is SLE or Sjogren's syndrome.

[0039] Also disclosed herein is a method for treating SLE or Sjögren's syndrome, comprising administering to a subject a composition of an inclusion optimized dinuclease fusion protein in an amount effective to degrade immune complexes containing RNA, DNA, or both RNA and DNA. In some aspects, the composition comprises a pharmaceutically acceptable carrier and an optimized dinuclease fusion protein as described herein. In other aspects, the composition comprises an optimized dinuclease fusion protein having an amino acid sequence shown in SEQ ID NO: 1.

[0040] In another aspect, the invention relates to an optimized dinuclease fusion protein for treating diseases characterized by defective clearance or processing of apoptotic cells and cell debris, such as SLE. In some embodiments, the optimized dinuclease fusion protein comprises the amino acid sequences shown in SEQ ID NO:4 and 5.

[0041] In another aspect, the present invention relates to the use of the optimized dinuclease fusion protein for preparing a medicament for treating a disease characterized by defects in the clearance or processing of apoptotic cells and cell debris, such as SLE. In some embodiments, the optimized dinuclease fusion protein comprises the amino acid sequences shown in SEQ ID NO: 5 and 6.

[0042] In another aspect, the present invention relates to the use of the optimized dinuclease fusion protein for preparing a medicament for treating a disease characterized by defects in the clearance or processing of apoptotic cells and cell debris, such as SLE. In some embodiments, the optimized dinuclease fusion protein comprises the amino acid sequences shown in SEQ ID NO: 7 and 8.

[0043] On the other hand, the present invention relates to the use of the optimized dinuclease fusion protein for preparing a medicament for treating a disease characterized by defects in the clearance or processing of apoptotic cells and cell debris, such as SLE. In some embodiments, the optimized dinuclease fusion protein comprises the amino acid sequences shown in SEQ ID NO: 13 and 14.

[0044] In another aspect, the present invention relates to the use of the optimized dinuclease fusion protein for preparing a medicament for treating a disease characterized by defects in the clearance or processing of apoptotic cells and cell debris, such as SLE. In some embodiments, the optimized dinuclease fusion protein comprises the amino acid sequences shown in SEQ ID NO: 15 and 16. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] These and other features, aspects and advantages of the present invention will be better understood with reference to the following description and accompanying drawings, in which:

[0046] Figure 1 is a description of an exemplary optimized dinuclease fusion protein.

[0047] Figure 2 is a graph showing RNase activity measured by OD260.

[0048] Figure 3 Display by OD 620 (Left) and IC 50 (Right) Measured DNase activity. DETAILED DESCRIPTION

[0049] Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease characterized by the presence of high titers of autoantibodies to self-nucleoproteins. There is strong evidence that defects in the clearance or processing of dead and dying cells in SLE lead to disease, primarily through the accumulation of ribonucleoproteins and deoxyribonucleoproteins (abbreviated nucleoproteins). Nucleoproteins cause damage by three mechanisms: i) activating the innate immune system to produce inflammatory cytokines; ii) serving as antigens to produce circulating immune complexes; and iii) serving as antigens to produce in situ complex formation at local sites such as the kidney.

[0050] The present invention provides a method for treating diseases characterized by defects in the clearance or processing of apoptotic cells and cell debris (e.g., SLE and Sjögren's syndrome) by administering an effective amount of long-acting nuclease activity to degrade extracellular RNA and DNA-containing immune complexes. This treatment can inhibit the production of type I interferon (IFN), which is an important cytokine in SLE and is strongly associated with disease activity and nephritis.

[0051] The present invention relates in part to providing such long-acting nucleases. In particular, the present invention relates to an optimized dinuclease fusion protein, such as a tandem dinuclease 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, wherein DNase1 is operably connected to RNase1 in series from the N-terminus to the C-terminus with or without a linker, and RNase1 is operably connected to the N-terminus or the C-terminus of the Fc region.

[0052] In other embodiments, the present invention relates to an optimized dinuclease fusion protein, such as a heterodimeric dinuclease 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, wherein DNase1 is operably linked to the N- or C-terminus of the Fc region with or without a linker, and RNase1 is operably linked to the N- or C-terminus of the Fc region with or without a linker, thereby forming a heterodimer.

[0053] In some respects, the optimized two nucleic acid enzyme fusion proteins show enhanced pharmacokinetic activity relative to the first or second nucleic acid enzyme domain alone. This optimized two nucleic acid enzyme fusion proteins show (for example, improved) serum half-life of change relative to the first or second nucleic acid enzyme domain alone.

[0054] In some aspects, the present invention provides an optimized dinuclease fusion protein comprising human DNase1, human RNase1, and mutant human IgG1 Fc, wherein human DNase1 is operably linked to human RNase1 from N-terminus to C-terminus via a linker (e.g., a gly-ser linker), and wherein human RNase1 is operably linked to a mutant human IgG1 Fc domain via a linker, wherein the mutant human IgG1 has a mutated hinge region (e.g., cysteine ​​substitutions, such as substitutions with serine, such as SCC)) and one or more CH2 mutations to reduce Fcγ receptor binding (e.g., P238S, P331S, or both P238S and P331S, numbered according to the EU index). In one embodiment, the optimized dinuclease fusion protein comprises human DNase1 (N-terminus-DNase1-linker-RNase1-C-terminus) operably linked to human RNase1 via a peptide linker (e.g., a gly-ser linker), and human RNase1 is operably linked to a mutant human IgG1 Fc domain having a mutant hinge region, an SCC hinge, and P238S and P331S mutations via a peptide linker (e.g., a gly-ser linker).

[0055] Thus, in one embodiment, a subject suffering from a disease characterized by defective clearance or processing of apoptotic cells and cellular debris is treated by administering an optimized di-nuclease fusion protein (which includes both DNase1 and RNase1) such that the optimized di-nuclease fusion protein has increased bioavailability and / or serum half-life relative to the unconjugated nuclease domain.

[0056] definition

[0057] Unless otherwise stated, the terms used in the claims and specification are defined as follows.

[0058] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, 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, i.e., carbon bound to a hydrogen, carboxyl, amino, and R groups, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid but function in a manner similar to naturally occurring amino acids.

[0059] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0060] "Amino acid substitution" refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (the amino acid sequence of the starting polypeptide) with a second, different "replacement" amino acid residue. "Amino acid insertion" refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. Although insertions typically consist of the insertion of one or two amino acid residues, larger "peptide insertions" can be formed, for example, insertions of about 3 to about 5 or even up to about 10, 15 or 20 amino acid residues. The inserted residues can be naturally occurring or non-naturally occurring, as described above. "Amino acid deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0061] "Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0062] "Nucleic acid" refers to a polymer of deoxyribonucleotides or ribonucleotides and single-stranded or double-stranded forms thereof. Unless otherwise specified, the term includes nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (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, the modification at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0063] The polynucleotides of the present invention can be composed of any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, the polynucleotides can be composed of single-stranded and double-stranded DNA, DNA as a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA as a mixture of single-stranded and double-stranded regions, comprising DNA and RNA that can be single-stranded or more generally double-stranded or a mixture of single-stranded and double-stranded regions. In addition, the polynucleotides can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. The polynucleotides can also contain one or more modified bases or DNA or RNA backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. DNA and RNA can be variously modified; therefore, "polynucleotides" include chemically, enzymatically or metabolically modified forms.

[0064] As used herein, the term "operably linked" or "operably coupled" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.

[0065] As used herein, the term "glycosylation" or "glycosylated" refers to the process or result of adding sugar moieties to a molecule (eg, an optimized di-nuclease fusion protein).

[0066] As used herein, the term "altered glycosylation" refers to non-glycosylated, deglycosylated or hypoglycosylated molecules.

[0067] As used herein, "glycosylation site" refers to sites that potentially may accept carbohydrate moieties as well as sites within a protein to which carbohydrate moieties are actually attached, and includes any amino acid sequence that can act as an acceptor for oligosaccharides and / or carbohydrates.

[0068] As used herein, the term "non-glycosylated" or "non-glycosylated" refers to the production of a molecule (e.g., an optimized dinuclease fusion protein) in an unglycosylated form (e.g., by engineering the optimized dinuclease fusion protein to lack amino acid residues that serve as acceptors for glycosylation). Alternatively, the optimized dinuclease fusion protein can be expressed in, for example, E. coli to produce a non-glycosylated optimized dinuclease fusion protein.

[0069] As used herein, the term "deglycosylation" or "deglycosylated" refers to the process or result of enzymatic removal of sugar moieties on a molecule.

[0070] As used herein, the term "hypoglycosylation" or "hypoglycosylated" refers to a molecule in which one or more carbohydrate structures normally present if produced in a mammalian cell have been omitted, removed, altered or masked.

[0071] As used herein, the terms "Fc region" and "Fc domain" are formed by the corresponding Fc domains (or Fc parts) of its two heavy chains without the part of the natural immunoglobulin of the variable region that binds the antigen. In some embodiments, the Fc domain starts in the hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Therefore, the complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc domain comprises at least one of the following: a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, part, or fragment thereof. In other embodiments, the Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In one embodiment, the Fc domain comprises a hinge domain (or part thereof) fused to a CH3 domain (or part thereof). In another embodiment, the Fc domain comprises a CH2 domain (or part thereof) fused to a CH3 domain (or part thereof). In another embodiment, the Fc domain consists of a CH3 domain or a portion thereof. In another embodiment, the Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In another embodiment, the Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In another embodiment, the Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In one embodiment, the Fc domain lacks at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). In one embodiment, the Fc domain of the present invention comprises at least a portion of the Fc molecule required for FcRn binding known in the art. In one embodiment, the Fc domain of the present invention comprises at least a portion of the Fc molecule required for protein A binding known in the art. In one embodiment, the Fc domain of the present invention comprises at least a portion of the Fc molecule required for protein G binding known in the art. The Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising complete CH1, hinge, CH2 and / or CH3 domains and fragments of such peptides comprising only, for example, hinge, CH2 and CH3 domains. The Fc domain can be derived from an immunoglobulin of any species and / or any subtype (including but not limited to human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies). The Fc domain includes natural Fc and Fc variant molecules. Like Fc variants and natural Fc, the term Fc domain includes molecules in monomeric or multimeric form, whether digested from a complete antibody or produced by other means.

[0072] As described herein, those of ordinary skill in the art will appreciate that any Fc domain can be modified such that its amino acid sequence is altered from that of a native Fc domain of a naturally occurring immunoglobulin molecule.

[0073] The Fc domain of the optimized dinuclease fusion protein of the present disclosure can be derived from different immunoglobulin molecules. For example, the Fc domain of the optimized dinuclease fusion protein can include CH2 and / or CH3 domains derived from IgG1 molecules and hinge regions derived from IgG3 molecules. In another example, the Fc domain can include a chimeric hinge region derived in part from IgG1 molecules and in part from IgG3 molecules. In another example, the Fc domain can include a chimeric hinge derived in part from IgG1 molecules and in part from IgG4 molecules. The wild-type human IgG1 Fc domain has the amino acid sequence shown in SEQ ID NO: 45.

[0074] As used herein, the term "serum half-life" refers to the time required for the in vivo serum concentration of the optimized dinuclease fusion protein to decrease by 50%. The shorter the serum half-life of the optimized dinuclease fusion protein, the shorter the time it takes to exert its therapeutic effect.

[0075] As used herein, the term "optimized dinuclease fusion protein" refers to a polypeptide comprising at least two nuclease domains operably connected to an Fc domain or a variant or fragment thereof with or without a joint, and nucleic acids encoding these polypeptides. In some embodiments, the optimized dinuclease fusion protein is a tandem dinuclease fusion protein, such as one or more DNase1 domains and one or more RNase 1 domains connected in series to the N- or C-terminus of one or more Fc domains. In some embodiments, the optimized dinuclease fusion protein is a heterodimer dinuclease fusion protein.

[0076] As used herein, the term "tandem two-nuclease fusion protein" refers to a polypeptide comprising at least two nuclease domains and an Fc domain or its variant or fragment connected in series (from N-terminal to C-terminal), and a nucleic acid encoding such a polypeptide. For example, in one embodiment, the tandem two-nuclease fusion protein is a polypeptide comprising at least one DNase1 domain and at least one RNase1 domain operably connected in series with at least one Fc domain. As another example, the tandem two-nuclease fusion protein comprises a DNase1 domain, a first joint, an RNase1 domain, a second joint and an Fc domain or its variant or fragment from N-terminal to C-terminal.

[0077] As used herein, the term "heterodimer dinuclease fusion protein" refers to a heterodimer comprising a first and a second polypeptide (which together comprises at least two nuclease domains and two Fc domains, variants or fragments thereof), and nucleic acids encoding these polypeptides. In some embodiments, the heterodimer dinuclease fusion protein is a heterodimer comprising at least one DNase1 domain and at least one RNase1 domain operably connected to at least one Fc domain, wherein the DNase1 domain is operably connected to the N- or C- end of the first Fc domain with or without a joint, and the RNase1 domain is operably connected to the N- or C- end of the same Fc domain (the first Fc domain) or different Fc domains (the second Fc domain) with or without a joint, so that the DNase 1 domain and the RNase 1 domain are located at the opposite end (N- or C- end) of the same Fc domain (the first Fc domain) or different Fc domains (the second Fc domain). In some embodiments, the heterodimer is included in a DNase 1 domain operably connected to the N- or C-terminal of the first Fc domain with or without a joint, and an RNase 1 connected to the N- or C- of the second Fc domain with or without a joint, so that the DNase 1 and RNase 1 domains are located in series at the same end (N- or C-terminal) of the heterodimer. In some embodiments, the heterodimer is included in a DNase 1 domain operably connected to the N- or C-terminal of the first Fc domain with or without a joint, and an RNase 1 operably connected to the C-terminal of the first Fc domain with or without a joint. In some embodiments, RNase 1 is operably connected to the N-terminal of the first Fc domain with or without a joint, and DNase 1 is operably connected to the C-terminal of the first Fc domain with or without a joint.

[0078] As used herein, the term "variant" refers to a polypeptide derived from a wild-type nuclease or Fc domain and differing from the wild type in one or more changes (i.e., substitution, insertion and / or deletion) at one or more positions. Substitution refers to replacing an amino acid occupying a certain position with a different amino acid. Deletion means removing an amino acid occupying a certain position. Insertion means adding 1 or more, such as 1-3 amino acids, to an amino acid adjacent to an amino acid occupying a certain position. Variant polypeptides must have less than 100% sequence identity or similarity with wild-type polypeptides. In some embodiments, variant polypeptides, for example, will have an amino acid sequence identity or similarity of about 75% to less than 100% with the amino acid sequence of the wild-type polypeptide over the entire length of the variant polypeptide, or about 80% to less than 100%, or 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%.

[0079] In some aspects, the optimized two nucleic acid enzyme fusion protein adopts one or more "joint domains", such as polypeptide joints. As used herein, the term "joint domain" refers to one or more amino acids connecting two or more peptide domains in a linear polypeptide sequence. As used herein, the term "polypeptide joint" refers to two or more polypeptide domain peptides or polypeptide sequences (for example, synthetic peptides or polypeptide sequences) in a linear amino acid sequence connecting a protein. For example, a polypeptide joint can be used to operably connect the first and second nucleic acid enzyme domains to each other, or to operably connect the first or second nucleic acid enzyme domain to an Fc domain. In some embodiments, such polypeptide joints provide flexibility for polypeptide molecules. In some embodiments, a polypeptide joint is used to connect DNase1 to RNase1 and / or RNase1 to an Fc domain (for example, genetic fusion). The optimized two nucleic acid enzyme fusion protein may include more than one joint domain or peptide joint. Various peptide joints are known in the art.

[0080] As used herein, the term "gly-ser polypeptide linker" refers to a peptide consisting of glycine and serine residues. An exemplary gly / ser polypeptide linker comprises the amino acid sequence (Gly4Ser)n (SEQ ID NO: 58). In some embodiments, n is 1 or more, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (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 more, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more (e.g., Ser(Gly4Ser)10) (SEQ ID NO: 61).

[0081] As used herein, the terms "coupled," "linked," "fused," or "fused" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains by any means, including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional cross-linking agents) are known in the art.

[0082] A polypeptide or amino acid sequence "derived from" a specified polypeptide or protein refers to the source of the polypeptide. Preferably, a polypeptide or amino acid sequence derived from a specific sequence has an amino acid sequence substantially identical to that sequence or a portion thereof (wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids), or it is otherwise identifiable to one of ordinary skill in the art as having its sequence origin. A polypeptide derived from another peptide may have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues that have been replaced by another amino acid residue or have one or more amino acid residue insertions or deletions.

[0083] In one embodiment, there is one amino acid difference between the starting polypeptide sequence and the sequence derived therefrom. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues (i.e., identical residues) in the candidate sequence that are identical to the starting amino acid residue, after aligning the sequences and introducing spaces (if necessary) to achieve maximum percent sequence identity.

[0084] In one embodiment, the polypeptide of the present disclosure consists of, consists essentially of, or comprises an amino acid sequence and functionally active variants thereof listed in the sequence listing or sequence table disclosed herein. In one embodiment, the polypeptide comprises an amino acid sequence that is at least 80%, such as 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 96%, at least 97%, at least 98% or at least 99% identical to an amino acid sequence listed in the sequence listing or sequence table disclosed herein. In some embodiments, the polypeptide comprises a continuous amino acid sequence that is at least 80%, such as 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 continuous amino acid sequence listed in the sequence listing or sequence table disclosed herein. In some embodiments, the polypeptide comprises an amino acid sequence having at least 10, such as 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 these numbers) of continuous amino acids of the amino acid sequence listed in the sequence listing or sequence table disclosed herein.

[0085] In some embodiments, the optimized dinuclease fusion protein of the present disclosure is encoded by a nucleotide sequence. The nucleotide sequence of the present disclosure can be used for many applications, including: cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of the host required, antibody production for passive immunity, PCR, primer and probe generation, siRNA design and generation (see, for example, Dharmacon siDesign website) etc. In some embodiments, the nucleotide sequence of the present disclosure comprises a nucleotide sequence encoding the amino acid sequence of the optimized dinuclease fusion protein selected from a sequence table or a sequence table, is composed of it or is substantially composed of it. In some embodiments, the nucleotide sequence includes at least 80% of the nucleotide sequence encoding the amino acid sequence of the sequence table or a sequence table disclosed herein, 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 96%, at least 97%, at least 98% or at least 99% identical nucleotide sequence. In some embodiments, the nucleotide sequence comprises a contiguous nucleotide sequence that is at least 80%, e.g., 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 a contiguous nucleotide sequence encoding an amino acid sequence listed in a sequence listing or sequence listing disclosed herein. In some embodiments, the nucleotide sequence includes a nucleotide sequence having at least 10, such as at least 15, such as 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 these numbers) consecutive nucleotides of a nucleotide sequence encoding an amino acid sequence listed in a sequence listing or sequence listing disclosed herein.

[0086] It will also be understood by those of ordinary skill in the art that the optimized dinuclease fusion protein can be changed so that they change in sequence from the natural existence or native sequence derived from its components (for example, nuclease domain, joint domain and Fc domain), while retaining the required activity of the native sequence.For example, the conservative substitution or the nucleotide or amino acid substitution causing the change at the "non-essential" amino acid residue can be carried out. The isolated nucleic acid molecule encoding non-natural variants can be produced by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of the optimized dinuclease fusion protein, so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Sudden changes can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0087] The optimized dinuclease fusion protein can comprise conservative amino acid replacement at one or more amino acid residues, for example, at essential or non-essential amino acid residues." conservative amino acid replacement " is the amino acid replacement that wherein amino acid residue is replaced by the amino acid residue with similar side chain. The family of amino acid residue with similar side chain has been defined in this area, including basic side chain (for example, lysine, arginine, histidine), acidic side chain (for example, aspartic acid, glutamic acid), uncharged polar side chain (for example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chain (for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chain (for example, threonine, valine, isoleucine) and aromatic side chain (for example, tyrosine, phenylalanine, tryptophan, histidine). Therefore, the non-essential amino acid residue in the optimized dinuclease fusion protein is preferably replaced by another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and / or in the composition of side chain family members. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be incorporated into optimized dinuclease fusion proteins and screened for their ability to bind to the desired target.

[0088] The term "amelioration" refers to any beneficial outcome in the treatment of a disease state, such as an autoimmune disease state (eg, SLE, Sjögren's syndrome), including prevention, reduction in severity or progression, regression, or cure thereof.

[0089] The term "in situ" refers to a process occurring in living cells grown isolated from a living organism, for example grown in tissue culture.

[0090] The term "in vivo" refers to processes that occur in a living organism.

[0091] As used herein, the term "mammal" or "subject" or "patient" includes both humans and non-humans, and includes, but is not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0092] In the context of two or more nucleic acid or polypeptide sequences, the term percent "identity" refers to two or more sequences or subsequences having a specified percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence (e.g., using one of the sequence comparison algorithms described below (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 percent "identity" can exist over a region of the sequences being compared, for example, over a functional domain, or, alternatively, over the entire length of the two sequences being compared.

[0093] For sequence comparison, usually one sequence serves as a reference sequence to be compared with a test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified (if necessary), and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

[0094] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv Appl Math 1981; 2: 482, by the homology alignment algorithm of Needleman & Wunsch, J Mol Biol 1970; 48: 443, by the similarity search method of Pearson & Lipman, PNAS 1988; 85: 2444, by computerized 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 generally Ausubel et al., below).

[0095] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J Mol Biol 1990; 215: 403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.

[0096] The term "sufficient amount" refers to an amount sufficient to produce the desired effect.

[0097] The term "therapeutically effective amount" is an amount effective to ameliorate symptoms of a disease. A therapeutically effective amount can be a "prophylactically effective amount" since prevention can be considered therapy.

[0098] The term "about" will be understood by an ordinary skilled artisan and will vary to some extent depending on the context in which it is used. If the use of the term is unclear to an ordinary skilled artisan given the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0099] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0100] Optimized dinuclease fusion protein

[0101] The optimized two-nuclease fusion protein of the present invention comprises an Fc domain or a variant or fragment thereof, which changes the serum half-life of the nuclease molecule fused thereto compared with the nuclease molecule not fused thereto.

[0102] In some embodiments, the compositions of the present disclosure include an optimized di-nuclease fusion protein. In some embodiments, the optimized di-nuclease fusion protein includes a nuclease domain operably coupled to an Fc domain or a variant or fragment thereof.

[0103] In some embodiments, the nuclease domain is operably coupled to the Fc domain or a variant or fragment thereof via a joint domain. In some embodiments, the joint domain is a joint peptide. In some embodiments, the joint domain is a joint nucleotide.

[0104] In some embodiments, the optimized dinuclease fusion protein includes a leader molecule, such as a leader peptide. In some embodiments, the leader molecule is a leader peptide located at the N-terminal of the nuclease domain. In some embodiments, the optimized dinuclease fusion protein of the present invention comprises a leader peptide at the N-terminal of the molecule, wherein the leader peptide is subsequently cut from the optimized dinuclease fusion protein. The method of generating the nucleic acid sequence encoding the leader peptide fused to the recombinant protein is well known in the art. In some embodiments, any optimized dinuclease fusion protein of the present invention can be expressed with or without a leader peptide fused to its N-terminal. Those skilled in the art can predict and / or infer the protein sequence of the optimized dinuclease fusion protein of the present invention after the leader peptide cutting of the fusion.

[0105] In some embodiments, the leader peptide is a VK3 leader peptide (VK3LP), wherein the leader peptide is fused to the N-terminal of the optimized dinuclease fusion protein. This leader sequence can improve the synthesis and secretion level of the optimized dinuclease fusion protein in mammalian cells. In some embodiments, the leader sequence is cut, thereby producing the optimized dinuclease fusion protein. In some embodiments, the optimized dinuclease fusion protein of the present invention is expressed without a leader peptide fused to its N-terminal, and the resulting optimized dinuclease fusion protein has an N-terminal methionine.

[0106] In some embodiments, the optimized di-nuclease fusion protein includes two nuclease domains operably coupled to each other in tandem and further operably coupled to the N- or C-terminus of the same or different Fc domains, or variants or fragments thereof.

[0107] Figure 1 Exemplary configurations of optimized di-nuclease fusion proteins are shown, and the sequence table provides the sequences of exemplary optimized di-nuclease fusion proteins of various configurations.

[0108] In some embodiments, the optimized di-nuclease fusion protein is a multi-nuclease protein (e.g., both an RNase and a DNase or two RNA or DNA nucleases with different substrate specificities) fused to the same or different Fc domains, or variants or fragments thereof, which specifically binds to extracellular immune complexes.

[0109] In one embodiment, the nuclease domain is operably coupled (e.g., chemically conjugated or genetically fused (e.g., directly or through a polypeptide linker)) to the N-terminal of an Fc domain or a variant or fragment thereof. In another embodiment, the nuclease domain is operably coupled (e.g., chemically conjugated or genetically fused (e.g., directly or through a polypeptide linker)) to the C-terminal of an Fc domain or a variant or fragment thereof. In other embodiments, the nuclease domain is operably coupled (e.g., chemically conjugated or genetically fused (e.g., directly or through a polypeptide linker)) via the amino acid side chains of an Fc domain or a variant or fragment thereof.

[0110] In certain embodiments, the disclosed two nucleic acid enzyme fusion proteins of optimization comprise two or more nucleic acid enzyme domains and at least one Fc domain or its variant or fragment.For example, the nucleic acid enzyme domain can be operably coupled to the N-terminal and C-terminal of identical or different Fc domain or its variant or fragment, has the optional joint between the nucleic acid enzyme domain and the Fc domain, its variant or fragment.In some embodiments, the nucleic acid enzyme domain is identical, for example RNase and RNase, or DNase1 and DNase1.In other embodiments, the nucleic acid enzyme domain is different, for example DNase and RNase.

[0111] In some embodiments, two or more nuclease domains are operably coupled to each other in series (for example, through a polypeptide linker), and the tandem array of nuclease domains is operably coupled (for example, chemically conjugated or genetically fused (for example, directly or through a polypeptide linker)) to the C-terminal or N-terminal of the same or different Fc domains or variants or fragments thereof. In other embodiments, the tandem array of nuclease domains is operably coupled to both the N-terminal and C-terminal of the same Fc domains or variants or fragments thereof. In some embodiments, the nuclease domains are operably connected (for example, N-DNase-RNase-C or N-RNase-DNase-C) to the N- or C-terminal of the same or different Fc domains in series with or without a joint. In some embodiments, the tandem two nuclease fusion proteins form homodimers or heterodimers.

[0112] In other embodiments, one or more nuclease domains can be inserted between two Fc domains or variants or fragments thereof.For example, one or more nuclease domains can form all or part of the polypeptide linker of the optimized two nuclease fusion proteins of the present disclosure.

[0113] In some embodiments, the optimized di-nuclease fusion protein comprises at least two nuclease domains (eg, RNase and DNase), at least one linker domain, and at least one Fc domain, or variants or fragments thereof.

[0114] In some embodiments, as described above, the optimized di-nuclease fusion protein of the present disclosure comprises an Fc domain or a variant or fragment thereof, thereby increasing the serum half-life and bioavailability of the optimized di-nuclease fusion protein.

[0115] In some embodiments, the optimized di-nuclease fusion protein comprises one or more polypeptides, such as a polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-17.

[0116] Those skilled in the art will appreciate that other configurations of the nuclease domain and the Fc domain are possible, including optional joints between the nuclease domains and / or between the nuclease domain and the Fc domain. It will also be appreciated that the domain orientations may be altered, as long as the nuclease domain is active in the particular configuration tested.

[0117] In certain embodiments, the optimized dinuclease fusion protein of the present disclosure has at least one nuclease domain specific to the target molecule that mediates biological effect.In another embodiment, the combination of the optimized dinuclease fusion protein of the present invention and target molecule (for example, DNA or RNA) causes the target molecule to be reduced or eliminated from, for example, cell, tissue or from circulation.

[0118] In other embodiments, the optimized dinuclease fusion proteins of the present disclosure can be assembled together or assembled with other polypeptides to form a binding protein ("multimer") having two or more polypeptides, wherein at least one polypeptide of the multimer is an optimized dinuclease fusion protein of the present invention. Exemplary multimeric forms include altered binding proteins of dimers, trimers, tetramers and hexamers, etc. In one embodiment, the polypeptides of the multimers are identical (i.e., homopolymeric altered binding proteins, such as homodimers, homotetramers). In another embodiment, the polypeptides of the multimers are different (e.g., heteropolymeric).

[0119] In some embodiments, the serum half-life of the optimized dinuclease fusion protein is increased by at least about 1.5 times, such as 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 relative to the corresponding nuclease molecule not fused to the Fc domain or its variant or fragment. In other embodiments, the serum half-life of the optimized dinuclease fusion protein 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 lower relative to the corresponding nucleic acid enzyme molecule not fused with the Fc domain or its variant or fragment. Conventional art-recognized methods can be used for determining the serum half-life of the optimized dinuclease fusion protein of the present disclosure.

[0120] In some embodiments, the activity of RNase in the optimized dinuclease fusion protein is not less than about 10 times of the activity of the control RNase molecule, for example, 9 times lower, 8 times lower, 7 times lower, 6 times lower, 5 times lower, 4 times lower, 3 times lower or 2 times lower. In some embodiments, the activity of RNase in the optimized dinuclease fusion protein is approximately equal to the activity of the control RNase molecule.

[0121] In some embodiments, the activity of DNase in the optimized dinuclease fusion protein is not less than about 10 times of the activity of the control DNase molecule, for example low 9 times, low 8 times, low 7 times, low 6 times, low 5 times, low 4 times, low 3 times or low 2 times.In some embodiments, the activity of DNase in the optimized dinuclease fusion protein is approximately equal to the activity of the control DNase molecule.

[0122] In some embodiments, the optimized di-nuclease fusion protein can be active against extracellular immune complexes containing DNA and / or RNA (eg, in soluble form or deposited as insoluble complexes).

[0123] In some embodiments, the activity of the optimized dinuclease fusion protein can be detected in vitro and / or in vivo. In some embodiments, the optimized dinuclease fusion protein binds to cells, malignant cells or cancer cells and interferes with their biological activity.

[0124] In another aspect, a multifunctional RNase or DNase molecule is provided, which is linked to another enzyme or antibody with binding specificity (e.g., a scFv targeting RNA or DNA) or a second nuclease domain with the same or different specificity as the first domain.

[0125] In some embodiments, the joint domain includes (gly4ser) 3, 4 or 5 variants, which change the length of the joint according to 5 amino acid increments. In another embodiment, the joint domain length is about 18 amino acids and includes a glycosylation site that N-connects, which may be sensitive to in vivo protease cutting. In some embodiments, the glycosylation site that N-connects can protect the optimized dinuclease fusion protein from cutting in the joint domain. In some embodiments, the glycosylation site that N-connects can help separate the folding of the independent functional domains separated by the joint domain.

[0126] In some embodiments, the linker domain is an NLG linker (VDGASSPVNVSSPSVQDI) (SEQ ID NO: 41).

[0127] In some embodiments, the optimized dinuclease fusion protein includes substantially all or at least enzymatically active fragments of DNase. In some embodiments, DNase is a type I secreted DNase, preferably a human DNase, such as mature human pancreatic DNase 1 (UniProtKB accession number P24855, SEQ ID NO: 20). In some embodiments, naturally occurring variant allele A114F (SEQ ID NO: 21) (which shows reduced sensitivity to actin) is included in the dinuclease fusion protein optimized by DNase1 (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 high DNase activity relative to wild-type DNase1) is included in the DNase1 optimized dinuclease fusion protein (see Yasuda et al., Int J Biochem Cell Biol 2010; 42: 1216-25). In some embodiments, the mutation is introduced into the optimized dinuclease fusion protein to produce a more stable derivative of human DNase1. In some embodiments, the DNase is human wild-type DNase1 or human DNase1 A114F (i.e., human DNase1 N18S / N106S / A114F, SEQ ID NO: 24) that has been mutated to remove all potential N-linked glycosylation sites (i.e., the asparagine residues at positions 18 and 106 of the DNase1 domain shown in SEQ ID NO: 20, which correspond to the asparagine residues at positions 40 and 128 of the full-length pancreatic DNase1 (SEQ ID NO: 23) with a natural leader sequence).

[0128] In some embodiments, DNase is human DNase1, which comprises one or more alkaline (i.e. positively charged) amino acid replacements to increase DNase functionality and chromatin cutting. In some embodiments, basic amino acids are introduced into human DNase1 at the DNA binding interface to enhance the combination with the negatively charged phosphate on the DNA substrate (see US 7407785; US 6391607). This overactive DNase1 can be called "chromatin cutter".

[0129] In some embodiments, 1, 2, 3, 4, 5 or 6 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 aforementioned amino acids are replaced by basic amino acids such as arginine, lysine and / or histidine. For example, mutant human DNase may include one or more of the following substitutions: Q9R, E13R, T14K, H44K, N74K, N110R, T205K. In some embodiments, mutant human DNase1 also includes A114F substitution, which reduces sensitivity to actin (see US 6348343). In one embodiment, mutant human DNase1 includes the following substitutions: E13R, N74K, A114F and T205K.

[0130] In some embodiments, the mutant human DNase1 further comprises mutations to remove potential glycosylation sites, such as the asparagine residues at positions 18 and 106 of the DNase1 domain shown in SEQ ID NO: 20, which correspond to the asparagine residues at positions 40 and 128 of the full-length pancreatic DNase1 with a native leader sequence, respectively. In one embodiment, the mutant human DNase1 comprises the following substitutions: E13R / N74K / A114F / T205K / N18S / N106S.

[0131] In some embodiments, the DNase is a DNase 1-like (DNaseL) enzyme, 1-3 (UniProtKB Accession No. Q13609; SEQ ID NO: 46). In some embodiments, the DNase is 3-prime repair exonuclease 1 (TREX1; UniProtKB Accession No. Q9NSU2; SEQ ID NO: 47). In some embodiments, the DNase is DNase2. In some embodiments, DNase2 is DNAse2α (i.e., DNase2; UnitProtKB Accession No. O00115; SEQ ID NO: 48) or DNase2β (i.e., DNase2-like acidic DNase; UnitProtKB Accession No. 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 a 20 or 25aa linker domain is prepared.

[0132] In some embodiments, the optimized dinuclease fusion protein includes RNase1, preferably human pancreatic RNase1 of the RNase A family (UniProtKB accession number P07998; SEQ ID NO: 27). In some embodiments, human RNase1 is mutated to remove all potential N-linked glycosylation sites, i.e., asparagine residues at positions 34, 76, and 88 of the RNase1 domain shown in SEQ ID NO: 27 (human RNase1 N34S / N76S / N88S, SEQ ID NO: 28), which correspond to asparagine residues at positions 62, 104, and 116 of full-length pancreatic RNase1 with a natural leader sequence (SEQ ID NO: 29), respectively. In some embodiments, RNase1-linker-Fc containing a linker domain of 20 or 25 aa is prepared.

[0133] In some embodiments, the optimized dinuclease fusion protein includes DNase-linker-RNase-Fc, wherein the RNase1 domain is located on the COOH side of Fc. In other embodiments, the optimized dinuclease fusion protein includes DNase-linker-RNase-Fc, wherein the RNase1 domain is located on the NH2 side of Fc. In some embodiments, the optimized dinuclease fusion protein includes: DNase-Fc and RNase-Fc; DNase1-Fc-linker-RNase and Fc domain; DNase1-Fc and Fc-linker-RNase; Fc-linker-DNase1 and Fc-linker-RNase; RNase-Fc-linker-DNase and Fc domain; Fc-linker-DNase and RNase-Fc; and RNase-Fc-linker-DNase.

[0134] In some embodiments, the fusion junction between the enzymatic domain and other domains of the optimized di-nuclease fusion protein is optimized.

[0135] In some embodiments, the target of the RNase enzymatic activity of the optimized dinuclease fusion protein is mainly extracellular, composed of, for example, RNA contained in the immune complex with anti-RNP autoantibodies and RNA expressed on the cell surface experiencing apoptosis. In some embodiments, the optimized dinuclease fusion protein has activity in the acidic environment of endocytic vesicles. In some embodiments, the optimized dinuclease fusion protein comprising Fc domains or its variants or fragments is adapted to have activity in the extracellular and endocytic environment. In some respects, this allows the optimized dinuclease fusion protein including wild-type Fc domains or its variants or fragments to terminate the TLR7 signaling of the RNA activating TLR7 after the immune complex previously engulfed or by virus infection. In some embodiments, the wild-type RNase of the optimized dinuclease fusion protein is not resistant to the inhibition of RNase cytoplasm inhibitors. In some embodiments, the wild-type RNase of the optimized dinuclease fusion protein is not active in the cytoplasm of cells.

[0136] In some embodiments, the optimized dinuclease fusion protein comprises both DNase and RNase.In some embodiments, these optimized dinuclease fusion proteins improve the treatment of SLE because they digest or degrade the immune complex containing RNA, DNA or RNA and DNA combination, and are active outside the cell.

[0137] Fc domain

[0138] In some embodiments, the polypeptide comprising one or more nuclease domains is operably coupled to an Fc domain, which serves as a scaffold and a means of increasing the serum half-life of the polypeptide. In some embodiments, one or more nuclease domains and / or Fc domains are non-glycosylated, deglycosylated, or hypoglycosylated.

[0139] Suitable Fc domains are well known in the art, and include, but are not limited to, Fc and Fc variants, such as those disclosed in WO2011 / 053982, WO02 / 060955, WO02 / 096948, WO05 / 047327, WO05 / 018572, and US2007 / 0111281 (the contents of the foregoing documents are incorporated herein by reference). Those skilled in the art can use conventional methods (e.g., cloning, conjugation) to introduce Fc domains into optimized dinuclease fusion proteins disclosed herein (with or without altered glycosylation).

[0140] In some embodiments, the Fc domain is a wild-type human IgG1 Fc, such as that shown in SEQ ID NO:45.

[0141] In some embodiments, the Fc domain is changed or modified, for example, by mutations that cause 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., amino acid substitution) compared to the wild-type Fc from which the Fc domain is derived. For example, in the case where the Fc domain is derived from human IgG1 antibodies, the variant comprises at least one amino acid mutation (e.g., substitution) compared to the wild-type amino acids at the corresponding positions in the human IgG1 Fc region. The amino acid substitutions of Fc variants can be located within the Fc domain and are referred to as positions corresponding to the position number (according to the EU index number) given in the Fc region of the antibody for the residue.

[0142] In one embodiment, the Fc variant comprises one or more amino acid replacements at amino acid positions located in the hinge region or a portion thereof. In another embodiment, the Fc variant comprises one or more amino acid replacements at amino acid positions located in the CH2 domain or a portion thereof. In another embodiment, the Fc variant comprises one or more amino acid replacements at amino acid positions located in the CH3 domain or a portion thereof. In another embodiment, the Fc variant comprises one or more amino acid replacements at amino acid positions located in the CH4 domain or a portion thereof.

[0143] In some embodiments, the Fc region has a mutation at N83 (i.e., N297 according to Kabat numbering), thereby generating an aglycosylated Fc region (e.g., Fc N83S; SEQ ID NO: 50). In some embodiments, the Fc domain includes a mutation in one or more of the three hinge region cysteines (residues 220, 226, and 229, numbered according to the EU index). In some embodiments, one or more of the three hinge cysteines of the Fc domain can be mutated to SCC (SEQ ID NO: 51) or SSS (SEQ ID NO: 52), wherein "S" indicates that the cysteine ​​is replaced with an amino acid of serine. Thus, "SCC" indicates that only the first of the three hinge region cysteines (residues 220, 226, and 229, numbered according to the EU index) is replaced with serine, while "SSS" indicates that all three hinge region cysteines (residues 220, 226, and 229, numbered according to the EU index) are replaced with serine.

[0144] In some aspects, the Fc domain is a mutant human IgG1 Fc domain. In some aspects, the mutant Fc domain comprises one or more mutations in the hinge, CH2 and / or CH3 domains.

[0145] CH2 substitution

[0146] In some aspects, mutation Fc domain includes P238S mutation.In some aspects, mutation Fc domain includes P331S mutation.In some aspects, mutation Fc domain includes P238S mutation and P331S mutation.In some aspects, mutation Fc domain includes P238S and / or P331S, and can include one or more mutations of three hinge cysteines (residues 220,226 and 229), according to EU index numbering.In some aspects, mutation Fc domain includes P238S and / or P331S, and / or one or more mutations in three hinge cysteines (residues 220,226 and 229), according to EU index numbering.In some aspects, mutation Fc domain includes P238S and / or P331S, and / or mutations in hinge cysteines to SCC or in three hinge cysteines to SSS. In some aspects, the mutant Fc domain comprises P238S and P331S and a mutation in at least one of the three hinge cysteines. In some aspects, the mutant Fc domain comprises P238S and P331S and SCC. In some aspects, the mutant Fc domain comprises P238S and P331S and SSS. In some aspects, the mutant Fc domain comprises P238S and SCC or SSS. In some aspects, the mutant Fc domain comprises P331S and SCC or SSS. (All numberings are according to EU index).

[0147] In some aspects, the mutant Fc domain includes a mutation at an N-linked glycosylation site, such as N297, for example, asparagine replaces another amino acid such as serine, such as N297S. In some aspects, the mutant Fc domain includes a mutation at an N-linked glycosylation site (such as N297, for example, asparagine replaces another amino acid such as serine, such as N297S) and a mutation in one or more of the three hinge cysteines. In some aspects, the mutant Fc domain includes a mutation at an N-linked glycosylation site (such as N297, for example, asparagine replaces another amino acid such as serine, such as N297S) and one of the three hinge cysteines mutates to SCC or all three cysteines mutate to SSS. In some aspects, the mutant Fc domain includes a mutation at an N-linked glycosylation site (such as N297, for example, asparagine replaces another amino acid such as serine, such as N297) and one or more mutations in the CH2 domain (which reduce FcγR binding and / or complement activation), such as a mutation at P238 or P331 or both, such as P238S or P331S or both P238S and P331S. In some aspects, such mutant Fc domains may also include a mutation in the hinge region, such as SCC or SSS. (All numbering is according to the EU index.) In some aspects, the mutant Fc domain is as shown in the sequence listing or sequence table herein.

[0148] CH3 substitution

[0149] Heterodimers can be preferentially formed by mutations in the CH3 domain of the Fc domain on the heterodimer dinuclease fusion protein disclosed herein. The heavy chain is first engineered for heterodimerization using the "knobs-into-holes" strategy (Rigway B et al., Protein Eng., 9 (1996) pp.617-621). The term "knobs-into-holes" refers to a technique for guiding two polypeptides to pair together in vitro or in vivo by introducing a protrusion (knob) in one polypeptide and a cavity (hole) in another polypeptide at the interface where the two polypeptides interact. See, for example, WO 96 / 027011, WO 98 / 050431, US 5,731,168, US2007 / 0178552, WO2009089004, US20090182127. In particular, combinations of mutations in the CH3 domain can be used to preferentially form heterodimers, for example, S354C, T366W in the "knob" heavy chain and Y349C, T366S, L368A, Y407V in the "hole" heavy chain. In some embodiments, the heterodimeric dinuclease fusion proteins disclosed herein include a first CH3 domain with a knob mutation T366W and a second CH3 domain with a hole mutation T366S, L368A and Y407V. (Numbering according to the EU index.)

[0150] In some embodiments, the CH3 mutations are those described by Zymeworks (US2012 / 0149876A1, incorporated by reference; and Von Kreudenstein, TS et al. mABs, 5 (2013), pp. 646-654) and include the following mutations: T350V, L351Y, F405A, and Y407V (first CH3 domain); and T350V, T366L, K392L, T394W (second CH3 domain). In some embodiments, the heterodimeric dinuclease fusion protein disclosed herein includes a first CH3 domain having T350V, L351Y, F405A, and Y407V mutations and a second CH3 domain having T350V, T366L, K392L, T394W mutations. (Numbering according to the EU index.)

[0151] In some embodiments, the CH3 mutations are those described by Moore, GL et al. (mABs, 3 (2011), pp. 546-557) and include the following mutations: S364H and F405A (first CH3 domain); and Y349T and T394F (second CH3 domain). In some embodiments, the heterodimeric dinuclease fusion protein disclosed herein includes a first CH3 domain with S364H and F405A mutations and a second CH3 domain with Y349T and T394F mutations. (Numbering according to the EU index.)

[0152] In some embodiments, the CH3 mutations are those described by Gunasekaran, K. et al. (J. Biol. Chem., 285 (2010), pp. 19637-19646) and include the following mutations: K409D and K392D (first CH3 domain); and D399K and E365K (second CH3 domain). In some embodiments, the heterodimeric dinuclease fusion protein disclosed herein includes a first CH3 domain having K409D and K392D mutations and a second CH3 domain having D399K and E365K mutations. (Numbering according to the EU index.)

[0153] The optimized dinuclease fusion proteins disclosed herein can use art-recognized Fc variants, which are known to produce changes in effector function and / or FcR binding. For example, International PCT Publications WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO03 / 074569A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351 A2, WO04 / 074455A2, WO04 / 099249A2, WO05 / 040217A2, WO04 / 044859, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350A2 and WO06 / 085967A2; US Patent Publication Nos. US2007 / 0231329, US2007 / 0231329, US2007 / 0237765, US2007 / 0237766, US2007 / 0237767, US2007 / 024318 8, changes (e.g., substitutions) at one or more amino acid positions disclosed in US20070248603, US20070286859, US20080057056, or US Patent Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784 and 7,317,091, each of which is incorporated herein by reference. In one embodiment, a specific change can be made at one or more disclosed amino acid positions (e.g., a specific substitution of one or more amino acids disclosed in the art). In another embodiment, a different change can be made at one or more disclosed amino acid positions (e.g., a different substitution at one or more amino acid positions disclosed in the art).

[0154] Other amino acid mutations in the Fc domain are expected to reduce binding to Fcγ receptors and Fcγ receptor subtypes. The assignment of amino acid residue numbers to the Fc domain is in accordance with Kabat's definition. See, e.g., Sequences of Proteins of Immunological Interest (Table of Contents, Introduction and Constant Region Sequences sections), 5th edition, Bethesda, MD: NIH vol. 1: 647-723 (1991); Kabat et al., "Introduction" Sequences of Proteins of Immunological Interest, USDept of Health and Human Services, NIH, 5th edition, Bethesda, MD vol. 1: xiii-xcvi (1991); Chothia & Lesk, J. Mol. Biol. 196: 901-917 (1987); Chothia et al., Nature 342: 878-883 (1989), each of which is incorporated herein by reference for all purposes.

[0155] For example, 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 303, 307, 312, 315, 322, 324, 327, 329, 330 Mutations at positions 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 as described in U.S. Pat. No. 6,737,056, issued May 18, 2004, the entire contents of which are incorporated herein by reference. The patent reports that changing Pro331 in IgG3 to Ser resulted in a 6-fold lower affinity compared to unmutated IgG3, indicating that Pro331 is involved in FcγRI binding. In addition, amino acid modifications at positions 234, 235, 236 and 237, 297, 318, 320 and 322 are disclosed as potentially altering receptor binding affinity in US Pat. No. 5,624,821, issued Apr. 29, 1997, which is incorporated herein by reference in its entirety. (Numbering according to the EU index.)

[0156] Further mutations contemplated for use include, for example, those described in U.S. Patent Application Publication No. 2006 / 0235208, published on October 19, 2006, the entire contents of which are incorporated herein by reference. This disclosure describes Fc variants that exhibit reduced binding to Fcγ receptors, reduced antibody-dependent cell-mediated cytotoxicity, or reduced complement-dependent cytotoxicity, comprising at least one amino acid modification in the Fc region, including 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, 327 R, 328R, 329K, 330I, 330L, 330N, 330P, 330R and 331L (numbering according to the EU index), and the 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. Other mutations contemplated for use as described in the disclosure include 227G, 234D, 234E, 234G, 234I, 234Y, 235D, 235I, 235S, 236S, 239D, 246H, 255Y, 258H, 260H, 264I, 267D, 267E, 268D, 268E, 272H, 272I, 272R, 281D, 282G, 283H, 284E, 293R, 295E, 304T, 324G, 324I, 327D, 327A, 328A, 328D, 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, insertion of G between positions 297 and 298, insertion of A between positions 297 and 298, insertion of S between positions 297 and 298, insertion of D between positions 297 and 298, insertion of G between positions 326 and 327, insertion of A between positions 326 and 327, insertion of T between positions 326 and 327, insertion of D between positions 326 and 327 and insertion of E between positions 326 and 327 (numbering is according to the EU index). In addition, the mutations described in U.S. Patent Application Publication No. 2006 / 0235208 include 227G / 332E, 234D / 332E, 234E / 332E, 234Y / 332E, 234I / 332E, 234G / 332E, 235I / 332E, 235S / 332E, 235D / 332E, 235E / 332E, 236S / 332E, 236A / 332E, 236S / 332D, 236A / 332D, 239D / 268E, 246H / 332E, 255Y / 332E, and 256Y / 332E. 32E, 258H / 332E, 260H / 332E, 264I / 332E, 267E / 332E, 267D / 332E, 268D / 332D, 268E / 332D, 268E / 332E, 268D / 332E, 268E / 3 30Y, 268D / 330Y, 272R / 332E, 272H / 332E, 283H / 332E, 284E / 332E, 293R / 332E, 295E / 332E, 304T / 332E, 324I / 332E, 324G / 33 2E, 324I / 332D, 324G / 332D, 327D / 332E, 328A / 332E, 328T / 332E, 328V / 332E, 328I / 332E, 328F / 332E, 328Y / 332E, 328M / 33 2E, 328D / 332E, 328E / 332E, 328N / 332E, 328Q / 332E, 328A / 332D, 328T / 332D, 328V / 332D, 328I / 332D, 328F / 332D, 328Y / 332 D. 328M / 332D, 328D / 332D, 328E / 332D, 328N / 332D, 328Q / 332D, 330L / 332E, 330Y / 332E, 330I / 332E, 332D / 330Y, 335D / 332E , 239D / 332E, 239D / 332E / 330Y, 239D / 332E / 330L, 239D / 332E / 330I, 239D / 332E / 268E, 239D / 332E / 268D, 239D / 332E / 327D,239D / 332E / 284E, 239D / 268E / 330Y, 239D / 332E / 268E / 330Y, 239D / 332E / 327A, 239D / 332 E / 268E / 327A, 239D / 332E / 330Y / 327A, 332E / 330Y / 268E / 327A, 239D / 332E / 268E / 330Y / 32 7A, insert G>297-298 / 332E, insert A>297-298 / 332E, insert S>297-298 / 332E, insert D>297-298 / 332E, insert G>326-327 / 332E, insert A>326-327 / 332E, insert T>326-327 / 332E, insert D>326-327 / 332E, insert E>326-3 27 / 332E, insert G>235-236 / 332E, insert A>235-236 / 332E, insert S>235-236 / 332E, insert T>235-236 / 332E, insert N>235-236 / 332E, insert D>235-236 / 332E, insert V>235-236 / 332E, insert L>235-236 / 332E, insert G> 235-236 / 332D, insertion A>235-236 / 332D, insertion S>235-236 / 332D, insertion T>235-236 / 332D, insertion N>235-236 / 332D, insertion D>235-236 / 332D, insertion V>235-236 / 332D, insertion L>235-236 / 332D (numbered according to the EU index), which is intended for use. Mutant L234A / L235A is described, for example, in U.S. Patent Application Publication No. 2003 / 0108548, published on June 12, 2003, the entire contents of which are incorporated herein by reference. In an embodiment, the modifications described include, alone or in combination. (numbered according to the EU index.),

[0157] Linker domain

[0158] In some embodiments, the optimized dinuclease fusion protein includes a joint domain. In some embodiments, the optimized dinuclease fusion protein includes a plurality of joint domains. In some embodiments, the joint domain is a polypeptide linker. In some aspects, it is desirable to use a polypeptide linker to fuse Fc or its variant or fragment with one or more nuclease domains to form the optimized dinuclease fusion protein.

[0159] In one embodiment, the polypeptide linker is synthetic. As used herein, the term "synthetic" with respect to a polypeptide linker is included in a linear sequence of amino acids that contains a peptide (or polypeptide) that is connected to a sequence (which may or may not be naturally occurring) that is not naturally connected thereto (e.g., an Fc sequence). For example, a polypeptide linker may include a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., including mutations such as additions, substitutions, or deletions) or that includes a first amino acid sequence (which may or may not be naturally occurring). For example, the polypeptide linker of the present invention can be used to ensure that Fc or its variants or fragments are juxtaposed to ensure the formation of correctly folded and functional Fc or its variants or fragments. Preferably, a polypeptide linker compatible with the present invention is relatively non-immunogenic and does not inhibit any non-covalent binding between the monomer subunits of the binding protein.

[0160] In certain embodiments, the optimized di-nuclease fusion protein uses an NLG linker as shown in SEQ ID NO:41.

[0161] In certain embodiments, the optimized dinuclease fusion protein of the present disclosure uses a polypeptide linker to connect any two or more domains in the same frame in a single polypeptide chain. In one embodiment, two or more domains can be independently selected from any Fc domain or variant or fragment thereof discussed herein, or a nuclease domain. For example, in certain embodiments, a polypeptide linker can be used to fuse the same Fc fragment, thereby forming a homodimer Fc region. In other embodiments, a polypeptide linker can be used to fuse different Fc fragments, thereby forming a heterodimer Fc region. In other embodiments, a polypeptide linker of the present invention can be used to genetically fuse the C-terminal of the first Fc fragment to the N-terminal of the second Fc fragment to form a complete Fc domain.

[0162] In one embodiment, the polypeptide linker comprises a portion of an Fc domain or a variant or fragment thereof. For example, in one embodiment, the polypeptide linker may comprise an Fc fragment (e.g., a C or N domain), or different portions of an Fc domain or a variant thereof.

[0163] In another embodiment, the polypeptide linker comprises a gly-ser linker or is composed of a gly-ser linker. As used herein, the term "gly-ser linker" refers to a peptide consisting of glycine and serine residues. An exemplary gly / ser linker comprises an amino acid sequence of 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 can be inserted between two other sequences of polypeptide linkers (e.g., any polypeptide linker sequence described herein). In other embodiments, a gly-ser linker is connected to one or both ends of a polypeptide linker (e.g., any polypeptide linker sequence described herein) of another sequence. In still other embodiments, two or more gly-ser linkers are incorporated in tandem in a polypeptide linker.

[0164] In other embodiments, the polypeptide linker of the present invention comprises a biologically relevant peptide sequence or a sequence portion thereof. For example, a biologically relevant peptide sequence may include, but is not limited to, a sequence derived from an anti-rejection or anti-inflammatory peptide. The anti-rejection or anti-inflammatory peptide may be selected from a cytokine inhibitory peptide, a cell adhesion inhibitory peptide, a thrombin inhibitory peptide, and a platelet inhibitory peptide. In a preferred embodiment, the polypeptide linker comprises a peptide sequence selected from the group consisting of an IL-1 inhibitory or antagonist peptide sequence, an erythropoietin (EPO)-mimicking peptide sequence, a thrombopoietin (TPO)-mimicking peptide sequence, a G-CSF mimicking peptide sequence, a TNF-antagonist peptide sequence, an integrin binding peptide sequence, a selectin antagonist peptide sequence, an anti-pathogenic peptide sequence, a vasoactive intestinal peptide (VIP) mimicking peptide sequence, a calmodulin antagonist peptide sequence, a mast cell antagonist, an SH3 antagonist peptide sequence, a urokinase receptor (UKR) antagonist peptide sequence, a somatostatin or cortistatin mimicking peptide sequence, and a macrophage and / or T cell inhibitory peptide sequence. Exemplary peptide sequences, any of which may be used as polypeptide linkers, are disclosed in U.S. Pat. No. 6,660,843, which is incorporated herein by reference.

[0165] Other linkers suitable for use in optimized dinuclease fusion proteins are known in the art, for example, the serine-rich linkers disclosed in US 5,525,491, the helix-forming peptide linkers 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 linkers disclosed in Chen et al., Mol Pharm 2011; 8: 457-65, i.e., the dipeptide linker LE, the thrombin-sensitive disulfide cyclic peptide linker and the α-helix-forming linker LEA(EAAAK)4ALEA(EAAAK)4ALE (SEQ ID NO: 53).

[0166] Other exemplary linkers include GS linkers (i.e., (GS)n) (SEQ ID NO: 66), GGSG (SEQ ID NO: 70) linkers (i.e., (GGSG)n) (SEQ ID NO: 67), GSAT linkers (SEQ ID NO: 44), SEG linkers, and GGS linkers (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 optimized dinuclease 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 domain-interconnected linkers in multifunctional enzymes used as potential linkers in new fusion proteins (see, e.g., George et al., Protein Engineering 2002; 15: 871-9).

[0167] It should be understood that variant forms of these exemplary polypeptide linkers can be produced by introducing one or more nucleotide substitutions, additions or deletions in the nucleotide sequence encoding the polypeptide linker, so that one or more amino acid replacements, additions or deletions are introduced into the polypeptide linker. Sudden changes can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0168] The polypeptide linker length of the present disclosure is at least one amino acid, and can have different lengths. In one embodiment, the polypeptide linker length of the present disclosure is about 1 to about 50 amino acids. As used in this case, the term "about" indicates + / - two amino acid residues. Since the linker length must be a positive integer, the length of about 1 to about 50 amino acids refers to a length of 1 to 48-52 amino acids. In another embodiment, the polypeptide linker length of the present disclosure is about 10-20 amino acids. In another embodiment, the polypeptide linker length of the present disclosure is about 15 to about 50 amino acids.

[0169] In another embodiment, the polypeptide linker of the present disclosure is about 20 to about 45 amino acids in length. In another embodiment, the polypeptide linker of the present disclosure is about 15 to about 25 amino acids in length. In another embodiment, the polypeptide linker of the present 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 acids in length.

[0170] Polypeptide linkers can be introduced into the polypeptide sequence using techniques known in the art. Modifications can be confirmed by DNA sequence analysis. Plasmid DNA can be used to transform host cells to stably produce the produced polypeptide.

[0171] Exemplary Optimized Dinuclease Fusion Proteins

[0172] The optimized dinuclease fusion protein of the present invention is modular and can be configured to merge various single domains. For example, in one embodiment, the optimized dinuclease fusion protein may include the mutant human DNase1 A114F domain shown in (SEQ ID NO:21). In another embodiment, the optimized dinuclease fusion protein may include the mutant human DNase1 N18S / N106S / A114F domain shown in SEQ ID NO:24. In another embodiment, the optimized dinuclease fusion protein may include the human wild-type RNase1 domain shown in SEQ ID NO:27. In another embodiment, the optimized dinuclease fusion protein may include the human mutant RNase1 N34S / N76S / N88S domain shown in SEQ ID NO:28. In another embodiment, the optimized dinuclease fusion protein may include the (Gly4Ser) 3 joint domain shown in SEQ ID NO:30. In another embodiment, the optimized dinuclease fusion protein may include the NLG joint shown in SEQ ID NO:41. In another embodiment, the optimized dinuclease fusion protein may include a VK3LP leader sequence (SEQ ID NO:54). Those skilled in the art will appreciate that these individual domains can be operably coupled to each other in any order to form an optimized dinuclease fusion protein with enzymatic activity. For example, as described in detail in the specific embodiments below, RNase1 can be operably coupled to the Fc domain. In another example, RNase1 can be operably coupled to the Fc domain via a (Gly4Ser)3 (SEQ ID NO:30) linker domain. In yet another example, DNase1 A114F can be operably coupled to the Fc domain. In yet another example, DNase1 A114F can be operably coupled to the Fc domain via a (Gly4Ser)3 (SEQ ID NO:30) linker domain. Figure 1 The SEQ ID NOs: 1 and 2 have non-limiting exemplary configurations disclosed herein, and various other configurations are possible.

[0173] In some embodiments, the optimized dinuclease fusion protein comprises a wild-type human RNase1 domain operably coupled to a mutant Fc domain or a fragment thereof comprising an SCC hinge and CH2 mutations P238S and P331S, and a mutant human DNase1 domain operably coupled to human RNase1, thereby forming a tandem homodimer. In some embodiments, DNase1 is connected to RNase1 via a peptide linker, such as an NLG linker disclosed herein. In some embodiments, RNase1 is operably connected to the N-terminus of the Fc domain with or without a linker. In some embodiments, the optimized dinuclease fusion protein comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1. In some embodiments, RNase1 is operably connected to the C-terminus of the Fc domain with or without a linker. In some embodiments, the optimized dinuclease fusion protein comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 2. In some embodiments, the optimized dinuclease fusion protein is homodimeric or heterodimeric.

[0174] In some embodiments, the optimized dinuclease fusion protein is a heterodimer comprising a mutant human DNase 1 domain operably coupled to a first mutant Fc domain (having an SCC hinge, CH2 mutations P238S, P331S and CH3 mutations T350V, L351Y, F405A and Y407V) or a variant or fragment thereof, with or without a linker, and a wild-type human RNase 1 domain operably coupled to a second mutant Fc domain (comprising an SCC hinge, CH2 mutations P238S and P331S and CH3 mutations T350V, T366L, K392L and T394W) or a fragment thereof, with or without a linker. In some embodiments, both DNase 1 and RNase 1 are connected to the N-terminus of their respective Fc domains. In some embodiments, the optimized di-nuclease fusion protein is a heterodimer comprising a polypeptide comprising the amino acid sequence shown in SEQ ID NO:3 and a polypeptide comprising the amino acid sequence shown in SEQ ID NO:4.

[0175] In some embodiments, the optimized dinuclease fusion protein is a heterodimer comprising a mutant human DNase 1 domain and a wild-type human RNase 1 domain, both of which are operably coupled with a first mutant Fc domain (comprising an SCC hinge, CH2 mutations P238S and P331S and CH3 mutations T350V, T366L, K392L and T394W) or a fragment thereof and a second mutant Fc domain (having mutations T350V, T366L, K392L and T394W) or a fragment thereof, with or without a linker. In some embodiments, DNase1 and RNase1 are connected to the N-terminus and C-terminus of the first and second Fc domains, respectively. In some embodiments, the optimized dinuclease fusion protein is a heterodimer comprising a polypeptide comprising the sequence shown in SEQ ID NO: 5 and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6.

[0176] In some embodiments, the optimized dinuclease fusion protein is a heterodimer comprising a mutant human DNase 1 domain operably coupled to a mutant Fc domain (comprising SCC hinge, CH2 mutations P238S, P331S and CH3 mutations T350V, L351Y, F405A and Y407V) or a fragment thereof with or without a linker, and a wild-type human RNase 1 domain operably coupled to a mutant Fc domain (comprising SCC hinge, CH2 mutations P238S, P331S and CH3 mutations T350V, T366L, K392L and T394W) or a fragment thereof with or without a linker. In some embodiments, DNase 1 is connected to the N-terminus of the Fc domain and RNase 1 is connected to the C-terminus of the Fc domain. In some embodiments, the optimized di-nuclease fusion protein is a heterodimer comprising a polypeptide comprising the sequence shown in SEQ ID NO:7 and a polypeptide comprising the amino acid sequence shown in SEQ ID NO:8.

[0177] In some embodiments, the optimized dinuclease fusion protein is a heterodimer comprising a mutant human DNase 1 domain operably coupled to a mutant Fc domain (comprising SCC hinge, CH2 mutation P238S, P331S and CH3 mutation T350V, L351Y, F405A and Y407V) or a fragment thereof with or without a linker, and a wild-type human RNase 1 domain operably coupled to a mutant Fc domain (comprising SCC hinge, CH2 mutation P238S, P331S and CH3 mutation T350V, T366L, K392L and T394W) or a fragment thereof with or without a linker. In some embodiments, both DNase 1 and RNase 1 are connected to the C-terminus of their respective Fc domains. In some embodiments, the optimized dinuclease fusion protein is a heterodimer comprising a polypeptide comprising the sequence shown in SEQ ID NO: 9 and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 10. In some embodiments, the optimized di-nuclease fusion protein is a heterodimer comprising a polypeptide comprising the sequence shown in SEQ ID NO:11 and a polypeptide comprising the amino acid sequence shown in SEQ ID NO:12.

[0178] In some embodiments, the optimized dinuclease fusion protein is a heterodimer comprising a mutant human DNase1 domain and a wild-type human RNase1 domain, both of which are operably coupled with a mutant Fc domain or fragment thereof comprising an SCC hinge, CH2 mutations P238S, P331S and CH3 mutations T350V, L351Y, F405A and Y407V, and a mutant Fc domain or fragment thereof comprising an SCC hinge, CH2 mutations P238S, P331S and CH3 mutations T350V, T366L, K392L and T394W. In some embodiments, DNase1 and RNase1 are connected to the C-terminus and N-terminus of the Fc domain, respectively. In some embodiments, the optimized dinuclease fusion protein is a heterodimer comprising a polypeptide comprising the sequence shown in SEQ ID NO: 13 and a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 14.

[0179] In some embodiments, the optimized dinuclease fusion protein is a heterodimer comprising a mutant human DNase 1 domain operably coupled to a mutant Fc domain (comprising SCC hinge, CH2 mutations P238S, P331S and CH3 mutations T350V, L351Y, F405A and Y407V) or a fragment thereof with or without a linker and a wild-type human RNase 1 domain operably coupled to a mutant Fc domain (comprising SCC hinge, CH2 mutations P238S, P331S and CH3 mutations T350V, T366L, K392L and T394W) or a fragment thereof with or without a linker. In some embodiments, DNase 1 is linked to the C-terminus of the Fc domain, and RNase 1 is linked to the N-terminus of the Fc domain. In some embodiments, the optimized di-nuclease fusion protein is a heterodimer comprising a polypeptide comprising the sequence shown in SEQ ID NO:15 and a polypeptide comprising the amino acid sequence shown in SEQ ID NO:16.

[0180] In some embodiments, the optimized dinuclease fusion protein comprises a polypeptide having an amino acid sequence at least 80% identical to any one of SEQ ID NOs: 1-17, such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97, 98%, 99% or at least 99.5% identical to any one of SEQ ID NOs: 1-17. In some embodiments, the polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-17.

[0181] In some embodiments, the aforementioned optimized di-nuclease fusion protein has a leader sequence.

[0182] Those of ordinary skill in the art will appreciate that the leader sequence and linker sequence are optional and are not limited to those described in the above embodiments. For example, the RNase and / or DNase domains can be directly fused to the N- and / or C-terminal of Fc or its variants or fragments; the leader domain can be any of those domains known in the art that can be used for its intended purpose, for example, for increasing protein expression and / or secretion (e.g., Gaussia luciferase signal peptide (MGVKVLFALICIAVAEA; SEQ ID NO: 31)); the linker can be any linker known in the art, such as (Gly4Ser) n (SEQ ID NO: 58), NLG (VDGASSPVNVSSPSVQDI; SEQ ID NO: 41), LE, thrombin-sensitive disulfide cyclic peptide linker, LEA (EAAAK) 4ALEA (EAAAK) 4 (SEQ ID NO: 32) or a disulfide linker that can be cleaved in vivo, as described herein. It should also be understood that it is within the capabilities of those skilled in the art that the amino acid sequence of the optimized dinuclease fusion protein is changed accordingly using conventional cloning and recombination methods. It should also be understood that the asparagine residues in the nuclease domain (i.e. N34, N76 and N88 in RNase1, and N18 and N106 in DNase1) can be replaced by amino acids other than serine (e.g. glutamine), as long as the amino acid is not used as an N-linked glycosylated receptor.

[0183] Method for preparing optimized dinuclease fusion protein

[0184] The optimized dinuclease fusion protein disclosed herein can be mainly prepared in transformed or transfected host cells using recombinant DNA technology. For this reason, recombinant DNA molecules encoding peptides are prepared. The method for preparing such DNA molecules is well known in the art. For example, suitable restriction enzymes can be used to excise the sequence of the encoding peptide from DNA. Alternatively, chemical synthesis techniques can be used to synthesize DNA molecules, such as the phosphoramidate method. Moreover, a combination of these technologies can be used.

[0185] The present invention also includes vectors capable of expressing peptides in suitable hosts. The vector comprises a DNA molecule encoding the peptide operably coupled to a suitable expression control sequence. Methods for affecting such operative connection are well known before or after the DNA molecule is inserted into the vector. Expression control sequences include promoters, activators, enhancers, operators, ribosomal nuclease domains, start signals, stop signals, cap signals, polyadenylation signals, and other signals related to transcription or translation control.

[0186] The resulting vector with the DNA molecule thereon is used to transform or transfect a suitable host.Such transformation or transfection can be performed using methods well known in the art.

[0187] Any of a large number of available and well-known host cells can be used in the implementation of the present invention. The selection of a specific host depends on many factors recognized in the art. These include, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, the rate of conversion or transfection, the recovery convenience of the peptide, expression characteristics, biosafety and cost. The balance of these factors must be reached under the understanding that not all hosts may be equally effective for the expression of a specific DNA sequence. In these general guidelines, useful microbial hosts include bacteria (e.g., Escherichia coli), yeast (e.g., yeast) and other fungi, insects, plants, mammalian (including human) cells or other hosts known in the art in culture. In a preferred embodiment, the optimized dinuclease fusion protein is produced in CHO cells.

[0188] Next, the transformed or transfected host is cultured and purified. The host cell can be cultured under conventional fermentation or culture conditions to express the desired compound. These 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.

[0189] 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 and include 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 Phase Peptide Synthesis; US 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: those described in 257-527. Solid phase synthesis is the preferred technique for preparing a single peptide because it is the most cost-effective method for preparing small peptides. Compounds containing derivatized peptides or containing non-peptide groups can be synthesized by well-known organic chemistry techniques.

[0190] Other molecular expression / synthesis methods are generally known to those of ordinary skill in the art.

[0191] Optimized dinuclease fusion proteins with altered glycosylation

[0192] Glycosylation (e.g., O-linked or N-linked glycosylation) can, for example, affect the serum half-life of the optimized dinuclease fusion protein of the present disclosure by minimizing their removal from the circulation through mannose and asialoglycoprotein receptors and other lectin-like receptors. Therefore, in some embodiments, the optimized dinuclease fusion protein of the present invention is prepared in a non-glycosylated, deglycosylated or hypoglycosylated form. Preferably, the N-linked glycosylation is changed and the optimized dinuclease fusion protein is non-glycosylated.

[0193] In some embodiments, all asparagine residues in the optimized dinuclease fusion protein that conform to the consensus sequence Asn-X-Ser / Thr (X can be any other naturally occurring amino acid except Pro) are mutated to residues that do not serve as acceptors for N-linked glycosylation (e.g., serine, glutamine), thereby eliminating glycosylation of the optimized dinuclease fusion protein when synthesized in a cell that glycosylates proteins.

[0194] In some embodiments, an optimized dinuclease fusion protein lacking an N-linked glycosylation site is produced in a mammalian cell. In one embodiment, the mammalian cell is a CHO cell. Therefore, in a specific embodiment, a non-glycosylated optimized dinuclease fusion protein is produced in a CHO cell.

[0195] In other embodiments, reduction or absence of N-glycosylation is achieved by producing the optimized dinuclease fusion protein, for example, in a host (e.g., bacteria such as E. coli), a mammalian cell engineered to lack one or more enzymes important for glycosylation, or a mammalian cell treated with an agent that prevents glycosylation, such as tunicamycin (an inhibitor of Dol-PP-GlcNAc formation).

[0196] In some embodiments, optimized di-nuclease fusion proteins are produced in lower eukaryotic organisms engineered to produce glycoproteins with complex N-glycans rather than high mannose-type sugars (see, eg, US 2007 / 0105127).

[0197] In some embodiments, the glycosylated optimized dinuclease fusion protein (e.g., those produced in mammalian cells such as CHO cells) is 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 mask one or more carbohydrate residues. Such modification or masking can reduce the binding of the optimized dinuclease fusion protein to the mannose receptor and / or the asialoglycoprotein receptor and / or other lectin-like receptors. Chemical deglycosylation can be achieved by treating the optimized dinuclease fusion protein with trifluoromethanesulfonic acid (TFMS), as disclosed in, for example, Sojar et al., JBC 1989; 264: 2552-9 and Sojar et al., Methods Enzymol 1987; 138: 341-50, or by treating the optimized dinuclease fusion protein with hydrogen fluoride, as disclosed in Sojar et al. (1987, supra). The enzymatic removal of N-linked carbohydrates from the optimized dinuclease fusion protein can be achieved by treating the optimized dinuclease fusion protein with protein N-glycosidase (PNGase) A or F, as disclosed in Thotakura et al. (Methods Enzymol 1987; 138: 350-9). Other suitable commercially available deglycosylation enzymes recognized in the art include endo-α-N-acetyl-galactosidase, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3, and endoglycosidase H. In some embodiments, one or more of these enzymes can be used to deglycosylate the optimized dinuclease fusion protein of the present invention. Alternative methods for deglycosylation are disclosed, for example, in US8,198,063.

[0198] In some embodiments, the dinuclease fusion protein of optimization is partially deglycosylated.Partial deglycosylation can be achieved by treating the dinuclease fusion protein of optimization with endoglycosidase (e.g., endoglycosidase H), which cuts the high mannose carbohydrates connected by N- instead of complex carbohydrates, thereby leaving a single GlcNAc residue connected to asparagine. The dinuclease fusion protein of optimization treated with endoglycosidase H lacks high mannose carbohydrates, resulting in a reduction in interaction with the liver mannose receptor. Although the receptor recognizes terminal GlcNAc, the possibility of productive interaction with the single GlcNAc on the protein surface is not as large as a complete high mannose structure.

[0199] In other embodiments, the glycosylation of the optimized dinuclease fusion protein is modified, for example, by oxidation, reduction, dehydration, substitution, esterification, alkylation, sialylation, carbon-carbon bond cleavage, etc., to reduce the clearance of the optimized dinuclease fusion protein from the blood. In some embodiments, the optimized dinuclease fusion protein is treated with periodate and sodium borohydride to modify the carbohydrate structure. Periodate treatment oxidizes vicinal diols, thereby cleaving carbon-carbon bonds and replacing hydroxyls with aldehyde groups; borohydrides reduce aldehydes to hydroxyls. Many sugar residues include vicinal diols and are therefore cleaved by this treatment. An example of extending serum half-life with periodate 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). Hickman et al., BBRC 1974; 57: 55-61 discloses a method of treatment with periodate and sodium borohydride. Thorpe et al. Eur J Biochem 1985; 147: 197-206 discloses a method of treatment with periodate and cyanoborohydride, which increases the serum half-life and tissue distribution of ricin toxin.

[0200] In one embodiment, the carbohydrate structure of the optimized di-nuclease fusion protein can be masked by adding one or more additional moieties (e.g., carbohydrate groups, phosphate groups, alkyl groups, etc.) that interfere with recognition of the structure by mannose or asialoglycoprotein receptors or other lectin-like receptors.

[0201] In some embodiments, one or more potential glycosylation sites are removed by mutation of the nucleic acid encoding the optimized dinuclease fusion protein, so that when synthesized in a cell (e.g., a mammalian cell such as a CHO cell) that makes protein glycosylation, the glycosylation (hypoglycosylation) of the optimized dinuclease fusion protein is reduced. In some embodiments, if, for example, the optimized dinuclease fusion protein of hypoglycosylation shows increased activity or contributes to increased serum half-life, it may be necessary to selectively make the nuclease domain of the optimized dinuclease fusion protein hypoglycosylated by mutating the potential N-linked glycosylation site in the nuclease domain of the dinuclease fusion protein. In other embodiments, if, for example, this modification improves the serum half-life of the optimized dinuclease fusion protein, it may be necessary to make the part of the optimized dinuclease fusion protein hypoglycosylated so that the region outside the nuclease domain lacks N-glycosylation. Alternatively, other amino acids near the glycosylation receptor can be modified, thereby destroying the recognition motif of the glycosylation enzyme without having to change the amino acids that are usually glycosylated.

[0202] In some embodiments, the glycosylation of the optimized dinuclease fusion protein can be changed by introducing a glycosylation site. For example, the amino acid sequence of the optimized dinuclease fusion protein can be modified to introduce a consensus sequence of N-linked glycosylation of Asp-X-Ser / Thr (X is any amino acid except proline). Additional N-linked glycosylation sites can be added anywhere in the entire amino acid sequence of the optimized dinuclease fusion protein. Preferably, the glycosylation site is introduced into the amino acid sequence at a position that does not substantially reduce the nuclease (e.g., RNase and / or DNase) activity of the optimized dinuclease fusion protein.

[0203] It has been reported that the addition of O-linked glycosylation sites changes the serum half-life of proteins such as growth hormone, follicle stimulating hormone, IGFBP-6, factor IX and many other proteins (e.g., as disclosed in 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; US 2011 / 0154516). Therefore, in some embodiments, the O-linked glycosylation (on serine / threonine residues) of the optimized dinuclease fusion protein is altered. Methods for altering O-linked glycosylation are routine in the art and can be, for example, by β-elimination (see, e.g., Huang et al., Rapid Communications in Mass Spectrometry 2002; 16: 1199-204; Conrad, Curr Protoc Mol Biol 2001; Chapter 17: Unit 17.15A; Fukuda, Curr Protoc Mol Biol 2001; Chapter 17; Unit 17.15B; Zachara et al., Curr Protoc Mol Biol 2011; Unit 17.6); by using commercially available kits (e.g., GlycoProfile TMBeta-Elimination Kit, Sigma); or by treating the optimized dinuclease fusion protein with a series of exoglycosidases (such as, but not limited to, β1-4 galactosidase and β-N-acetylglucosaminidase) until only Galβ1-3GalNAc and / or GlcNAcβ1-3GalNAc are retained, and then treated with, for example, endo-α-N-acetylgalactosaminidase (i.e., O-glycosidase). These enzymes are commercially available from, for example, New England Biolabs. In yet other embodiments, the optimized dinuclease fusion protein is changed to introduce O-linked glycosylation in the optimized dinuclease fusion protein, as described in, for example, Okada et al. (supra), Weenen et al. (supra), US2008 / 0274958, and US2011 / 0171218. In some embodiments, one or more O-linked glycosylation consensus sites are introduced into the optimized dinuclease fusion protein, such as CXXGGT / SC (SEQ ID NO: 33) (van den Steen et al., 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). Additional O-linked glycosylation sites can be added anywhere throughout the amino acid sequence of the optimized dinuclease fusion protein. Preferably, the glycosylation site is introduced into the amino acid sequence at a position that does not substantially reduce the nuclease (e.g., RNase and / or DNase) activity of the optimized dinuclease fusion protein. Alternatively, O-linked sugar moieties are introduced by chemically modifying the amino acids in the optimized di-nuclease fusion protein as described in, for example, WO 87 / 05330 and Aplin et al., CRC Crit Rev Biochem 1981; 259-306.

[0204] In some embodiments, both N-linked and O-linked glycosylation sites are introduced into the optimized di-nuclease fusion protein, preferably at positions in the amino acid sequence that do not significantly reduce the nuclease (e.g., RNase and / or DNase) activity of the optimized di-nuclease fusion protein.

[0205] 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 di-nuclease fusion protein and determine whether such a change in glycosylation state increases or decreases the nuclease activity or serum half-life of the optimized di-nuclease fusion protein using routine methods in the art.

[0206] In some embodiments, the optimized di-nuclease fusion protein can comprise altered glycoforms (eg, hypofucosylated or fucose-free glycans).

[0207] In some embodiments, the optimized dinuclease fusion protein with altered glycosylation has an increase of at least about 1.5 times, such as at least 3 times, at least 5 times, at least 10 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 of the serum half-life relative to the optimized dinuclease fusion protein of the corresponding glycosylation (e.g., wherein the potential N-linked glycosylation site is not mutated). Conventional methods recognized in the art can be used to determine the serum half-life of the optimized dinuclease fusion protein with an altered glycosylation state.

[0208] In some embodiments, an optimized dinuclease fusion protein with altered glycosylation (e.g., a non-glycosylated, deglycosylated, or hypoglycosylated optimized dinuclease fusion protein) retains at least 50%, e.g., 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 a corresponding glycosylation-optimized dinuclease fusion protein (e.g., an optimized dinuclease fusion protein in which the potential N-linked glycosylation site is not mutated).

[0209] In some embodiments, changing the glycosylation state of the optimized dinuclease fusion protein can increase the nuclease activity by directly increasing the enzyme activity or by improving the bioavailability (e.g., serum half-life). Therefore, in some embodiments, the nuclease activity of the optimized dinuclease fusion protein with changed glycosylation is increased by at least 1.3 times relative to the corresponding glycosylated optimized dinuclease fusion protein (e.g., wherein the optimized dinuclease fusion protein in which the potential N-linked glycosylation site is not mutated), 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.

[0210] Those skilled in the art can easily determine the glycosylation state of the optimized dinuclease fusion protein using methods recognized in the art. In a preferred embodiment, the glycosylation state is determined using mass spectrometry. In other embodiments, the interaction with concanavalin A (Con A) can be evaluated to determine whether the optimized dinuclease fusion protein is low glycosylated. When compared with the corresponding glycosylated optimized dinuclease fusion protein, the low glycosylated optimized dinuclease fusion protein is expected to show reduced binding to Con A-agarose. SDS-PAGE analysis can also be used to compare the mobility of low glycosylated proteins and corresponding glycosylated proteins. Compared with glycosylated proteins, low glycosylated proteins are expected to have greater mobility in SDS-PAGE. Other suitable methods recognized in the art for analyzing the glycosylation state of proteins are disclosed in, for example, Roth et al., International Journal of Carbohydrate Chemistry 2012; 1-10.

[0211] The pharmacokinetics, such as serum half-life, of optimized dinuclease fusion proteins with different glycosylation states can be determined using conventional methods, for example, by introducing the optimized dinuclease fusion protein into mice (e.g., intravenously), collecting blood samples at predetermined time points, and determining and comparing the levels and / or enzymatic activities of the optimized dinuclease fusion protein in the samples.

[0212] Pharmaceutical composition

[0213] In certain embodiments, the optimized dinuclease fusion protein is administered alone. In certain embodiments, the optimized dinuclease fusion protein is administered before administering at least one other therapeutic agent. In certain embodiments, the optimized dinuclease fusion protein is administered simultaneously with the administration of at least one other therapeutic agent. In certain embodiments, the optimized dinuclease fusion protein is administered after administering at least one other therapeutic agent. In other embodiments, the optimized dinuclease fusion protein is administered before administering at least one other therapeutic agent. As will be appreciated by those skilled in the art, in some embodiments, the optimized dinuclease fusion protein is combined with other agents / compounds. In some embodiments, the optimized dinuclease fusion protein and other agents are administered simultaneously. In some embodiments, the optimized dinuclease fusion protein and other agents are not administered simultaneously, wherein the optimized dinuclease fusion protein is administered before or after administering the agent. In some embodiments, the subject receives both the optimized dinuclease fusion protein and other agents while preventing the occurrence of the disease and / or during treatment.

[0214] The pharmaceutical composition of the present invention can be used in combination therapy, i.e., in combination with other agents. In certain embodiments, the combination therapy comprises an optimized dinuclease fusion protein in combination with at least one other agent. Agents include, but are not limited to, chemical compositions, antibodies, antigen binding regions, combinations thereof, and conjugates prepared by in vitro synthesis. In certain embodiments, agents can be used as agonists, antagonists, allosteric modulators, or toxins.

[0215] In certain embodiments, the present invention provides a pharmaceutical composition comprising an optimized dinuclease fusion protein and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0216] In certain embodiments, the present invention provides pharmaceutical compositions comprising an optimized dinuclease fusion protein and a therapeutically effective amount of at least one additional therapeutic agent, as well as a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant.

[0217] In certain embodiments, acceptable formulation materials are preferably non-toxic to the recipient at the dosages and concentrations used. In some embodiments, formulation materials are used for sc and / or IV administration. In certain embodiments, pharmaceutical compositions may contain formulation materials that are used to change, maintain or preserve, for example, pH, osmolality, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., gelatin); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptide; salt-forming counter ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tylofloxacin); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company (1995)). In some embodiments, the formulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl and / or 10 mM NaOAC, pH 5.2, 9% sucrose.

[0218] In certain embodiments, the optimized dinuclease fusion protein and / or therapeutic molecule are connected to a half-life extension medium known in the art. Such media include, but are not limited to, polyethylene glycol, glycogen (e.g., glycosylation of the optimized dinuclease fusion protein) and dextran. Such media are described, for example, in U.S. Patent Application Serial No. 09 / 428,082, now U.S. Patent No. 6,660,843 and disclosed PCT Application No. WO 99 / 25044.

[0219] In certain embodiments, one skilled in the art will determine the optimal pharmaceutical composition based on, for example, the intended route of administration, delivery form, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antibodies of the invention.

[0220] In certain embodiments, the main medium or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, in certain embodiments, a suitable medium or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, which may be supplemented with other materials common in the composition of parenteral administration. In some embodiments, saline comprises isotonic phosphate buffered saline. In certain embodiments, the pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5, or an acetate buffer of about pH 4.0-5.5, which may further include sorbitol or its suitable substitute. In certain embodiments, the composition comprising the optimized dinuclease fusion protein (with or without at least one other therapeutic agent) can be prepared by mixing the selected composition with the desired purity level with the optional formulation reagent (Remington's Pharmaceutical Sciences, the same) in the form of lyophilized cake or aqueous solution. In addition, in certain embodiments, the composition comprising the optimized dinuclease fusion protein (with or without at least one other therapeutic agent) can be prepared as a lyophilisate using a suitable excipient such as sucrose.

[0221] In certain embodiments, pharmaceutical compositions can be selected for parenteral delivery. In certain embodiments, compositions can be selected for suction or delivered by the digestive tract, for example, orally. The preparation of such pharmaceutically acceptable compositions is within the capabilities of those skilled in the art.

[0222] In certain embodiments, the formulation components are present at concentrations acceptable to the site of administration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8.

[0223] In certain embodiments, when considering parenteral administration, therapeutic composition can be the form of pyrogen-free, parenterally acceptable aqueous solution, which comprises the required optimized dinuclease fusion protein in a pharmaceutically acceptable medium with or without other therapeutic agent. In certain embodiments, the medium for parenteral injection is sterile distilled water, wherein the optimized dinuclease fusion protein (with or without at least one other therapeutic agent) is formulated into a sterile isotonic solution of suitable preservation. In certain embodiments, preparation can include the preparation of required molecules with the reagent of controlled or sustained release that can provide product, such as injectable microspheres, biologically erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), pearls or liposomes, and then the product can be delivered by accumulating injection. In certain embodiments, hyaluronic acid can also be used, and the effect of maintaining the duration in promoting circulation can be there. In certain embodiments, implantable drug delivery device can be used for introducing required molecules.

[0224] In certain embodiments, pharmaceutical compositions can be formulated for inhalation. In certain embodiments, the optimized dinuclease fusion protein with or without at least one additional therapeutic agent can be formulated into a dry powder for inhalation. In certain embodiments, an inhalation solution (with or without at least one additional therapeutic agent) comprising the optimized dinuclease fusion protein can be formulated for aerosol delivery with a propellant. In certain embodiments, the solution can be atomized. Pulmonary administration is further described in PCT application no. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0225] In certain embodiments, it is expected that the preparation can be orally administered. In certain embodiments, the optimized dinuclease fusion protein (with or without at least one other therapeutic agent) used in this way can be prepared with or without those carriers that are generally used for the preparation of solid dosage forms such as tablets and capsules. In certain embodiments, the capsule can be designed to release the active part of the preparation at the point in the gastrointestinal tract when the bioavailability is maximized and the degradation before the system is minimized. In certain embodiments, at least one other reagent may be included to promote the absorption of the optimized dinuclease fusion protein and / or any other therapeutic agent. In certain embodiments, diluents, flavoring agents, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants and adhesives can also be used.

[0226] In certain embodiments, the pharmaceutical composition may include an effective amount of an optimized dinuclease fusion protein in a mixture with a non-toxic excipient suitable for preparing a tablet, with or without at least one additional therapeutic agent. In certain embodiments, the solution may be prepared in unit dosage form by dissolving the tablet in sterile water or another suitable medium. 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 adhesives such as starch, gelatin or gum arabic; or lubricants such as magnesium stearate, stearic acid or talc.

[0227] Other pharmaceutical compositions are obvious to those skilled in the art, including formulations involving optimized dinuclease fusion proteins (with or without at least one additional therapeutic agent) in sustained or controlled delivery formulations. In certain embodiments, the technology for preparing various other sustained or controlled delivery modes, such as liposome carriers, bioerodible microparticles or porous beads and deposit injections, is also known to those skilled in the art. See, for example, PCT application number PCT / US93 / 00829, which describes the controlled release of porous polymer microparticles for delivery of pharmaceutical compositions. In certain embodiments, sustained release formulations may include semipermeable polymer matrices in the form of shaped articles (e.g., films or microcapsules). Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers 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., supra) or poly-D (-) -3-hydroxybutyric acid (EP 133,988). In certain embodiments, sustained release compositions may also include liposomes, which may be prepared by any of several methods known in the art. See, e.g., Eppstein et al., PNAS, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

[0228] Pharmaceutical compositions for in vivo administration are usually sterile. In certain embodiments, this can be accomplished by filtration through a sterile filtration membrane. In certain embodiments, where the composition is freeze-dried, sterilization using the method can be performed before or after freeze-drying and reconstruction. In certain embodiments, compositions for parenteral administration can be stored in freeze-dried form or in solution. In certain embodiments, parenteral compositions are typically placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle.

[0229] In certain embodiments, once the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid or as a dehydrated or lyophilized powder. In certain embodiments, such preparations can be stored in a ready-to-use form or in a form (e.g., lyophilized) reconstituted before administration.

[0230] In certain embodiments, kits for producing single-dose administration units are provided. In certain embodiments, the kit may comprise a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments, kits containing single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyosyringes) are included.

[0231] In certain embodiments, the effective amount of the pharmaceutical composition comprising the optimized dinuclease fusion protein (with or without at least one additional therapeutic agent) used in treatment will depend on, for example, the treatment context and purpose. It will be understood by those skilled in the art that, according to certain embodiments, the appropriate dosage level for treatment will therefore vary in part according to the delivered molecule, the indications being used for the optimized dinuclease fusion protein (with or without at least one additional therapeutic agent), the route of administration, and the patient's body shape (weight, body surface or organ size) and / or condition (age and general health). In certain embodiments, clinicians can determine the dosage and change the route of administration to obtain the best therapeutic effect. In certain embodiments, depending on the above factors, a typical dosage range can be from about 0.1 μg / kg to a maximum of about 100 mg / kg or higher. In certain embodiments, the dosage range can be from 0.1 μg / kg to about 100 mg / kg; or 1 μg / kg to about 100 mg / kg; or 5 μg / kg to about 100 mg / kg.

[0232] In certain embodiments, the frequency of administration will take into account the pharmacokinetic parameters of the dinuclease fusion protein optimized in the formulation used and / or any other therapeutic agent. In certain embodiments, the clinician will apply the composition until the dosage for achieving the desired effect is reached. In certain embodiments, the composition can therefore be used as a single dose, or over time as two or more dosages (which may or may not include the desired molecule of the same amount), or as a continuous infusion by an implant or catheter. The further optimization of suitable doses is routinely performed by those of ordinary skill in the art, and within the scope of their routinely performed tasks. In certain embodiments, suitable dosage can be determined by using appropriate dose-response data.

[0233] In certain embodiments, the route of administration of the pharmaceutical composition is consistent with known methods, for example, oral, by injection via intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, subcutaneous, intraocular, intraarterial, intraportal or intralesional routes; by sustained release system or by implant device. In certain embodiments, the composition can be administered by bolus injection or by infusion or continuously administered by implant device.

[0234] In certain embodiments, the composition can be administered locally by implanting a membrane, sponge, or another suitable material into which the desired molecule has been absorbed or encapsulated. In certain embodiments in which an implant device is used, the device can be implanted in any suitable tissue or organ, and the desired molecule can be delivered by diffusion, timed release bolus, or continuous administration.

[0235] In certain embodiments, it may be desirable to use a pharmaceutical composition comprising an optimized dinuclease fusion protein (with or without at least one additional therapeutic agent) in an ex vivo manner. In these cases, cells, tissues and / or organs removed from a patient are exposed to a pharmaceutical composition comprising an optimized dinuclease fusion protein (with or without at least one additional therapeutic agent), after which cells, tissues and / or organs are subsequently implanted back into the patient's body.

[0236] In certain embodiments, the optimized dinuclease fusion protein and / or any other therapeutic agent can be delivered by using methods as described herein to implant certain cells of genetic engineering to express and secrete polypeptides. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous or xenogeneic. In certain embodiments, cells can be immortalized. In certain embodiments, in order to reduce the chance of immune response, cells can be encapsulated to avoid the penetration of surrounding tissues. In certain embodiments, encapsulating materials are typically biocompatible, semipermeable polymer shells or membranes, which allow the release of protein products but prevent the patient's immune system or other harmful factors from surrounding tissues from destroying cells.

[0237] In vitro analysis

[0238] Various in vitro assays known in the art can be used to assess the efficacy of the optimized di-nuclease fusion proteins of the present invention.

[0239] For example, in the presence or absence of optimized dinuclease fusion protein, human PBMC or lupus patient PBMC from normal people's culture are separated, cultured and processed with various stimulants (for example, TLR ligands, co-stimulatory antibodies, immune complexes and normal or autoimmune serum). The cytokine production of stimulated cells can be measured using commercially available reagents, such as from Biolegend (San Diego, CA) for various cytokines (for example, IL-6, IL-8, IL-10, IL-4, IFN-γ and TNF-α) antibody pair kit. Harvest culture supernatant to determine the influence of optimized dinuclease fusion protein on cytokine production at each different time point suitable for determination (for example, 24, 48 hours or later time point). Using for example anti-human IFN-α antibody and the standard curve reagent measurement IFN-α that can be obtained from PBL interferon source (Piscataway, NJ) produces. Similar assays are performed using human lymphocyte subsets (isolated monocytes, B cells, pDCs, T cells, etc.); purification is performed using commercially available magnetic bead-based separation kits such as those available from Miltenyi Biotech (Auburn, CA).

[0240] Multicolor flow cytometry can be used to assess the effects of optimized dinuclease fusion proteins on immune cell activation by measuring the expression of lymphocyte activation receptors such as CD5, CD23, CD69, CD80, CD86 and CD25 in PBMCs or isolated cell subsets at various time points after stimulation using routine art-recognized methods.

[0241] The efficacy of the optimized dinuclease fusion protein can also be tested by incubating SLE patient serum with normal human pDC to activate IFN output, as described in, 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. Without being bound by theory, immune complexes containing circulating nucleic acids in SLE patient serum promote the entry of nucleic acid antigens into pDC endosomes through Fc receptor-mediated endocytosis, and then the nucleic acids bind and activate endosomal TLRs 7, 8 and 9. In order to evaluate the impact of the optimized dinuclease fusion protein, SLE patient serum or plasma was pretreated with the optimized dinuclease fusion protein and then added to pDC cell cultures isolated from healthy volunteers. The level of IFN-α produced was then determined at multiple time points. By degrading nucleic acid-containing immune complexes, it is expected that the effective optimized dinuclease fusion protein will reduce the amount of IFN-α produced.

[0242] The effectiveness of the optimized dinuclease fusion protein is demonstrated by comparing the assay results of cells treated with the optimized dinuclease fusion protein disclosed herein with the assay results of cells treated with a control formulation. After treatment, relative to the marker levels present before treatment or relative to the levels measured in the control group, the levels of the various markers (e.g., cytokines, cell surface receptors, proliferation) described above are generally improved in the effective optimized dinuclease fusion protein treatment group.

[0243] Treatment

[0244] The optimized two nucleic acid enzyme fusion proteins of the present invention are particularly effective in treating autoimmune diseases or abnormal immune responses. At this point, it should be understood that the optimized two nucleic acid enzyme fusion proteins of the present disclosure can be used for controlling, inhibiting, regulating, treating or eliminating the unwanted immune response to both external and self antigens.

[0245] On the other hand, the optimized dinuclease fusion protein is adapted to prevent (preventative) or treat (therapeutic) a disease or condition in a mammal, such as an autoimmune disease, by administering the optimized dinuclease fusion protein to a mammal in need in a therapeutically effective amount or a sufficient amount, wherein the disease is prevented or treated. The present invention contemplates any route of administration (e.g., intravenous, intramuscular, subcutaneous) suitable for achieving the desired effect. Treatment of a disease condition can result in alleviation of symptoms associated with the condition, which may be long-term or short-term, or even transient beneficial effects.

[0246] Many disease conditions are suitable for treatment with the optimized dinuclease fusion proteins of the present disclosure. For example, in some aspects, the disease or disorder is an autoimmune disease or cancer. In some such aspects, the autoimmune disease is insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental forms of uveitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes, Goodpasture syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, lenticular uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis HBs-ve, cryptogenic cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid LE, SLE, or connective tissue disease.

[0247] In a specific embodiment, the optimized dinuclease fusion protein is used to prevent or treat SLE or Sjögren's syndrome.The effectiveness of the optimized dinuclease fusion protein is demonstrated by comparing the IFN-α level, IFN-α response gene level, autoantibody titer, renal function and pathology and / or circulating immune complex level in a mammal treated with the optimized dinuclease fusion protein disclosed herein and a mammal treated with a control formulation.

[0248] For example, select or identify a human subject (for example, a patient meeting the SLE standard of the American College of Rheumatology, or a patient meeting the American-European Consensus Sjogren's Classification Criteria) who needs treatment. The subject may need, for example, to alleviate the cause of disease or symptoms of SLE or Sjogren's syndrome. The identification of the subject can be carried out in a clinical setting or elsewhere, for example, by the subject himself using a self-test kit in the subject's home.

[0249] At time zero, the dinuclease fusion protein of the optimization of the suitable first dosage is applied to the subject.The dinuclease fusion protein of optimization is prepared as described herein.After a period of time after the first dosage, for example 7 days, 14 days and 21 days, the condition of the subject is assessed, for example by measuring IFN-α level, IFN-α response gene level, autoantibody titer, renal function and pathology and / or circulating immune complex level.Other relevant standards can also be determined.The quantity and intensity of dosage are adjusted according to the needs of the subject.After treatment, the IFN-α level, IFN-α response gene level, autoantibody titer, renal function and pathology and / or circulating immune complex level of the subject are relative to the level existing before treatment before treatment or relative to the level measured in the subject of similar illness but untreated / control to reduce and / or improve.

[0250] In another example, select or identify a rodent subject (see, e.g., Example 7) that needs treatment. The identification of the subject can be carried out in a laboratory environment or elsewhere. At time zero, the optimized dinuclease fusion protein of the first dosage is applied to the subject. The optimized dinuclease fusion protein is prepared as described herein. After a period of time after the first dosage, e.g., 7 days, 14 days, and 21 days, the subject's condition is assessed, e.g., by measuring IFN-α levels, IFN-α response gene levels, autoantibody titers, renal function, pathology, and / or circulating immune complex levels. Other relevant standards can also be determined. The quantity and intensity of the dosage are adjusted according to the needs of the subject.

[0251] Following treatment, the subject's IFN-α levels, IFN-α responsive gene levels, autoantibody titers, renal function and pathology, and / or circulating immune complex levels are reduced and / or improved relative to levels present prior to treatment or relative to levels measured in similarly ill but untreated / control subjects.

[0252] Another aspect of the present invention is to use gene therapy methods to treat or prevent disorders, diseases and conditions with one or more optimized dinuclease fusion proteins. The gene therapy method involves introducing optimized dinuclease fusion protein nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into animals in need to achieve expression of one or more polypeptides of the present disclosure. The method can include introducing one or more polynucleotides encoding the optimized dinuclease fusion proteins of the present disclosure, which are operably coupled to promoters and any other genetic elements necessary for target tissue expression of the polypeptide.

[0253] In gene therapy applications, the optimized dinuclease fusion protein gene is introduced into cells to achieve in vivo synthesis of therapeutically effective genetic products. "Gene therapy" includes conventional gene therapy and the administration of gene therapy agents (which involve one or repeated administration of therapeutically effective DNA or mRNA) in which a lasting effect is achieved by a single treatment. Oligonucleotides can be modified to enhance their uptake, for example, by replacing their negatively charged phosphodiester groups with uncharged groups.

[0254] Example

[0255] The following are examples for implementing specific embodiments of the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to employed numerals (e.g., amount, temperature, etc.), but some experimental errors and deviations should certainly be allowed.

[0256] Unless otherwise indicated, the practice of the present invention will employ conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the skill of the art. These techniques are fully described in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3 rd Ed. (Plenum Press) Vols A and B (1992).

[0257] Example 1

[0258] Generation of optimized dinuclease fusion protein encoding expression vector

[0259] Various embodiments of the optimized dinuclease fusion protein disclosed herein are shown in Figure 1 As an exemplary optimized dinuclease fusion protein, a dinuclease fusion protein having Figure 1 The dinuclease fusion protein of the configuration shown. Specifically, starting from the amino acid sequence of the optimized dinuclease fusion protein, the codon optimization of Genescript (Genescript, Piscatawy, NJ) is used to directly synthesize the polynucleotides encoding the optimized dinuclease fusion protein so as to be optimally expressed in mammalian cells. The optimized process involves, for example, avoiding regions with very high (>80%) or very low (<30%) GC content when possible, and avoiding cis-acting sequence motifs, such as internal TATA boxes, chi-sites and ribosome entry sites, AT-rich or GC-rich sequence extensions, RNA instability motifs, repetitive sequences and RNA secondary structures and recessive splicing donors and acceptor sites in higher eukaryotic organisms. The DNA encoding the optimized dinuclease fusion protein is cloned into pcDNA3.1+ mammalian expression vector. Produce the optimized dinuclease fusion protein with the following configuration.

[0260] The tandem homodimer RSLV-145 (SEQ ID NO: 1) has a DNase-linker-RNase-Fc configuration, wherein the wild-type human RNase1 domain (SEQ ID NO: 27) is operably coupled to the N-terminus of the mutant Fc region (SEQ ID NO: 55) comprising an SCC hinge and CH2 mutations P238S, P331S without 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).

[0261] To preferentially form heterodimers, each Fc domain in the following constructs includes complementary CH3 mutations: T350V, L351Y, F405A, and Y407V; and T350V, T366L, K392L, and T394W (numbered according to the EU index.)

[0262] The tandem heterodimer RSLV-147 has a DNase-Fc (SEQ ID NO:3) and RNase-Fc (SEQ ID NO:4) configuration, wherein a mutant human DNase1 domain (SEQ ID NO:25) is operably coupled to the N-terminus of a first mutant Fc region comprising an SCC hinge, CH2 mutations P238S, P331S, and CH3 mutations, and wherein a wild-type human RNase1 domain (SEQID NO:27) is operably coupled to the N-terminus of a second mutant Fc region comprising an SCC hinge, CH2 mutations P238S, P331S, and CH3 mutations.

[0263] The heterodimeric RSLV-148 has the configuration DNase-first Fc domain-linker-RNase (SEQ ID NO:5) and a mutant second Fc domain comprising CH2 mutations P238S, P331S and CH3 mutations (N-terminal truncation, including the first cysteine ​​in the CCC hinge) (SEQ ID NO:6), wherein a mutant human DNase1 domain (SEQ ID NO:25) is operably coupled to the N-terminus of the first mutant Fc region comprising an SCC hinge, CH2 mutations P238S, P331S and CH3 mutations, and wherein a 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.

[0264] The heterodimeric RSLV-149 has a DNase-Fc (SEQ ID NO:7) and Fc-linker-RNase (SEQ ID NO:8) configuration, wherein a mutant human DNase1 domain (SEQ ID NO:25) is operably coupled to the N-terminus of a first mutant Fc region comprising an SCC hinge, CH2 mutations P238S, P331S, and CH3 mutations, and wherein a wild-type human RNase1 domain (SEQ ID NO:27) is operably coupled to the C-terminus of a second mutant Fc region comprising CH2 mutations P238S, P331S, and CH3 mutations via an NLG linker (SEQ ID NO:6).

[0265] The heterodimeric RSLV-152 has the configuration RNase-first mutant Fc-linker-DNase (SEQ ID NO: 13) and a second mutant Fc domain comprising CH2 mutations P238S, P331S and CH3 mutations (SEQ ID NO: 14), wherein a wild-type human RNase1 domain (SEQ ID NO: 27) is operably coupled to the N-terminus of the first mutant Fc region comprising an SCC hinge and CH2 mutations P238S, P331S and CH3 mutations, and wherein a 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.

[0266] The heterodimeric RSLV-153 has a configuration of Fc-linker-DNase (SEQ ID NO: 15) and RNase-Fc (SEQ ID NO: 16), wherein a mutant human DNase1 domain (SEQ ID NO: 25) is operably coupled to the C-terminus of a first mutant Fc region comprising CH2 mutations P238S, P331S, and CH3 mutations via an NLG linker, and wherein a wild-type human RNase1 domain (SEQ ID NO: 27) is operably coupled to the N-terminus of a second mutant Fc region comprising an SCC hinge, CH2 mutations P238S, P331S, and CH3 mutations.

[0267] Constructs RLSV-327 (containing a dinuclease of RNase 1 and DNase 1 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.

[0268] Example 2

[0269] Stable mammalian cell lines expressing optimized dinuclease fusion proteins for transient expression

[0270] For transient expressions, use FreeStyle TM The expression vector from Example 1 containing the optimized dinuclease fusion protein insert is transiently transfected into Chinese hamster ovary (CHO) cells, such as CHO-S cells (e.g., FreeStyle TM CHO-S cells, Invitrogen). CHO-S cells were maintained in FreeStyle medium containing 2 mM L-glutamine and penicillin-streptomycin. TM CHO expression medium.

[0271] A stable CHO-S cell line expressing the optimized dinuclease fusion protein is generated using conventional methods known in the art. For example, CHO-S cells can be transfected with a nucleic acid sequence comprising the optimized dinuclease fusion protein and a coding marker (e.g., GFP, a surface marker selectable by magnetic beads) using, for example, flow cytometry or magnetic bead separation (e.g., MACSelect TM Alternatively, CHO-S cells are transfected using any transfection method known in the art (e.g., electroporation (Lonza) or FreeStyle as described above). TM The cells are transfected with a vector containing the nucleic acid sequence of the optimized dinuclease fusion protein and a selection marker, and then selected using, for example, flow cytometry. The selection marker can be added to the same vector as the encoding optimized dinuclease fusion protein or a separate vector.

[0272] By using a column filled with protein-A agarose beads to capture molecules, then washing in a column wash buffer (e.g., 90mMTris, 150mM NaCl, 0.05% sodium azide) and using a suitable elution buffer (e.g., 0.1M citrate buffer, pH 3.0) to release molecules from the column, the optimized dinuclease fusion protein is purified from the culture supernatant. The eluted material is further concentrated by using a Centricon concentrator in PBS through a series of centrifugal buffer exchanges, and then filtered through a 0.2 μm filter device. The concentration of the optimized dinuclease fusion protein is determined using standard spectrophotometry (e.g., Bradford, BCA, Lowry, Biuret assay).

[0273] Example 3

[0274] Nuclease activity of purified optimized dinuclease fusion protein

[0275] The optimized dinuclease fusion proteins were analyzed for RNase activity in mouse serum. Proteins were added at doses of 12.5-100 ng to 2.5 mg / ml poly-IC (Sigma) in 50 mM Hepes and 100 mM NaCl at pH 7.3 and incubated at 37°C for 50 min. TCA was added to a final concentration of 5% and placed on ice. Samples were filtered to remove precipitate and the filtrate was collected for OD 260 The result is displayed on Figure 2In. RSLV-132 and RSLV-145 (both containing 2 RNase parts per mole equivalent construct) are all active, and RSLV-145 is more active than RSLV-132. Other constructs (RSLV-147, RSLV-152, RSLV-153 and RSLV-327) that only contain an RNase part per mole are suitable and consistent with RSLV-132. Surprisingly, RSLV-148 and RSLV-149 have higher activity than RSLV-132 and other single RNase constructs. These constructs contain the RNase part that is connected to the C-terminal of Fc domain separately.

[0276] ODN-2006-G5 (InvivoGen, tlrl-2006g5), a DNA oligonucleotide agonist of TLR9, was used to measure the DNase1 activity of optimized dinuclease fusion proteins containing DNase1 domains. Proteins ranging from 0.24ng / ml to 500ng / ml were incubated with ODN-2006-G5 for 1 hour at 37°C in DMEM containing 25mM Hepes and 10% FBS. The reaction mixture was applied to hTLR9 HEKBlue cells (which are engineered to secrete alkaline phosphatase (SEAP) in response to TLR9 agonists) at 37°C overnight. The culture medium was harvested and SEAP was determined using a colorimetric substrate, and then at OD 620 Readings were taken using GraphPad IC50 values ​​were calculated using software version 6.0e. Figure 3 All six constructs (RSLV-145, RSLV-147, RSLV-148, RSLV-149, RSLV-152, and RSLV-153) had potent DNase activity and were at least 5000-fold more active than recombinant huDNase1. RSLV-145 appeared to be approximately 2-fold more active than the other heterodimeric constructs and RSLV-327, likely due to the presence of 2 DNase domains in RSLV-145 compared to 1 DNase domain in the other constructs. RSLV-152 was consistently the least active of the constructs.

[0277] Example 4

[0278] Optimized in vitro potency of dinuclease fusion proteins

[0279] Effects of optimized dinuclease fusion proteins on cytokine expression

[0280] Human PBMC is separated from normal patients and lupus patients and cultivated.With or without the dinuclease fusion protein of the optimization of embodiment 2, various irritating TLR ligands, co-stimulatory antibodies, immune complexes and normal or autoimmune serum are used to process cells.At each different time point (for example, 6 hours, 12 hours, 24 hours, 48 ​​hours, etc.), culture supernatant is collected, and the level of a group of cytokines (including human IL-6, IL-8, IL-10, IL-4, IFN-γ, IFN-α and TNF-α) is measured using the commercially available ELISA kit from, for example, Thermo Fisher Scientific, Inc..Effective dinuclease fusion protein of optimization is expected to reduce the cytokine level produced by stimulated PBMC relative to control.

[0281] Effects of optimized dinuclease fusion protein on lymphocyte activation receptor expression

[0282] Human PBMC is separated from normal patients and lupus patients and cultivated.With or without the dinuclease fusion protein of the optimization of embodiment 2, various irritating TLR ligands, co-stimulatory antibodies, immune complexes and normal or autoimmune serum are used to process cells.Then cells are subjected to polychromatic flow cytometry analysis to measure the expression of lymphocyte activation receptors CD5, CD23, CD69, CD80, CD86 and CD25 at various different time points (for example, 6 hours, 12 hours, 24 hours, 48 ​​hours, etc.) after stimulation using conventional art-recognized methods.Suitable antibodies for these receptors can be commercially available from, for example, BD / PharMingen.Expected effective dinuclease fusion protein of optimization reduces the expression of lymphocyte activation receptors in the PBMC stimulated relative to the control.

[0283] Effects of optimized dinuclease fusion protein on interferon output from plasmacytoid dendritic cells (pDCs)

[0284] Use art-recognized methods or commercially available kits such as EasySep TM pDCs from healthy volunteers were isolated using the Human EpCAM Positive Selection Kit (StemCell Technologies, Inc.). The isolated pDCs were cultured in, for example, 96-well flat-bottom plates in 0.1 ml of appropriate culture 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) at a density ranging from 5 × 10 4 Up to 2.5×10 5 / well. The cultured pDCs were activated by adding serum or plasma from individual SLE patients diluted with culture medium at a ratio of 1:5, and 0.1 ml of these samples were added to the wells containing cells (final patient serum concentration was 10%). The culture was incubated at 37 ° C for 40 hours, after which the conditioned medium was harvested and the IFNα content was assessed using a commercially available ELISA kit. Serum samples obtained from healthy volunteers were used as controls. In order to evaluate the effects of the optimized dinuclease fusion protein, SLE patient serum or plasma was pretreated with the optimized dinuclease fusion protein (1-10 μg / ml) for 30 minutes and added to the pDC culture. It is expected that the effective optimized dinuclease fusion protein reduces the amount of IFNα produced due to the degradation of nucleic acid-containing ICs.

[0285] ************************

[0286] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternative embodiments, it will be understood by those skilled in the relevant art that various changes in form and details may be made without departing from the spirit and scope of the invention.

[0287] All references, issued patents, and patent applications cited in the body of this specification are incorporated herein by reference in their entirety for all purposes.

[0288] Sequence Table

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

Claims

1. A heterodimer comprising a first nucleic acid enzyme domain, a second nucleic acid enzyme domain, a first Fc domain and a second Fc domain, wherein the first nucleic acid enzyme domain is DNase1 and the second nucleic acid enzyme domain is RNase1, wherein the DNase1 is operably connected to the N- or C-terminal of the first Fc domain with or without a joint, and the RNase1 is operably connected to the N- or C-terminal of the second Fc domain with or without a joint.

2. The heterodimer according to claim 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.

3. The heterodimer according to claim 1, wherein the DNase1 is operably connected to the N-terminus of the first Fc domain with a linker, and the RNase1 is operably connected to the N-terminus of the second Fc domain with a linker. 4 . The heterodimer according to claim 1 , wherein the DNase1 is operably linked to the N-terminus of the first Fc domain with a linker, and the RNase1 is operably linked to the C-terminus of the second Fc domain without a linker.

5. The heterodimer according to claim 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 C-terminus of the second Fc domain without a linker.

6. The heterodimer according to claim 1, wherein the DNase1 is operably linked to the N-terminus of the first Fc domain with a linker, and the RNase1 is operably linked to the C-terminus of the second Fc domain with a linker.

7. The heterodimer according to claim 1, wherein the DNase1 is operably linked to the C-terminus of the first Fc domain with a linker, and the RNase1 is operably linked to the C-terminus of the second Fc domain with a linker.

8. The heterodimer according to claim 1, wherein the DNase1 is operably linked to the C-terminus of the first Fc domain without a linker, and the RNase1 is operably linked to the C-terminus of the second Fc domain without a linker.

9. The heterodimer according to claim 1, wherein the DNase1 is operably linked to the C-terminus of the first Fc domain with a linker, and the RNase1 is operably linked to the N-terminus of the second Fc domain without a linker.

10. The heterodimer according to claim 1, wherein the DNase1 is operably linked to the C-terminus of the first Fc domain with a linker, and the RNase1 is operably linked to the N-terminus of the second Fc domain with a linker.

11. The heterodimer according to any one of claims 1-10, wherein the RNase is wild-type human RNase1 or a mutant RNase, such as non-glycosylated, hypoglycosylated or deglycosylated RNase1, such as human RNase1 N34S / N76S / N88S.

12. The heterodimer according to claim 11, wherein the RNase is wild-type human RNase1. 13 . The heterodimer according to claim 1 , wherein the DNase is wild-type human DNase1 or mutant human DNase1 A114F, or non-glycosylated, hypoglycosylated or deglycosylated mutant human DNase1 N18S / N106S / A114F.

14. A heterodimer according to any one of the preceding claims, wherein the first and second Fc domains comprise a hinge domain, a CH2 domain and a CH3 domain.

15. The heterodimer of claim 14, wherein the first and second Fc domains comprise replacement of one or more of the three hinge region cysteine ​​residues with serine.

16. The heterodimer of claim 15, wherein the first and second Fc domains comprise mutations selected from SCC, SSS (residues 220, 226 and 229), G236R, L328R, L234A and L235A numbered according to the EU index.

17. The heterodimer of claim 16, wherein the first and second Fc domains comprise SCC mutations numbered according to the EU index (residues 220, 226 and 229).

18. A heterodimer according to any one of the preceding claims, wherein the first and second Fc domains comprise P238S and P331 mutations numbered according to the EU index.

19. A heterodimer according to any of the preceding claims, wherein the first and second Fc domains comprise one or more CH3 mutations to preferentially form heterodimers.

20. The heterodimer of claim 19, wherein the first Fc domain comprises CH3 mutations T350V, L351Y, F405A and Y407V numbered according to the EU index, and the second Fc domain comprises CH3 mutations T350V, T366L, K392L, T394W.

21. A heterodimer according to any one of the preceding claims, wherein the linker domain is a polypeptide linker, such as a gly-ser linker or an NLG linker (vdgasspvnvsspsvqdi).

22. A heterodimer comprising a first and a second polypeptide sequence selected from the group consisting of: (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:3, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:3; and a second polypeptide comprising the amino acid sequence of SEQ ID NO:4, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:4, or (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO:7, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:7; and a second polypeptide comprising the amino acid sequence of SEQ ID NO:8, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:8, or (iii) a first polypeptide comprising the amino acid sequence of SEQ ID NO:9, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:9; and a second polypeptide comprising the amino acid sequence of SEQ ID NO:10, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:10, or (iv) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 11, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 11; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 12, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12, or (v) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 15, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 15; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 16, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

16.

23. A heterodimer comprising a first nuclease domain, a second nuclease domain, a first Fc domain and a second Fc domain, wherein the first nuclease domain is DNase1 and the second nuclease domain is RNase1, wherein (i) the DNase1 is operably linked to the N-terminus of the first Fc domain with or without a linker, and the RNase1 is operably linked to the C-terminus of the first Fc domain with or without a linker, or (ii) the RNase1 is operably linked to the N-terminus of the first Fc domain with or without a linker, and the DNase1 is operably linked to the C-terminus of the first Fc domain with or without a linker.

24. The heterodimer of claim 23, 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 first Fc domain with a linker.

25. The heterodimer of claim 23, wherein the DNase1 is operably linked to the N-terminus of the first Fc domain with a linker, and the RNase1 is operably linked to the C-terminus of the first Fc domain without a linker.

26. The heterodimer of claim 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 with a linker.

27. The heterodimer of claim 23, wherein the RNase1 is operably linked to the N-terminus of the first Fc domain with a linker, and the DNase1 is operably linked to the C-terminus of the first Fc domain with a linker.

28. The heterodimer according to any one of claims 23-27, wherein the RNase is wild-type human RNase1 or a mutant RNase, such as non-glycosylated, hypoglycosylated or deglycosylated RNase1, such as human RNase1 N34S / N76S / N88S.

29. The heterodimer according to claim 28, wherein the RNase is wild-type human RNase1.

30. The heterodimer according to any one of claims 23-29, wherein the DNase is wild-type human DNase1 or mutant human DNase1 A114F, or non-glycosylated, hypoglycosylated or deglycosylated mutant human DNase1 N18S / N106S / A114F.

31. A heterodimer according to any of the preceding claims, wherein the first and second Fc domains comprise a hinge domain, a CH2 domain and a CH3 domain.

32. The heterodimer of claim 31, wherein the first and second Fc domains comprise replacement of one or more of the three hinge region cysteine ​​residues with serine.

33. A heterodimer according to claim 32, wherein the first and second Fc domains comprise mutations selected from SCC, SSS (residues 220, 226 and 229), G236R, L328R, L234A and L235A numbered according to the EU index.

34. The heterodimer of claim 33, wherein the first and second Fc domains comprise SCC mutations numbered according to the EU index (residues 220, 226 and 229).

35. A heterodimer according to any of the preceding claims, wherein the first and second Fc domains comprise P238S and P331 mutations numbered according to the EU index.

36. A heterodimer according to any of the preceding claims, wherein the first and second Fc domains comprise one or more CH3 mutations to preferentially form a heterodimer.

37. A heterodimer according to claim 36, wherein the first Fc domain comprises CH3 mutations T350V, L351Y, F405A and Y407V numbered according to the EU index, and the second Fc domain comprises CH3 mutations T350V, T366L, K392L, T394W.

38. A heterodimer according to any one of the preceding claims, wherein the linker domain is a polypeptide linker, such as a gly-ser linker or an NLG linker (vdgasspvnvsspsvqdi).

39. A heterodimer comprising a first and a second polypeptide sequence selected from the group consisting of: (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO:5, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:5; and a second polypeptide comprising the amino acid sequence of SEQ ID NO:6, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:6, or (ii) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 13, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 13; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 14, or a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:

14.

40. A composition comprising the heterodimer of any one of the preceding claims and a pharmaceutically acceptable carrier.

41. A nucleic acid molecule encoding the heterodimer of any one of the preceding claims.

42. A recombinant expression vector comprising the nucleic acid molecule according to claim 41.

43. A host cell transformed with the recombinant expression vector according to claim 42.

44. A method for preparing the heterodimer according to any one of claims 1 to 39, include: Providing a host cell comprising a nucleic acid sequence encoding the heterodimer; and maintaining the host cell under conditions wherein the heterodimer is expressed.

45. A method for treating or preventing a disorder associated with an abnormal immune response, comprising administering to a subject an effective amount of the heterodimer of any one of claims 1-39.

46. ​​The method of claim 45, wherein the disorder is an autoimmune disease.

47. The method of claim 46, wherein the autoimmune disease is selected from the group consisting of insulin-dependent diabetes mellitus, multiple sclerosis, experimental autoimmune encephalomyelitis, rheumatoid arthritis, experimental autoimmune arthritis, myasthenia gravis, thyroiditis, experimental forms of uveoretinitis, Hashimoto's thyroiditis, primary myxedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, premature menopause, male infertility, juvenile diabetes, Goodpasture syndrome, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, lenticular uveitis, autoimmune hemolytic anemia, idiopathic leukopenia, primary biliary cirrhosis, active chronic hepatitis HBs-ve, cryptogenic cirrhosis, ulcerative colitis, Sjögren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid LE, systemic lupus erythematosus (SLE), and connective tissue disease.

48. The method of claim 47, wherein the autoimmune disease is SLE.

49. The method of claim 47, wherein the autoimmune disease is Sjögren's syndrome.

50. A method for treating SLE comprising administering to a subject an amount of a heterodimer effective to degrade immune complexes containing RNA, DNA, or both RNA and DNA, wherein the composition comprises a pharmaceutically acceptable carrier and the heterodimer of any one of claims 1-39.

51. A method for treating Sjögren's syndrome, comprising administering to a subject an amount of a heterodimer effective to degrade immune complexes containing RNA, DNA, or both RNA and DNA, wherein the composition comprises a pharmaceutically acceptable carrier and the heterodimer of any one of claims 1-39.

Images