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  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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  • C12N15/09—Recombinant DNA-technology
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  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
• • C—CHEMISTRY; METALLURGY
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  • C07K—PEPTIDES
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  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/27—Endoribonucleases producing 3'-phosphomonoesters (3.1.27)
  • C12Y301/27005—Pancreatic ribonuclease (3.1.27.5)

Definitions

• a hybrid nuclease molecule includes a modified Fc domain which is a mutant, IgGl Fc domain.
• a mutant Fc domain comprises one or more mutations in the hinge, CH2, and/or CH3 domains.
• the Fc domain comprises an amino acid sequence having one or more of the mutations P238S, P331S, SCC, SSS (residues 220, 226, and 229), G236R, L328R, L234A, and L235A.
• a mutant Fc domain includes a P238S mutation.
• a mutant Fc domain includes a P331S mutation.
• a nucleic acid encoding a mutant Fc domain is as shown in SEQ ID NO:59. In some aspects, a mutant Fc domain is as shown in SEQ ID NO:60. In some aspects, a nucleic acid encoding a mutant Fc domain is as shown in SEQ ID NO:71. In some aspects, a mutant Fc domain is as shown in SEQ ID NO:72. In some aspects, a nucleic acid encoding a mutant Fc domain is as shown in SEQ ID NO:73. In some aspects, a mutant Fc domain is as shown in SEQ ID NO:74. In some aspects, a nucleic acid encoding a mutant Fc domain is shown in SEQ ID NO: 75.
• a nucleic acid encoding a hybrid nuclease molecule is as shown in SEQ ID NO: 63, or 79. In some aspects, a hybrid nuclease molecule is as shown in SEQ ID NO: 64, or 79.
• a nucleic acid encoding a hybrid nuclease molecule is as shown in SEQ ID NO: 65, or 81.
• a hybrid nuclease molecule is as shown in SEQ ID NO: 66, or 82.
• a hybrid nuclease molecule comprises an amino acid sequence set forth in SEQ ID NO: 62, 64, 78, 80, 92, or 96, or a hybrid nuclease molecule comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 62, 64, 78, 80, 92, or 96.
• the activity of a hybrid nuclease molecule having a DNase is not less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or >30-fold less than the activity of a control DNase molecule. In some aspects, the activity of a hybrid nuclease molecule having a DNase is about equal to the activity of a control DNase molecule. In some aspects, the activity of a hybrid nuclease molecule having an RNase is not less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or >30-fold less than the activity of a control RNase molecule. In some aspects, the activity of a hybrid nuclease molecule having an RNase is about equal to the activity of a control RNase molecule.
• a hybrid nuclease molecule is a polypeptide, wherein the amino acid sequence of the first nuclease domain comprises a human, wild-type RNase amino acid sequence, wherein the first linker domain is (Gly4Ser)n, where n is 0, 1, 2, 3, 4 or 5, wherein the amino acid sequence of the Fc domain comprises a human, mutant IgGl Fc domain amino acid sequence, and wherein the first linker domain is coupled to the C- terminus of the first nuclease domain and the N-terminus of the Fc domain.
• a hybrid nuclease molecule is a polypeptide comprising or consisting of a sequence shown in Table 1.
• a hybrid nuclease molecule includes a leader sequence.
• the leader sequence is human VK3LP peptide from the human kappa light chain family, and the leader sequence is coupled to the N-terminus of the first nuclease domain.
• the VK3LP has the sequence set forth in SEQ ID NO: 100.
• the gly/ser peptide is of the formula (Gly 4 Ser) n , wherein n is a positive integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the gly/ser peptide includes (Gly 4 Ser) 3 . In some embodiments, the gly/ser peptide includes (Gly 4 Ser) 4 . In some embodiments, the gly/ser peptide includes (Gly 4 Ser) 5 . In some embodiments, the first linker domain includes at least one restriction site. In some embodiments, the first linker domain includes about 12 or greater nucleotides including at least one restriction site. In some embodiments, the first linker domain includes two or more restriction sites.
• the first nuclease domain is linked to the N-terminus of the Fc domain. In some embodiments, the first nuclease domain is linked to the C-terminus of the Fc domain.
• FIG. 4 shows the concentration of RSLV-124 in mouse serum as extrapolated from the RNase enzymatic activity of the molecule.
• FIG. 5 shows single radial enzyme diffusion (SRED) analysis of serum from two
• FIG. 7 shows survival of TLR7.1 Tg versus TLR7.1xRNaseA DTg mice
• FIG. 10 shows SRED analysis comparing aliquots of protein A purified proteins from RSLV transfected COS supernatants.
• a subject is treated by administering a nuclease activity which is a DNase or an RNase activity, preferably in the form of a hybrid nuclease molecule.
• a nuclease activity which is a DNase or an RNase activity, preferably in the form of a hybrid nuclease molecule.
• the nuclease activity is a first nuclease domain.
• the nuclease domain is coupled to a modified Fc domain such that the molecule has reduced cytotoxicity.
• a hybrid nuclease molecule includes a second nuclease domain.
• a method of treating SLE in which an effective amount of a nuclease-containing composition is administered to a subject.
• treatment results in degradation of immune complexes containing RNA, DNA or both RNA and DNA.
• treatment results in inhibition of Type I interferons, such as interferon-a in a subject.
• a method of treating a subject comprises administering an effective amount of a composition of a hybrid nuclease molecule comprising an amino acid sequence set forth in SEQ ID NO: 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, or 98.
• the composition is a hybrid nuclease molecule comprising an amino acid sequence set forth in SEQ ID NO: 96 or 98.
• Another exemplary gly/ser polypeptide linker comprises the amino acid sequence Ser(Gly 4 Ser)n.
• n l .
• n 2.
• n 3.
• n 4.
• n 5.
• n 6.
• a polypeptide includes a contiguous amino acid sequence at least 80%>, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous amino acid sequence set forth in Table 1.
• a polypeptide includes an amino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous amino acids of an amino acid sequence set forth in Table 1.
• sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
• test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
• sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
• the nuclease domain is linked to the Fc domain via a linker domain.
• the linker domain is a linker peptide.
• the linker domain is a linker nucleotide.
• the hybrid nuclease molecule includes a leader molecule, e.g., a leader peptide.
• the leader molecule is a leader peptide positioned at the N-terminus of the nuclease domain.
• a hybrid nuclease molecule of the invention comprises a leader peptide at the N-terminus of the molecule, wherein the leader peptide is later cleaved from the hybrid nuclease molecule.
• Methods for generating nucleic acid sequences encoding a leader peptide fused to a recombinant protein are well known in the art.
• any of the hybrid nuclease molecules of the present invention can be expressed either with or without a leader fused to their N-terminus.
• the protein sequence of a hybrid nuclease molecule of the present invention following cleavage of a fused leader peptide can be predicted and/or deduced by one of skill in the art.
• the mutations are P238S, P331 S or N297S, and may include mutations in one or more of the three hinge cysteines.
• the mutations in one or more of three hinge cysteines can be SCC or SSS.
• the molecules contain the SCC hinge, but are otherwise wild type for human IgGl Fc CH2 and CH3 domains, and bind efficiently to Fc receptors, facilitating uptake of the hybrid nuclease molecule into the endocytic compartment of cells to which they are bound.
• the molecule has activity against single and/or double-stranded RNA substrates.
• a hybrid nuclease molecule is as shown in any of SEQ ID NOs 62, 64, 66, 68, 70, 78, 80, 82, 84, 86, 92, 94, 96, or 98.
• a hybrid nuclease molecule comprises a wild-type, human RNasel domain linked to a mutant, human IgGl Fc domain comprising SCC, P238S, and P331S, or a mutant, human IgGl Fc domain comprising SSS, P238S, and P331S.
• a nucleic acid sequence encoding a hybrid nuclease molecule is shown in SEQ ID NO: 65, or 81. In some aspects, a hybrid nuclease molecule is shown in SEQ ID NO: 66, or 82.
• a nucleic acid sequence encoding a hybrid nuclease molecule is shown in SEQ ID NO: 67, or 83. In some aspects, a hybrid nuclease molecule is shown in SEQ ID NO: 68, or 84.
• a hybrid nuclease molecule comprises a wild-type, human RNasel domain linked to a mutant, human IgGl Fc domain comprising SCC, P238S, and P331S linked via a NLG linker domain to a human DNasel G105R Al 14F domain.
• a hybrid nuclease molecule comprises a wild-type, human RNasel domain linked to a mutant, human IgGl Fc domain comprising SSS, P238S, and P331 S linked via a NLG linker domain to a human DNasel G105R Al 14F domain.
• a nucleic acid sequence encoding a hybrid nuclease molecule is shown in SEQ ID NO: 69, 85, or 93.
• a hybrid nuclease molecule is shown in SEQ ID NO: 70, 86, 94, or 98.
• the domain orientation can be altered to construct an Ig-RNase molecule or an Ig-DNase molecule or an RNase-Ig molecule or an RNase-Ig molecule that retains FcR binding and has active nuclease domains.
• linker domains include (gly4ser) 3, 4 or 5 variants that alter the length of the linker by 5 amino acid progressions.
• a linker domain is approximately 18 amino acids in length and includes an N-linked glycosylation site, which can be sensitive to protease cleavage in vivo.
• an N-linked glycosylation site can protect the hybrid nuclease molecules from cleavage in the linker domain.
• an N-linked glycosylation site can assist in separating the folding of independent functional domains separated by the linker domain.
• hybrid nuclease molecules include DNaselL3-Ig- linker-RNase constructs.
• a hybrid nuclease molecule includes a DNase 1-Ig-linker-RNase construct or an RNase-Ig-linker-DNase construct.
• fusion junctions between enzyme domains and the other domains of the hybrid nuclease molecule is optimized.
• a hybrid nuclease molecule includes TREX1.
• a TREX1 hybrid nuclease molecule can digest chromatin.
• further optimization of the fusion junction chosen for truncation of a TREXl enzyme can be used to improve expression of the hybrid nuclease molecules.
• the hybrid nuclease molecule is a TREXl -tandem hybrid nuclease molecule.
• an alternative method for facilitating head-to-tail folding of TREXl is to generate a TREXl -TREXl -I g hybrid hybrid nuclease molecule that incorporates two TREXl domains in tandem, followed by a linker domain and an Ig Fc domain.
• positioning of TREXl cassettes in a head-to-tail manner can be corrected for head-to tail folding on either arm of the immunoenzyme and introduce a single TREXl functional domain into each arm of the molecule.
• each immunoenzyme of a hybrid nuclease molecule has two functional TREXl enzymes attached to a single IgG Fc domain.
• the hybrid nuclease molecule includes 1L3-Ig.
• the 1L3 DNase is constructed from a murine sequence and expressed.
• the enzyme is active.
• a murine 1L3 DNase-Ig-RNase hybrid nuclease is constructed and expressed.
• the molecule includes human 1L3-Ig, human lL3-Ig-RNase, and/or human RNase-Ig-lL3.
• a mutant Fc domain comprises P238S and/or P331S, and/or one or more mutations in the three hinge cysteines. In some aspects, a mutant Fc domain comprises P238S and/or P331 S, and/or mutations in a hinge cysteine to SCC or in the three hinge cysteines to SSS. In some aspects, a mutant Fc domain comprises P238S and P331S and mutations in at least one of the three hinge cysteines. In some aspects, a mutant Fc domain comprises P238S and P331S and SCC. In some aspects, a mutant Fc domain comprises P238S and P331S and SSS.
• the hybrid nuclease molecule of the invention comprises at least one Fc domain which comprises at least one CH4 domain or portion thereof. In another embodiment, the hybrid nuclease molecule of the invention comprises at least one Fc domain which comprises at least one hinge domain or portion thereof and at least one CH2 domain or portion thereof (e.g, in the hinge-CH2 orientation). In another embodiment, the hybrid nuclease molecule of the invention comprises at least one Fc domain which comprises at least one CH2 domain or portion thereof and at least one CH3 domain or portion thereof (e.g, in the CH2-CH3 orientation).
• a hybrid nuclease molecule of the invention comprises at least one Fc domain comprising a complete CH3 domain. In another embodiment, a hybrid nuclease molecule of the invention comprises at least one Fc domain comprising a complete CH2 domain. In another embodiment, a hybrid nuclease molecule of the invention comprises at least one Fc domain comprising at least a CH3 domain, and at least one of a hinge region, and a CH2 domain. In one embodiment, a hybrid nuclease molecule of the invention comprises at least one Fc domain comprising a hinge and a CH3 domain.
• an Fc domain employed in a hybrid nuclease molecule of the invention is altered or modified, e.g., by amino acid mutation (e.g., addition, deletion, or substitution).
• the term "Fc domain variant" refers to an Fc domain having at least one amino acid modification, such as an amino acid substitution, as compared to the wild-type Fc from which the Fc domain is derived.
• a variant comprises at least one amino acid mutation (e.g., substitution) as compared to a wild type amino acid at the corresponding position of the human IgGl Fc region.
• the Fc variant confers an improvement in at least one effector function imparted by an Fc domain comprising said wild-type Fc domain (e.g., an improvement in the ability of the Fc domain to bind to Fc receptors (e.g. FcyRI, FcyRII, or FcyRIII) or complement proteins (e.g. Clq), or to trigger antibody-dependent cytotoxicity (ADCC), phagocytosis, or complement-dependent cytotoxicity (CDCC)).
• Fc receptors e.g. FcyRI, FcyRII, or FcyRIII
• complement proteins e.g. Clq
• an Fc domain includes changes in the region between amino acids 234-238, including the sequence LLGGP at the beginning of the CH2 domain.
• an Fc variant alters Fc mediated effector function, particularly ADCC, and/or decrease binding avidity for Fc receptors.
• sequence changes closer to the CH2-CH3 junction, at positions such as K322 or P331 can eliminate complement mediated cytotoxicity and/or alter avidity for FcR binding.
• an Fc domain incorporates changes at residues P238 and P331, e.g., changing the wild type prolines at these positions to serine.
• alterations in the hinge region at one or more of the three hinge cysteines, to encode CCC, SCC, SSC, SCS, or SSS at these residues can also affect FcR binding and molecular homogeneity, e.g., by elimination of unpaired cysteines that may destabilize the folded protein.
• hybrid nuclease molecules of the invention may employ art-recognized Fc variants which are known to impart an improvement in effector function and/or FcR binding.
• a hybrid nuclease molecule of the invention may include, for example, a change (e.g., a substitution) at one or more of the amino acid positions disclosed in International PCT Publications WO88/07089A1, W096/14339A1, WO98/05787A1, W098/23289A1,
• the specific change (e.g., the specific substitution of one or more amino acids disclosed in the art) may be made at one or more of the disclosed amino acid positions.
• a different change at one or more of the disclosed amino acid positions (e.g., the different substitution of one or more amino acid position disclosed in the art) may be made.
• a hybrid nuclease molecule of the invention comprises an Fc variant comprising an amino acid substitution which alters the antigen-dependent effector functions of the polypeptide, in particular ADCC or complement activation, e.g., as compared to a wild type Fc region.
• said hybrid nuclease molecules exhibit altered binding to an Fc gamma receptor (e.g., CD16).
• Fc gamma receptor e.g., CD16
• the hybrid nuclease molecule exhibits altered binding to an activating FcyR (e.g. Fcyl, Fcylla, or FcyRIIIa). In another embodiment, the hybrid nuclease molecule exhibits altered binding affinity to an inhibitory FcyR (e.g. FcyRIIb). Exemplary amino acid substitutions which altered FcR or complement binding activity are disclosed in International PCT Publication No. WO05/063815 which is incorporated by reference herein.
• At least two of the Fc domains may differ at about 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acid positions), about 10 positions, about 15 positions, about 20 positions, about 30 positions, about 40 positions, or about 50 positions).
• Linker Domains e.g., 1, 2, 3, 4, or 5 amino acid positions
• a polypeptide linker can comprise at least a portion of an immunoglobulin hinge region.
• a polypeptide linker comprises an upper hinge domain (e.g., an IgGl, an IgG2, an IgG3, or IgG4 upper hinge domain).
• a polypeptide linker comprises a middle hinge domain (e.g., an IgGl, an IgG2, an IgG3, or an IgG4 middle hinge domain).
• a polypeptide linker comprises a lower hinge domain (e.g., an IgGl, an IgG2, an IgG3, or an IgG4 lower hinge domain).
• polypeptide linkers can be constructed which combine hinge elements derived from the same or different antibody isotypes.
• the polypeptide linker comprises a chimeric hinge comprising at least a portion of an IgGl hinge region and at least a portion of an IgG2 hinge region.
• the polypeptide linker comprises a chimeric hinge comprising at least a portion of an IgGl hinge region and at least a portion of an IgG3 hinge region.
• a polypeptide linker comprises a chimeric hinge comprising at least a portion of an IgGl hinge region and at least a portion of an IgG4 hinge region.
• variant forms of these exemplary polypeptide linkers can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence encoding a polypeptide linker such that one or more amino acid substitutions, additions or deletions are introduced into the polypeptide linker.
• mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
• the invention also includes a vector capable of expressing the peptides in an appropriate host.
• the vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of affecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known.
• Expression control sequences include promoters, activators, enhancers, operators, ribosomal nuclease domains, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.
• a hybrid nuclease molecule is administered alone. In certain embodiments, a hybrid nuclease molecule is administered prior to the administration of at least one other therapeutic agent. In certain embodiments, a hybrid nuclease molecule is administered concurrent with the administration of at least one other therapeutic agent. In certain embodiments, a hybrid nuclease molecule is administered subsequent to the administration of at least one other therapeutic agent. In other embodiments, a hybrid nuclease molecule is administered prior to the administration of at least one other therapeutic agent. As will be appreciated by one of skill in the art, in some embodiments, the hybrid nuclease molecule is combined with the other agent/compound. In some embodiments, the hybrid nuclease molecule and other agent are administered concurrently. In some
• the invention provides for pharmaceutical compositions comprising a hybrid nuclease molecule together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
• suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or
• hydroxypropyl-beta-cyclodextrin fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters
• a hybrid nuclease molecule and/or a therapeutic molecule is linked to a half-life extending vehicle known in the art.
• vehicles include, but are not limited to, polyethylene glycol, glycogen (e.g., glycosylation of the hybrid nuclease molecule), and dextran.
• glycogen e.g., glycosylation of the hybrid nuclease molecule
• dextran e.g., dextran
• compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antibodies of the invention.
• a pharmaceutical composition can be formulated for inhalation.
• a hybrid nuclease molecule, with or without at least one additional therapeutic agent can be formulated as a dry powder for inhalation.
• an inhalation solution comprising a hybrid nuclease molecule, with or without at least one additional therapeutic agent, can be formulated with a propellant for aerosol delivery.
• solutions can be nebulized. Pulmonary administration is further described in PCT application no. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.
• a pharmaceutical composition can involve an effective quantity of a hybrid nuclease molecule, with or without at least one additional therapeutic agents, in a mixture with non-toxic excipients which are suitable for the manufacture of tablets.
• suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
• sustained-release preparations can include semipermeable polymer matrices in the form of shaped articles, e.g.
• sustained release compositions can also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.
• the effective amount of a pharmaceutical composition comprising a hybrid nuclease molecule, with or without at least one additional therapeutic agent, to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
• the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which a hybrid nuclease molecule, with or without at least one additional therapeutic agent, is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient.
• the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
• a typical dosage can range from about 0.1 ⁇ g/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In certain embodiments, the dosage can range from 0.1 ⁇ g/kg up to about 100 mg/kg; or 1 ⁇ g/kg up to about 100 mg/kg; or 5 ⁇ g/kg up to about 100 mg/kg.
• the composition can therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. In certain embodiments, appropriate dosages can be ascertained through use of appropriate dose-response data.
• the route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, subcutaneously, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices.
• the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.
• the composition can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated.
• the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.
• a hybrid nuclease molecule and/or any additional therapeutic agents can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides.
• such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic.
• the cells can be immortalized.
• the cells in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues.
• the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
• hybrid nuclease molecules of the instant invention are particularly effective in the treatment of autoimmune disorders or abnormal immune responses.
• the hybrid nuclease molecules of the present invention may be used to control, suppress, modulate, treat, or eliminate unwanted immune responses to both external and autoantigens.
• Plasmid DNA was prepared using Qiagen (Valencia, CA) QIAprep miniprep kits according to manufacturer's instructions, and eluted in 50 ul EB buffer. DNA concentrations were measured using a Nanodrop 1000 (Thermo Fisher Scientific,
• pDG a selectable, amplifiable plasmid
• the pDG vector is a modified version of pcDNA3 encoding the DHFR selectable marker with an attenuated promoter to increase selection pressure for the plasmid.
• Plasmid DNA was prepared using Qiagen maxiprep kits, and purified plasmid was linearized at a unique Ascl site prior to phenol extraction and ethanol precipitation. Salmon sperm DNA (Sigma- Aldrich, St.
• Transfected cells were allowed to recover overnight in non-selective media prior to selective plating in 96 well flat bottom plates (Costar) at varying serial dilutions ranging from 125 cells/well to 2000 cells/well.
• Culture media for cell cloning was Excell 302 complete, containing 50 nM methotrexate. Once clonal outgrowth was sufficient, serial dilutions of culture supernatants from master wells were screened for expression of hybrid nuclease molecules by use of an -IgG sandwich ELISA.
• NUNC immulon II plates were coated overnight at 4°C with 7.5 microgram/ml F(ab'2) goat anti-mouse IgG (KPL Labs, Gaithersburg, MD) or 2 ug/ml goat anti-human or anti-mouse IgG (Jackson
• DTg mice had very high levels of RNase in their serum (equivalent to >13 U/ml RNase based on our standard with specific activity of 993 U /mg).
• RNaseA concentration in Tg and DTg mice was also measured by ELISA assay as shown in Fig. 6.
• the RNase A Tg and TLR7.1XRNaseA Dtg mice have RNase A serum
• Coat plate with anti-RnaseA Abeam Ab(ab6610) 2.5-10ug/ml O/N in 4C.
• IRGs including MX1 (Interferon-induced GTP-binding protein Mxl (UniProtKB
• RNA-to-cDNA kit (Applied Biosystems) using random primers.
• Relative quantification was calculated using the 2 "ddCT method with age matched wild type B6 mice as baseline to determine fold changes for each GOI.
• the dissociation curves for the reactions show a single melt peak for each gene.
• the standard curve showed similar amplification efficiencies for each gene and that template concentrations were within the linear dynamic range for each of primer set.
• Example 8 Construction and expression of DNasel-Ig single and dual enzyme hybrid nuclease molecules.
• Naturally occurring enzyme variants may thus be less immunogenic when administered to patients, since these isoforms occur in the human population.
• the combination of the actin resistant properties of alleles similar to Al 14F with the increased enzymatic activity of alleles like G105R would generate novel allelic variants of human DNase 1 that might show improved clinical activity in vitro and in vivo.
• ours is the first report of this new mutant form of DNase 1 generated from a combination of two naturally occurring variants G105R and Al 14F.
• DNA concentration was measured using the Nanodrop and an aliquot equivalent to 2.5 ug plasmid DNA used for each transfection reaction.
• Each DNaselg ) or RNase-Ig-DNase ) expression cassette was inserted into the mammalian expression vector pDG, a derivative of pcDNA3.1.
• Transfected cells were incubated for 72 hours at 37°C, 5% C02 prior to harvest of culture supernatants for further analysis. Culture supernatants were harvested, residual cells centrifuged from the solution, and the liquid transferred to new tubes.
• COS-7 cells were transiently transfected with plasmids containing human DNasel wild type or naturally occurring DNase 1 mutant alleles (G105R and/or Al 14F) ) fused to the wild type human IgGl Fc domain.
• This hinge-CH2-CH3 cassette contains a single C- ⁇ S mutation in the hinge region to eliminate the first cysteine in this domain since it is unpaired due to absence of its pairing partner present in the light chain of the antibody.
• more complex multi-nuclease fusion proteins were also expressed from COS cell transient transfections.
• Example 9 Isolation of human-Ig tails, introduction of mutations into the coding sequence, and construction of mutant nuclease molecules
• the P238S mutation and SSS substitutions for SCC were introduced by PCR mutagenesis using two overlapping 5' oligos in sequential PCR reactions.
• the first PCR reaction included the following 5' primer that incorporates the P238S mutation within its sequence: CS-P238S 5-1 : TCT CCA CCG AGC CCA GCA CCT GAA CTC CTG GGA GGA TCG TCA GTC TTC CTC TTC CCC C (58mer). (SEQ ID NO: 25)
• a 5' subfragment was amplified using the full-length wild type clone as template, and the 5' primer was SSSH-5-2: AGA TCT CGA GCC CAA ATC TTC TGA CAA AAC TCA CAC ATC TCC ACC GAG CCC AGC ACC T (58 mer), while the 3' primer was P331S-AS: TGG AGA TGG TTT TCT CGA TGG GGG CTG GGA GGG CTT TGT TGG AGA CC (47mer). (SEQ ID NO: 27)
• RSLV- 128 is a multinuclease fusion construct that incorporates an amino terminal wild type human RNase domain, fused to a (gly4ser)4 linker domain, followed by an SSS mutant hinge and P238S-P33 IS double mutant Fc domain fused to an NLG linker domain and followed by a C terminal mutant DNase (G105R/A114F) domain (SEQ ID NO:67-68).
• COS supernatants were harvested after 72 hours and 0.5 ml samples were immunoprecipitated overnight at 4°C with 100 ul protein A-agarose beads.
• Protein A beads were centrifuged and washed twice in PBS prior to resuspending in SDS- PAGE loading buffer, for NuPAGE gels— reducing or nonreducing LDS sample buffer (Invitrogen, Carlsbad, CA). Samples were heated according to manufacturer's instructions, protein A beads centrifuged to pellet, and sample buffer loaded onto 5-12% NuPAGE gradient gels. Samples were electrophoresed at 150 volts for 1.5-2 hours, and gels blotted to nitrocellulose membranes at 30 mAmp for 1 hour.
• Figure 11 shows results from a DNase nuclease activity assay performed on protein A purified protein from COS7 supematants transfected with the RSLV fusion plasmids in Example 10.
• Figure 11 shows fivee panels (11a, 1 lb, 11c), with each gel panel showing the digestion pattern with decreasing amounts of the indicated fusion protein and 1 microgram of plasmid DNA.
• Each protein was serially diluted in two-fold increments in nuclease free water from 500ng to 4ng of enzyme. To each sample, was added 1 ug PDG plasmid DNA, incubated for 30 minutes at 37 degrees.
• Figure 11c shows the resits of an in gel DNase enzyme activity assay using commercially available DNase 1 (Biolabs, Inc.).
• the far right lane is a negative control with the DNA alone and no enzyme, the lane to the left of that is another negative control, this is an RNase-Ig molecule which has RNase activity but no DNase activity, as expected the plasmid DNA remains intact and is not digested in both cases.
• the results demonstrate that commercially available DNase 1 enzyme is highly active and digests all the DNA at most of the concentrations tested.
• Figures 11a and 1 lb show the DNase activity of four different nuclease Fc fusion constructs.
• the top panel in Figure 11a shows the ability of a DNase-Ig fusion protein to digest the plasmid DNA, as is apparent from the gel, this enzyme digests all the DNA at all concentrations tested is as active, or more active than commercially available DNase 1.
• the lower panel of Figure 1 la is a bi-specific nuclease Fc fusion protein with the DNase on the amino terminus of the Fc (SED ID 65-66) and it also has robust DNase enzymatic activity, but a little less than the DNase-Ig in the upper panel of Figure 11a.
• This bi-specific nuclease also has good DNase activity but it appears to be somewhat less than the other two bi-specific nuclease Fc fusion proteins shown in this experiment.
• This data suggests that all of the bi-specific nucleases have good DNase activity, which is an unexpected given the past efforts of others in this regard (Dwyer et al. JBC; Vol 271, No. 14; pp 9738-9743).
• the position of the DNase in the construct as well as the linker length and composition connecting the DNase to the Fc are critical in creating a highly active DNase enzyme in the context of a bi-specific nuclease Fc fusion protein.
• Figures 12-13 show results from a kinetic fluorescence enzyme activity assay comparing the RNase enzyme activity of recombinant RNase A (Ambion), RSLV 125, RSLV 126, hRNase WT-SCCH-WThlgGl, and hRNaseG88D-SCCH-(P238S/K322S/P331S)hIgGl .
• RNase A recombinant RNase A
• RSLV 125 RSLV 126
• hRNase WT-SCCH-WThlgGl hRNaseG88D-SCCH-(P238S/K322S/P331S)hIgGl
• hRNaseG88D-SCCH-(P238S/K322S/P331S)hIgGl hRNaseG88D-SCCH-(P238S/K322S/P331S)hIgGl .
• Figure 12 shows the RFU (relative fluorescence units) versus time for each protein at equimolar concentrations, with the test proteins at 4.5 pg/ul or 4.5 ng/ml, and the recombinant RNaseA control at 1.3 pg/ul in the presence of 200 nM RNase Alert substrate.
• Figure 13 shows a Lineweaver Burk plot of the different molecules.
• RNase Alert kinetic fluorescence assays were set up using 105pM enzyme, and the substrate concentration was decreased from 200nM to 50 pM in four fold increments. Thus, the enzyme concentration was fixed and substrate concentration titrated in this series of experiments.
• the data show the Lineweaver Burk plots generated for the different fusion proteins under these conditions. Taken together the data in Figures 12 and 13 demonstrate that the RNase moieties are highly active in the three RNase Fc fusion proteins constructed and tested here.
• Example 14 Assessment of in vitro cytotoxicity against human THP-1 cell line
• Figure 15 presents the results of fluorescence- activated cell sorting (FACS) experiments to examine the cytotoxicity of RNase Fc fusions constructs with a wild type or mutant Fc domain (RNase-wtlgG or RNase-mtlgG
• IC immune complexes
• normal human serum does not stimulate the production of interferon.
• the IC are incubated with normal human peripheral blood mononuclear cells (PBMC's) as reporter cells.
• PBMC's peripheral blood mononuclear cells
• the production of interferon by the reporter cell sis measured using an interferon-a ELISA.
• Reporter cells were obtained from normal volunteers by Ficoll density gradient centrifugation. Lupus patient or healthy normal volunteer serum was obtained under University of Washington Institutional Rreview Board #HSD No. 3971, the serum was diluted 1/1000 and added to 10% (v/v) of necrotic cell extract (NCE) derived from cultured U937 cells as above.
• NCE necrotic cell extract
• Diluted lupus patient or normal volunteer serum was incubated with the NCE for 15 minutes at room temperature, the resulting IC's were incubated with or without various doses of RSLV-132, RSLV-124, or wild type RNase for 15 minutes then incubated with normal PBMC's for 20 hours in the presence of 500 U/mL Universal IFN followed by measurement of the amount of IFN secreted from the PBMC culture.
• Serum was obtained from three (3) different lupus patients with various degrees of disease activity ranging from mild to active.
• the NCE was incubated with either lupus patient serum or healthy normal volunteer serum at room temperature for 15 minutes followed by 20 hour incubation with PBMC's.
• the test article (RSLV-124 or RSLV-132) was added at concentrations of 0.16, 0.5, 1.6 and 5.0 ug/mL or RNAse was added at concentrations of 0.05, 0.16, 0.5 and 1.6 ug/mL (equimolar) to the NCE.
• the ability of the lupus patient serum to stimulate production of IFN from the PBMC's was reduced by approximately 50% with the addition of 5.0 ug/mL RSLV-124. This inhibition mirrored that of an equimolar quantity of RNAse.
• RSLV-132 binds to its molecular target, the RNA associated with the lupus patient IC's and potently degrades it, thereby preventing the stimulation of IFN from the PBMC's ( Figure 16).
• RSLV-132 appears to be more active than RSLV-124 in this assay.
• RSLV-132 is a potent in vivo inhibitor of RNA-induced Interferon activation.
• poly I:C polyinosinic:polycytidilic acid
• Poly(LC) is a mismatched double-stranded RNA with one strand being a polymer of inosinic acid, the other a polymer of cytidylic acid. It is know to interact with toll-like receptor 3 (TLR3) which is expressed in the membrane of B cells, macrophages and dendritic cells.
• TLR3 toll-like receptor 3
• Poly(LC) is available from Invitrogen. The effects of poly I:C can be quantitated by measuring the expression levels of interferon stimulated genes in the spleen of the mouse following administration.
• mice On day zero 10 B6 mice, three months of age, were treated with either RSLV-132 (250ug per mouse) or intravenous immunoglobulin (IVIG) (Privigen, Behring) (250ug per mouse) as a control, both via intraperitoneal injection. Twenty hours after injection of RSLV-132 or IVIG, poly(LC) was injected into the mice at 200ug per mouse intraperitoneally. Two hours later, the animals were sacrificed by C02 exposure, the spleens were collected into RNAlater (Qiagen) and stored at -80C for later study of the expression of Interferon stimulated genes (ISGs).
• ISGs Interferon stimulated genes
• RSLV-132 and RSLV-133 were transiently expressed in CHO cells and purified using protein-A.
• the RNase activity of these RNase Fc fusion proteins was quantitated using the RNaseAlert QC kit from Ambion (Cat # AM 1966).
• Various amounts of the RNase Fc fusion protein were used and the results shown in Figure 18 in relative fluorescence units (RFU's) over time.
• the results demonstrate that RSLV-132 is a highly active RNase enzyme, and has increased RNase activity relative to other RNase Fc fusion constructs such as RSLV-124 and wild type RNase For example using equal amounts (400pM) of RSLV132 and RSLV-124 yields more than twice the RFU's (80,000 vs.
• RSLV-132 vs. RSLV-124.
• two production lots were tested for their stability at 4C.
• RSLV- 132.1 was stored at 4C for 8 weeks prior to this experiment and RSLV-132.2 was stored at - 80C and thawed just prior to testing, demonstrating that the protein is stable at 4C for up to 2 months.
• the stability of the drug and increased catalytic activity may provide increased efficacy in a therapeutic setting.
• Figure 19 shows the RNase enzymatic activity in RFUs over time, comparing the amount of RNase activity of the bi-specific RSLV-133 molecule with the monospecific RSLV-132, and wild type RNase.
• the RSLV-133 molecule has significantly increased RNase activity relative to the monospecific RSLV-124 molecule or, an earlier bispecific nuclease Fc, RSLV-123, or wild type RNase, yielding greater than 2-fold more RFU's with an equal amount of protein.
• Figure 20 show the results of a DNase enzymatic activity assay of the RLSV-133 molecule in comparison to RSLV-123, a previous bi-specific nuclease construct, and wild type DNase.
• Figure 21 demonstrates the ability of the RSLV-133 molecule to digest DNA in an in-gel digestion experiment.
• the results show that RSLV-133 is able to digest DNA in this assay as effectively as wild type DNase (compare lanes 5&7). Given the relative molecular weights of RSLV-133 and wild type DNase it appears that RSLV-133 is more effective in digesting DNA in this assay as well.
• Example 18 RSLV-132 Demonstrates decreased Fc Receptor binding.
• RSLV124 wild type Fc domain
• RSLV-132 mutant Fc domain; P238S/P331S
• FACS fluorescence-activated cell sorting
• Example 19 In vitro assessment of hybrid nuclease molecule biological activity.
• hybrid nuclease molecules are purified, e.g., by affinity or ion exchange chromatography as previously described in the examples above.
• the hybrid nuclease molecule is a polypeptide.
• the hybrid nuclease molecule includes one or more sequences from Table 1.
• the hybrid nuclease molecule includes a nuclease domain linked to a mutant Fc domain.
• the hybrid nuclease molecule includes a mutant Fc domain.
• the mutant Fc domain comprises mutations in the hinge, CH2, and/or CH3 domains.
• the mutant Fc domain comprises P238S and/or P331S, and may include mutations in one or more of the three hinge cysteines. In some instances, the mutant Fc domain comprises P238S and/or P331S, and/or mutations in the three hinge cysteines. In some instances, the mutant Fc domain comprises P238S and/or P331S, and/or mutations in the three hinge cysteines to SSS. In some instances, the mutant Fc domain comprises P238S and P331 S and mutations in the three hinge cysteines. In some instances, the mutant Fc domain comprises P238S and P331S and SSS. In some instances the mutant Fc domain is shown in SEQ ID NOs 59, 60, 61.
• PBMC normal or patient human PBMC (approximately lxl 0e6 cells) are cultured for 24, 48, or 96 hours depending on the assay. PBMC are cultured in the presence of stimuli such as TLR ligands, costimulatory antibodies, immune complexes, and normal or autoimmune sera.
• stimuli such as TLR ligands, costimulatory antibodies, immune complexes, and normal or autoimmune sera.
• the effects of the molecules on cytokine production is measured using commercially available reagents, such as the antibody pair kits from Biolegend (San Diego, CA) for IL-6, IL-8, IL-10, IL-4, IFN-gamma, TNF-alpha.
• Culture supernatants from in vitro cultures are harvested at 24, 48 hours or later time points to determine the effects of the molcules on cytokine production.
• IFN-alpha production is measured using, e.g., anti-human IFN-alpha antibodies and standard curve reagents available from PBL interferon source (Piscataway, NJ). A similar set of assays is performed using human lymphocyte
• subpopulations isolated monocytes, B cells, pDCs, T cells, etc.
• purified using, e.g., commercially available magnetic bead based isolation kits available from Miltenyi Biotech (Auburn, CA).
• lymphocyte activation receptors such as CD5, CD23, CD69, CD80, CD86, and CD25
• PBMC or isolated cell subpopulations are subjected to multi-color flow cytometry to determine how these molecules affect the expression of different receptors associated with immune cell activation.
• Another assay will measure the cytotoxicity of one or more molecules. This assay will measure toxicity using Annexin 5 staining (e.g., Annexin 5-FITC).
• Cells of interest e.g., monocytes or a monocyte cell line
• a hybrid nuclease molecule of interest e.g., a hybrid nuclease molecule having a mutant Fc domain
• cells are separated from culture and stained with Annexin 5.
• the number of apoptotic or dead cells are then counted, e.g., using flow cytometry or a fluorescence microscope.
• Cells contacted with a hybrid nuclease molecule of interest show lower numbers of cells staining positive for Annexin 5 compared to positive controls.
• hybrid nuclease molecule The effectiveness of a hybrid nuclease molecule is demonstrated by comparing the results of an assay from cells treated with a hybrid nuclease molecule disclosed herein to the results of the assay from cells treated with control formulations. After treatment, the levels of the various markers (e.g., cytokines, cell-surface receptors, proliferation) described above are generally improved in an effective molecule-treated group relative to the marker levels existing prior to the treatment, or relative to the levels measured in a control group.
• markers e.g., cytokines, cell-surface receptors, proliferation
• Example 20 Administration of a hybrid nuclease molecule to a mammal in need thereof.
• the mutant Fc domain comprises mutations in the hinge, CH2, and/or CH3 domains. In some instances, the mutant Fc domain comprises P238S and/or P331S, and may include mutations in one or more of the three hinge cysteines. In some instances, the mutant Fc domain comprises P238S and/or P331 S, and/or mutations in the three hinge cysteines. In some instances, the mutant Fc domain comprises P238S and/or P331S, and/or mutations in the three hinge cysteines to SSS. In some instances, the mutant Fc domain comprises P238S and P331S and mutations in the three hinge cysteines. In some instances, the mutant Fc domain comprises P238S and P331 S and SSS.
• the mutant Fc domain is shown in SEQ ID NOs 59, 60, 61.
• the hybrid nuclease molecule is shown in SEQ ID NOs.
• Various linker domains can be used to link the Fc domains to nuclease domains.
• linker domains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more amino acids in length can be used.
• the hybrid nuclease molecule is formulated a pharmaceutically acceptable carrier.
• the molecule is formulated as described in the pharmaceutical compositions section above.
• the hybrid nuclease molecule targets RNase and/or DNase.
• a suitable first dose of a hybrid nuclease molecule is administered to the subject.
• the hybrid nuclease molecule is formulated as described herein.
• the subject's condition is evaluated, e.g., by measuring IFN-alpha levels, IFN-alpha response gene levels, autoantibody titers, kidney function and pathology, and/or circulating immune complex levels. Other relevant criteria can also be measured.
• the number and strength of doses are adjusted according to the subject's needs.
• the subject's IFN-alpha levels, IFN-alpha response gene levels, autoantibody titers, kidney function and pathology, and/or circulating immune complex levels are lowered and/or improved relative to the levels existing prior to the treatment, or relative to the levels measured in a similarly afflicted but untreated/control subject.
• a rodent subject in need of treatment is selected or identified.
• the identification of the subject can occur in a laboratory setting or elsewhere.
• a suitable first dose of a hybrid nuclease molecule is administered to the subject.
• the hybrid nuclease molecule is formulated as described herein.
• the subject's condition is evaluated, e.g., by measuring IFN-alpha levels, IFN-alpha response gene levels, autoantibody titers, kidney function and pathology, and/or circulating immune complex levels. Other relevant criteria can also be measured.
• the number and strength of doses are adjusted according to the subject's needs.
• the subject's IFN-alpha levels, IFN-alpha response gene levels, autoantibody titers, kidney function and pathology, and/or circulating immune complex levels are lowered and/or improved relative to the levels existing prior to the treatment, or relative to the levels measured in a similarly afflicted but untreated/control subject.
• RSLV125-2 atggaaacccctgcccagctgctgttcctgctgctgctgtggctgcccgacacc huVK3LP- accggtaaggaatcccgggccaagaaattccagcggcagcatatggactcagac wthRNase- agttcccccagcagcagctccacctactgtaaccaaatgatgaggcgccggaat atgacacaggggcggtgcaaaccagtgaacacctttgtgcacgagcccctggta SCC-mthlgGl
• RSLV127-2 metpaqllfllllwlpdttglkiaafniqtfgetkmsnatlvsyivqilsrydi huVK3LP- alvqevrdshltavgklldnlnqdapdtyhy vseplgrnsykerylfvyrpdq hDNasel vsavdsyyddgcepcrndtfnrepfivrffsrftevrefaivplhaapgdava eidalydvyldvqekwgledvmlmgdfnagcsyvrpsqwssirlwtsptfqwli 105/114- pdsadttatpthcaydrivvagmllrga vpdsalpfnfqaayglsdq
• RSLV128-2 metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspssstycnqmmrrrn huVK3LP- mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn hRNase WT- gsrypncayrtspkerhiivacegspyvpvhfdasvedstdlsggggsggggsg gggsggggstglepkssdkthtcppcpapellggssvflfppkpkdtlmisrtp (g4s) 4-SCC- evtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhq m
• RSLV129-2 metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspssstycnqmmrrrn huVK3LP- mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn hRNAseWT- gsrypncayrtspkerhiivacegspyvpvhfdas edstlepkssdkthtcpp scc- cpapellggssvflfppkpkdtlmisrtpevtcv vdvshedpevkfnwyvdgv evhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpasiekti mthlg
• RSLV132 metpaqllfllllwlpdttgkesrakkfqrqhmdsdsspssstycnqmmrrrn huVK3LP- mtqgrckpvntfvheplvdvqnvcfqekvtckngqgncyksnssmhitdcrltn wthRNase- gsrypncayrtspkerhiivacegspyvpvhfdasvedstlepkssdkthtcpp cpapellggssvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgv SCC-mthlgGl

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