JP2007312781A - Human dnase variants - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide amino acid sequence variants of human DNase I that have reduced binding affinity for actin. <P>SOLUTION: The invention provides nucleic acid sequences encoding such actin-resistant variants, thereby enabling the production of these variants in quantities sufficient for clinical use. The invention also relates to pharmaceutical compositions and therapeutic uses of actin-resistant variants of human DNase I. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to results obtained from studies on human deoxyribonuclease I (DNase I), a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid. The present invention generally relates to modified (mutant) forms of human DNase I and their preparation by recombinant DNA methods, pharmaceutical compositions by which their utility can be developed clinically, and these DNases. The invention relates to methods using I variants and compositions thereof.

  DNase I is a phosphodiesterase that can hydrolyze polydeoxyribonucleic acid. DNase I has been purified to varying degrees from many species.

Bovine DNase I has been extensively studied biochemically. See, for example, Moore, The Enzyme (Boyer, PD), pages 281-296, Academic Press, New York (1981). The complete amino acid sequence for bovine DNase I is known (Liao et al., J. Biol. Chem., 248 : 1489-1495 (1973); Oefner et al., J. Mol. Biol. 192 : 605-632 (1986). Lahm et al., J. Mol. Biol. Biol. 221 : 645-667 (1991), DNA encoding bovine DNase I has been cloned and expressed (Worrall et al., J. Biol. Chem. 265 : 21889-21895 (1990)) The structure of bovine DNase I has been determined by X-ray crystallography Suck et al., EMBO J. 3: 2423-2430 (1984); Suck et al., Nature 321 : 670-625 (1986); Oefner et al., J. Mol. Biol. 192 : 605-632 (1986)).

DNA encoding human DNase I has been isolated and sequenced, and the DNA has been expressed in a recombinant host cell, thereby allowing production of human DNase I in commercially useful quantities. To do. Shak et al., Proc. Nat. Acad. Sci. 87 : 9188-9192 (1990).

DNase I has a number of known uses and has been used for therapeutic purposes. Its main therapeutic use was to reduce the viscoelasticity of pulmonary secretion (mucus) in diseases such as pneumonia and cystic fibrosis (CF), thereby helping airway cleaning. For example, Lourenco et al., Arch. Intern., Med. 142 : 2299-2308 (1982); Shak et al., Proc. nat. Acad. Sci . 8 7: 9188-9192 (1990); Hubbard et al., New Engl. J. Med. 326 : 812-815 (1992); Fuchs et al., New Engl. J. Med. 321 : 637-642 (1994); Bryson et al., Drugs 48 : 894-906 (1994). Mucus also contributes to the prevalence of chronic bronchitis, asthmatic bronchitis, bronchiectasis, emphysema, acute and chronic sinusitis, and common colds.

  The pulmonary secretion of individuals with such diseases is a complex substance, which includes mucus glycoproteins, mucopolysaccharides, proteases, actin and DNA. Some of the substances in the pulmonary material are those of microorganisms (eg, strains of Pseudomonas, Pneumocossus, or Staphylococcus) or other stimulants (eg, tobacco smoking, pollen) Released from white blood cells (neutrophils) that infiltrate lung tissue in response to their presence. While reacting with such microorganisms or stimulants, leukocytes denature and release their contents, which contribute to the viscoelastic properties of pulmonary secretion.

DNase I's ability to reduce pulmonary secretion viscoelasticity has been attributed to its enzymatic degradation of large amounts of DNA released by neutrophils. Shak et al., Proc. Nat. Acad. Sci. 87 : 9188-9192 (1990)); Aitken et al., J. Biol. Am. Med. Assoc. 267 : 1947-1951 (1992)).

More recently, various mechanisms have been proposed for the mucolytic effect of DNase I including actin disaggregation. Vasconcellos et al., Science 263 : 969-971 (1994)). Actin is one of the most abundant proteins in eukaryotic cells (eg, actin consists of about 10% of total leukocyte protein) and has been extensively studied. Kabsch et al., Ann. Rev. Biophys. Biomol. Struct. 21 : 49-76 (1992); Sheterline et al., Proc. Profile 1 : 1-121 (1994). Actin exists in two forms, a monomeric form (G-actin) and a filamentous form assembled from G-actin monomers (F-actin). Actin polymer filaments are highly viscoelastic and contribute significantly to the viscosity of pulmonary secretion. Mornet et al., Proc. Nat. Acad. Sci. 81 : 368 0-3684 (1984); Newman et al., Biochemistry 24 : 1538-1544 (1985); Janmey et al., Biochemistry 27 : 8218-8226 (1988); Vasconcellos et al., Science 263 : 969-971 (1994).
Moore, The Enzyme (Boyer, PD), 281-296, Academic Press, New York (1981)

DNase I binds to actin (Lazarides et al., Proc. Nat. Acad. Sci. 71 : 4 742-4746 (1974); Kabsch et al., Nature 347 : 37-44 (1990)) and depolymerizes actin filaments. (As well as inhibiting the polymerization of G-actin into filaments) (Mannherz et al., FEBS Lett. 60 : 34-38 (1975); Hitchcock et al., Cell 7 : 531-542 (1976); Pinder et al., Biochemistry 21 : 4886-4890 (1982); Weber et al., Biochemistry 33 : 4780-4786 (1994)), so that the mucolytic effect of DNase I on sputum and other pulmonary secretion is rather than DNA hydrolysis Proposed by actin disaggregation (depolymerization). Vasconcellos et al., Science 263 : 969-971 (1994). Consistent with this view, it is known that the DNA-hydrolysis activity of DNase I is inhibited in the presence of actin. Lazarides et al., Proc. Nat. Acad. Sci. 71 : 4742-4746 (1974); Mannherz et al., Eur. J. Biochem. 104 : 367-379 (1980). Consistent with this view, it has been reported that actin-cleaving proteins (eg, glucolin) are effective in reducing the viscoelasticity of cystic fibrosis sputum. Vasconcellos et al., Science 263 : 969-971 1994); Stossel et al., PCT application publication WO 94/22465 (published October 13, 1994).

  The present invention is based, in part, on studies by the inventors that measure the biochemical basis of DNase I mucolytic activity. This study addresses the design and synthesis of various human DNase I mutants and their ability to hydrolyze DNA, bind to actin, and reduce the viscoelasticity of pupae in vitro. Of the assay. We have created several classes of human DNase I variants. A class of mutants (actin-resistant mutants) have reduced ability to bind to actin but still have mucolytic activity and in some cases reduced mucolysis compared to native human DNase I Had activity. These actin-resistant mutants have approximately the same DNA-hydrolysis activity as native human DNase I, but such activity was less sensitive to inhibition by actin. The second class of variants binds to actin with similar affinity as found in native human DNase I, but has reduced mucolytic activity and reduced DNA compared to native human DNase I. -It had hydrolytic activity.

  These results indicate that the therapeutic effect of human DNase I in reducing pulmonary secretion viscoelasticity is due to its catalytic DNA-hydrolysis activity rather than its ability to depolymerize filamentous actin. . Thus, variants of human DNase I that bind to actin with lower affinity than native human DNase I but still retain DNA-hydrolysis activity are particularly patients with pulmonary secretions consisting of relatively large amounts of actin. It should be a useful therapeutic agent in the treatment of Since such mutants have a reduced affinity for actin, their DNA hydrolytic activity is less inhibited in the presence of actin, and therefore these mutants are compared to native human DNase I, Has greater mucolytic activity in the presence of actin.

  Accordingly, it is an object of the present invention to provide a human DNase I variant that retains DNA-hydrolysis activity but binds to actin with a lower affinity than native human DNase I.

  Another object of the present invention is to provide nucleic acids encoding such actin-resistant mutants of human DNase I, recombinant vectors comprising such nucleic acids, recombinant host cells transformed with these nucleic acids or vectors, and sets It is to provide a method for producing a human DNA variant by a replacement DNA technique.

  The present invention is also directed to a pharmaceutical composition comprising a human DNase I actin-resistant mutant, optionally with a pharmaceutically acceptable excipient.

  The present invention is also directed to a method of reducing the viscoelasticity or viscosity consistency of a DNA-containing substance in a patient, comprising administering to the patient a therapeutically effective amount of an actin-resistant mutant of DNase I.

  The present invention particularly relates to cystic fibrosis, chronic bronchitis, pneumonia, bronchiectasis, emphysema, asthma, or administration comprising administering to a patient a therapeutically effective amount of an actin-resistant mutant of DNase I It is directed to a method of treating patients with illnesses such as systemic lupus erythematosus.

  The present invention also measures the amount of actin present and determines whether the patient is a suitable candidate for treatment with an actin-resistant DNase I variant (粘性) from the patient. Directed toward the use of actin-resistant mutants of human DNase I in in vitro diagnostic assays.

  These and other objects of the invention will be apparent to those skilled in the art when the specification is considered in its entirety.

  I. Definition

  As used herein, the terms “human DNase I”, “natural human DNase I”, and “wild-type DNase I” are polypeptides having the amino acid sequence of human mature DNase I described in FIG. Say.

A “variant” or “amino acid sequence variant” of human DNase I is a polypeptide consisting of an amino acid sequence different from that of native human DNase I. In general, variants have at least 80% sequence identity (homology) with native human DNase I, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, most preferably at least 98%. Possesses the same sequence identity. Percent sequence identity can be determined, for example, by aligning sequences so that maximum homology is provided, followed by Fitch et al., Proc. Nat. Acad. Sci. USA 80 : 1382-1386 (1983) Needleman et al. Mol. Biol. 48 : The version of the algorithm described by 443-453 (1970)).

  The terms “human DNase I-resistant mutant”, “actin-resistant mutant”, and “actin-resistant mutant of human DNase I” are (1) DNA-hydrolytic activity and (2) against actin. A variant of natural human DNase I with reduced binding affinity.

“DNA-hydrolyzing activity” refers to the enzymatic activity of natural human DNase I or a variant of human DNase I in the production of a 5′-phosphorylated oligonucleotide terminal product by hydrolyzing (cutting) the substrate DNA. Say. DNA-hydrolysis activity was determined by analytical polyacrylamide and agarose gel electrophoresis, increased light absorption assay (Kunitz, J. Gen. Physiol., 33 : 349-362 (1950); Kunitz, J. Gen. Physiol. 33 : 363 -377 (1950)), or methyl green assay (Kurnick, Arch. Biochem. 29 : 41-53 (1950); Sinicropi et al., Anal. Biochem. 222 ; 351-35 (1994)) Is easily measured by one of several different methods.

The “binding affinity” of natural human DNase I or actin-resistant mutants of human DNase I to actin refers to the ability of DNase I to bind to actin non-covalently. Binding affinity can be determined, for example, by Mannherz et al., Eur. J. Biochem. 104 : 367-379 (1980), which can be measured by any of a variety of methods known in the art. Alternatively, the relative binding affinity of different DNases (eg, native human DNase I and variants thereof) is determined by the binding of DNase to immobilized actin in an ELISA assay (described in Example 3). Determined by measuring or by comparing the DNA-hydrolysis activity of DNase in the presence or absence of actin (also described in Example 3). The methods described in the Examples are particularly convenient for screening variants of human DNase I to rapidly identify variants with reduced binding affinity for actin.

Human DNase I actin-resistant mutants with “reduced binding affinity for actin” are against actin which is relatively lower in affinity than native human DNase I binds to actin, as measured under comparable conditions. It has binding affinity. If the actin-binding ELISA assay described in Example 3 is used to determine the binding affinity of human DNase I (natural or mutant) to actin, an actin having “reduced binding affinity for actin” — Resistant variants are those that have an EC ₅₀ value greater than that of native human DNase I. In that assay, actin-resistant mutants typically have an EC ₅₀ value that is 5- to 100-fold greater than that of native human DNase; however, in particular, of the native human DNase I amino acid sequence. By modifying multiple amino acid residues (see FIGS. 5A and 5D), actin-resistant mutants with EC ₅₀ values that are 500-fold greater than that of native human DNase I are also readily produced.

“Mucinolytic activity” refers to, for example, a reduction in viscoelasticity (viscosity) of sputum or other biological material observed when the material is treated with natural human DNase I or a variant of human DNase I. Say. Mucolytic activity is determined by a sputum compression assay (PCT patent application WO94 / 10567 published on May 11, 1994), an assay using a torsion pendulum (Janmey. J. Biochem. Biophys. Methods 22 : 41-53 (1991) Or easily measured by any of a number of different methods known in the art, including other rheological methods.

  “Polymerase chain reaction” or “PCR” generally refers to a method for in vitro amplification of a desired sequence as described, for example, in US Pat. No. 4,683,195. In general, PCR methods involve repeated cycles of primer extension synthesis using an oligonucleotide that can preferentially hybridize to a template nucleic acid.

  “Cells”, “host cells”, “cell lines” and “cell cultures” are used interchangeably herein and such terms should be understood to include progeny obtained from cell growth or culture. It is. “Transformation” and “transfection” refer to the process of introducing DNA into a cell and are used interchangeably.

  “Operably linked” refers to the covalent linking of two or more DNA sequences by enzymatic ligation or other methods in mutual arrangement such that the normal function of the sequence can be performed. For example, the DNA for a presequence or secretory leader is operably linked to the polypeptide DNA if it is expressed as a preprotein involved in polypeptide secretion; the promoter or enhancer is responsible for the transcription of the sequence. If affected, it is operably linked to the coding sequence; or the ribosome binding site is operably linked to the coding sequence if it is positioned to facilitate transcription. In general, “operably linked” means that the DNA sequences to be linked are adjacent and, in the case of a secretory leader, are adjacent and in the reading phase. Ligation is achieved by ligation at normal restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are typically used in combination with recombinant DNA methods.

Here, amino acids are confirmed by the three-letter or single-letter display as follows.
Asp D Aspartate Ile I Isoleucine
Thr T Threonine Leu L Leucine
Ser S Serine Tyr Y Tyrosine
Glu E Glutamic acid Phe F Phenylalanine
Pro P Proline His H Histidine
Gly G Glycine Lys K Lysine
Ala A Alanine Arg R Arginine
Cys C Cysteine Trp W Tryptophan
Val V Valin Gln Q Glutamine
Met M Methionine Asn N Asparagine

  II. Selection of actin-resistant mutants

  The present invention is based on the study of the structure, actin binding properties, DNA-hydrolysis activity, and mucolytic activity of the amino acid sequence variant of human DNase I. The actin-resistant mutants of the present invention have DNA-hydrolysis activity but bind to actin with a lower affinity than native human DNase I. The decrease in actin binding is preferably, for example, natural human DNase I Glu13, His44, Leu45, Val48, Gly49, Leu52, Asp53, Asn56, Asp58, His64, Tyr65, Val66, Val67, Ser68, Glu69, Pro70, Ser94. , Tyr96 and Ala114 residues (numbers following the three letter amino acid designation indicate the specific position of the amino acid residues in the sequence of FIG. 1) within the natural human DNase I likely to affect actin binding. This is accomplished by making mutations at and / or around amino acid residues.

  There are various ways in which actin-resistant mutants of human DNase I can be generated. In one embodiment of the invention, the actin-resistant variant is a single at or adjacent to (ie within about 5 amino acid residues of) native human DNase I amino acids that affect actin binding. Or prepared by introducing multiple amino acid substitutions, insertions, and / or deletions. Some illustrative examples of such mutations are: D53R, D53K, D53Y, D53Y, D53A, Y65A, Y65E, Y65R, V67E, V67K, E69R, D53R: Y65A, D53R: E69R, H44A: D53R. : Y65A, H44A: Y65A: E69R (see FIG. 2-6).

  In another embodiment of the invention, the actin-resistant variant is new at or adjacent to (ie within about 5 amino acid residues of) native human DNase I amino acids that affect actin binding. Prepared by introducing mutations that result in glycosylation sites. For example, using site-directed mutagenesis, one tripeptide sequence, asparagine-X-serine or asparagine-X-threonine, which is a recognition sequence for enzyme attachment to the asparagine side chain of a carbohydrate site (where X introduces any amino acid except proline). Creghton, Proteins, pp. 76-78 (W. H. Freeman, 1984). The steric hindrance that occurs between the resulting N-glycosylated variant DNase I and the carbohydrate site of actin is inhibited as a result of actin binding and DNase I DNA-hydrolysis activity compared to native human DNase I. Reduce or hinder. Some illustrative examples of such mutations to introduce new glycosylation sites are as follows: H44N, D58S, D58S, V66N, H44N: T46S, H64N: V66S, H64N: V66T, Y65N: V67S, Y65N: V67T, V66N: S68T, V67N: E69S, V67N: E69T, S68N: P70S, S68N: P70T, S94N: Y96S, S94N: Y96T.

  Optionally, positions 18 and / or 106 within the native human DNase I amino acid sequence, depending on the degree of glycosylation desired in the actin-resistant mutant, in combination with such a mutation to create a new glycosylation site It is also possible to delete naturally occurring glycosylation sites in

  In a further embodiment of the invention, amino acid residues of native human DNase I involved in actin binding suitable for post-translational modification biologically or chemically (see below) using site-directed mutagenesis Or adjacent (ie within about 5 amino acid residues thereof). Means et al., “Chemical modification of proteins (Holden-Day, 1971); Glazer et al.,“ Chemical modification of proteins: selected methods and analytical techniques (Elsevier, 1975); Creighton, Proteins, 70-87 (WHFreeman, 1984). Lundblad, “Chemical Reagents for Protein Modification” (CRC Press, 1991). Such post-translational modifications can introduce steric hindrance or altered electrostatic properties to DNase I, which is a result of inhibition of actin binding and DNA-hydrolysis activity compared to native human DNase I. Reduce or hinder. For example, cysteine residues can be introduced at or adjacent to the residues of natural human DNase I involved in actin binding. The free thiol of the cysteine residue can form an intermolecular disulfide bond with another such DNase I variant to form a DNase I dimer, or it can be modified, for example, with a thiol-specific alkylating agent. . Some illustrative examples of such mutations are as follows: H44C, L45C, V48C, G49C, L52C, D53C, N56C, Y65C, V67C, E69C, A114C.

  For convenience, substitutions, insertions and / or deletions in the amino acid sequence of native human DNase I are usually nucleotide sequences corresponding to DNA encoding native human DNase I, for example by site-directed mutagenesis. Created by introducing mutations into Mutant DNA expression then results in a mutant human DNase I having the desired (non-natural) amino acid sequence.

Site-directed mutagenesis using any technique known in the art, such as disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, New York (1989)). Although it can be performed, oligonucleotide-specific mutagenesis is the preferred method for preparing the human DNase I variants of the present invention. Well known this process is in the art (Zoller, et al., Meth.Enz.100: 4668-500 (1983); Zoller et al., Meth.Enz 154:.. 329-350 ( 1987); Carter, Meth, Enz 154 : 382-403 (1987); Kunkel et al., Meth. Enzymol 154 : 367-382 (1987); Horwitz et al., Meth, Enz. 185 : 599-611 (1990)) are particularly useful for creating substitution mutants. Although suitable, it can also be used to prepare deletion and insertion mutants for convenience.

Site-directed mutagenesis typically uses phage vectors that exist in both single-stranded and double-stranded forms. Typical vectors useful in site-directed mutagenesis include M13 phage and plasmid vectors containing a single-stranded phage origin of replication (Messing et al., Meth. Enzymol. 101 : 20-78 (1983); Veria et al., .. Meth Enzymol 153:. 3-11 (1987); Short et al., Nuc.Acids.Res 16: 7583-7600 (1988) ). As a result of replication of these vectors in a suitable host cell, single-stranded DNA is synthesized that can be used in site-directed mutagenesis.

  Briefly, in performing site-directed mutagenesis of DNA encoding native human DNase I (or a variant thereof), an oligonucleotide encoding the desired mutation is first hybridized to a single strand of DNA. To modify the DNA. After hybridization, a DNA polymerase is used to synthesize the entire second strand using the hybridized oligonucleotide as a primer and a single strand of DNA as a template. Thus, an oligonucleotide encoding the desired mutation is incorporated into the resulting double stranded DNA.

Oligonucleotides used as hybridization probes or primers can be prepared by any suitable method, such as by purification of naturally occurring DNA or by in vitro synthesis. For example, oligonucleotides are described in Narang et al., Meth, Enzymol. 68 : 90-98 (1979); Brown et al., Meth. enzymol. 68 : 109-151 (1979); Caruthers et al., Meth. Enzymol. 154 : 287-313 (1985) and is readily synthesized using various techniques in organic chemistry. General approaches to suitable hybridization probes or primers are well known. Keller et al., DNA Probes, 11-18 (stockton Press, 1989). Typically, a hybridization probe or primer contains 10-25 or more nucleotides, includes at least 5 nucleotides on either side of the sequence encoding the desired mutation, and the oligonucleotide is single stranded. Ensure preferential hybridization to the desired position relative to the DNA template molecule.

  Of course, multiple substitution, insertion or deletion mutations can be introduced into the starting DNA using site-directed mutagenesis. If the sites to be mutated are located close to each other, the mutations can be introduced simultaneously using a single oligonucleotide that encodes all of the desired mutations. However, if the sites to be mutated are some distance from each other (more than about 10 nucleotides apart), it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. It is. Instead, one of two alternative methods can be used.

  In the first method, a separate oligonucleotide is generated for each desired mutation. The oligonucleotide is then annealed simultaneously to the single stranded template DNA and the second strand of DNA synthesized from the template will encode all of the desired amino acid substitutions.

  Another method involves two or more rounds of mutagenesis to generate the desired mutant. The first round is described as introducing a single mutation. The second round of mutagenesis uses the mutant DNA generated in the first round as a template. Thus, this template contains all of one or more mutations. Oligonucleotides encoding additional desired amino acid substitutions are then annealed to this template, and the resulting strand of DNA now encodes mutations from both first and second rounds of mutagenesis. This resulting DNA is used in the third round of mutagenesis.

PCR mutagenesis (Higuchi, PCR Protocols, pp. 177-183 (Academic Press, 1990); Vallette et al., Nuc. Acids Res. 17 : 723-733 (1989) also produces mutants of human DNase I. In short, when a small amount of template DNA is used as a starting material in PCR, a primer sequence that is slightly different from the corresponding region in the template DNA is used, and the template sequence is only at a position where the primer is different from the template. A relatively large amount of a specific DNA fragment is obtained that differs from 1. For introduction of a mutation into plasmid DNA, for example, one sequence of primers contains the desired mutation and one of the plasmid DNA at the position of the mutation Designed to hybridize to the strand; the sequence of the other primers must be identical to the nucleotide sequence in the opposite strand of the plasmid DNA, but this sequence is It can be located anywhere along the Sumito DNA, however, the sequence of the second primer is eventually 200 nucleotides from that of the first so that the entire amplified region of DNA bordering the primer can be easily sequenced. PCR amplification using primer pairs such as those just described results in a population of DNA fragments that differ at the location of the mutation specified by the primer, possibly at other positions of the template. Copy is somewhat error-prone, Wagner et al., PCR Topics, pages 69-71 (Springer-Verlag, 1991).

  If the ratio of template to product amplified DNA is extremely low, the majority of product DNA fragments incorporate the desired mutation. This product DNA is used to replace the corresponding region in the plasmid that serves as a PCR template using standard recombinant DNA methods. Mutations at different positions can be performed using a mutant second primer, or a second PCR can be performed with a different mutant primer, and two resulting PCR fragments can be simultaneously ligated in three (or more) partial ligations Can be introduced at the same time.

Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene 34 : 315-323 (1985). The starting material is a plasmid (or other vector) consisting of the DNA sequence to be mutated. Identify the codons in the starting DNA to be mutated. There must be a unique restriction endonuclease site on each side of the identified mutation site. If such restriction sites do not exist, they can be generated by introducing them into the appropriate positions in the DNA using the oligonucleotide-mediated mutagenesis method described above. The plasmid DNA is cut at these sites to linearize it. A double stranded oligonucleotide encoding the sequence of DNA between the restriction sites but containing the desired mutation is synthesized using standard techniques, wherein the two strands of the oligonucleotide are synthesized separately, They are then hybridized together using standard techniques. This double-stranded oligonucleotide is called a cassette. This cassette is designed to have 5 'and 3' ends that match the ends of the linearized plasmid so that it can be directly ligated to the plasmid. The resulting plasmid contains the mutated DNA sequence.

The presence of mutations in DNA is measured by methods well known in the art, including restriction mapping and / or DNA sequencing. A preferred method for DNA sequencing is described by Sanger et al., Proc. Nat. Acad. Sci. This is the dideoxy chain termination method of USA 72 : 3918-3921 (1979).

  DNA encoding the human DNase I variant is inserted into a replicable vector for further cloning or expression. A “vector” is a vector and other DNA that can replicate in a host cell and is useful in performing two functions in combination with a host cell that is itself compatible (vector-host system). One function is to facilitate cloning of the nucleic acid encoding the human DNase I variant, ie, to generate an available amount of nucleic acid. Other functions direct the expression of human DNase I variants. One or both of these functions are performed by the vector in the particular host cell used for cloning or expression. To generate human DNase I mutants that contain different elements depending on the function they perform, the expression vector consists of DNA encoding the aforementioned mutant operably linked to a promoter and a ribosome binding site. Become. The mutant is then either directly in recombinant cell culture or a single sequence or other polypeptide having a specific cleavage site in the linkage between a heterologous peptide, preferably a heterologous polypeptide and a human DNase I variant. Expressed as a fusion with

  Prokaryotes (eg, E. coli and other bacteria) are preferred host cells for the initial cloning steps of the present invention. They are particularly useful in the rapid production of large amounts of DNA, the production of single-stranded DNA templates for use in site-directed mutagenesis, and the DNA sequencing of the resulting mutants. Prokaryotic host cells can also be used to express DNA encoding the human DNase I mutant. Polypeptides produced in prokaryotic cells are typically not glycosylated.

  In addition, the human DNase I variants of the present invention can be used to eukaryotic microorganisms (eg, yeast) or cells derived from animals or other multicellular organisms (Chinese hamster ovary cells, and other mammalian cells). It can be expressed in eukaryotic host cells, or in live animals (eg, chickens, goats, sheep).

  Cloning and expression methods are well known in the art. Prokaryotic and eukaryotic host cells, and expression vectors useful for producing the human DNase I variants of the present invention are described, for example, in Shak, PCT patent application WO90 / 07572 (published 12 July 1990). ).

A preferred method of transfection of cells with DNA using prokaryotic cells or cells containing substantial cell wall architecture as a host is described in Cohen et al., Proc. Natl. Acad. Sci. 69 : 2110-2114 (1972) or Chung et al., Nuc. Acids. Res. 16 : 3580 (1988). If yeast is used as the host, transfection is generally performed by Hinnnen, Proc. Natl. Acad. Sci. Accomplished using polyethylene glycol as taught by USA, 75 : 1929-1933 (1978). If mammalian cells are used as host cells, transfection is generally performed by the calcium phosphate precipitation method. Graham et al., Virology, 52 : 546 (1978), Gorman et al., DNA and Protein Eng. tech. 2 : 3-10 (1990). Other known methods for introducing DNA into prokaryotic and eukaryotic cells, such as nuclear injection, electroporation, or protoplast fusion, are also suitable for use in the present invention.

Particularly useful in the present invention are expression vectors that provide for transient expression in mammalian cells of DNA encoding a human DNase I variant. In general, transient expression is an expression vector that can be effectively replicated in the host cell so that the host cell accumulates multiple copies of the expression vector and synthesizes the high level of the desired polypeptide encoded by the expression vector. Including the use of Transient expression systems consisting of appropriate expression vectors and resident cells allow convenient positive identification of polypeptides encoded by cloned DNA and rapid screening of such polypeptides for desired biological or physiological properties. Make it possible. Wong et al., Science 228 : 810-815 (1985); Lee et al., Proc. Nat. Acad. Sci. USA 82 : 4360-4364 (1985); Yang et al., Cell 47 : 3-10 (1986). Thus, the transient expression system is combined with an assay for identifying variants that bind to actin with a lower affinity than native human DNase I as well as an assay for measuring variants with DNA-hydrolyzing activity. And is conveniently used to express DNA encoding amino acid sequence variants of native human DNase I.

  The human DNase I variant is preferably secreted from the host cell in which it is expressed, in which case the variant is recovered from the culture medium in which the host cell is grown. In that case, it may be desirable to grow the cells in serum-free medium. This is because the absence of serum proteins and other serum components in the medium facilitates purification of the mutants. If it is not secreted, the human DNase I variant is recovered from the lysate of the host cell. If the variant is expressed in a host cell other than of human origin, the variant is completely free of proteins of human origin. In any case, in order to obtain a substantially homogeneous preparation of the human DNase I variant, it is necessary to purify the variant from the recombinant cell protein. For therapeutic use, the purified variant will preferably be greater than 99% pure (ie, any other protein will comprise less than 1% total protein in the purified composition). ).

  In general, purification of a human DNase I variant is accomplished by taking advantage of the different physicochemical properties of the variant compared to the contaminant with which it may associate. For example, as a first step, the culture medium or host cell lysate is centrifuged to remove particulate cell contaminants. Thereafter, for example, ammonium sulfate or ethanol precipitation, gel filtration (molecular exclusion chromatography), ion exchange chromatography, hydrophobic chromatography, immunoaffinity chromatography (eg anti-human DNase I antibody coupled to Sepharose ), Tentacle cation exchange chromatography (Frenz et al., PCT patent application WO 93/25670, December 23, 1993), reverse phase HPLC and / or gel electrophoresis to contaminate human DNase I variants Purify from soluble proteins and polypeptides.

  Of course, those skilled in the art will appreciate that the purification methods used with native human DNase I are useful for purifying human DNase I mutants and explain structural and other differences between native and mutant proteins. In order to do so, some modification is necessary. For example, in some host cells (especially bacterial host cells), the human DNase I variant can be expressed in an initially insoluble and associated form (referred to in the art as “refractor” or “inclusion body”). In this case, it will be necessary to solubilize and renature the human DNase I variant during this purification. Methods for solubilizing and restoring recombinant protein refractive bodies are known in the art (see, eg, Builder et al., US Pat. No. 4,511,502).

  In another embodiment of the invention, the human DNase I variant is prepared by making a covalent modification directly in the natural or mutant human DNase I protein. Such modifications affect actin binding or other properties of the protein (eg, stability, biological half-life, immunogenicity), which replaces the amino acid sequence substitutions, insertions and deletions described above. Or you can do it in addition.

  Covalent modifications can be introduced by reacting the targeted amino acid residue of natural or mutant human DNase I with an organic derivatizing agent capable of reacting with a selected amino acid side chain or N- or C-terminal residue. Suitable derivatizing agents and methods are well known in the art.

  For example, cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines) such as chloroacetic acid or chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues include bromotrifluoroacetone, α-bromo-β- (5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, and methyl 2-pyridyl sulfide. , P-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

  The histidyl residue is derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0. This is because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

  Ricinyl and amino terminal residues are reacted with succinic acid or other carboxylic anhydrides. Derivatization with these agents has the effect of reversing the charge of the ricinyl residue. Other suitable reagents for derivatizing α-amino containing residues are methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzene sulfonic acid, O-methylisourea, 2,4-pentanedione, And transaminase-catalyzed imidoesters such as reaction with glyoxylate. Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be carried out under alkaline conditions due to the high pKa of the guanidine functional group. In addition, these reagents can be reacted with lysine groups as well as arginine epsilon amino groups.

  The carboxyl side group (aspartyl or glutamyl) is a carbodiimide such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylphenyl) carbodiimide. It is selectively modified by reaction with (R′—N═C═N—R ′) wherein R and R ′ are different alkyl groups. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

  Covalent coupling of glycosides to protein amino residues can be used to modify or increase the number or profile of carbohydrate substituents, particularly at or adjacent to residues involved in actin binding. Depending on the coupling mode used, the sugars may be (a) arginine and histidine, (b) free carboxyl groups, (c) free sulnohydryl groups such as those of cysteine, (d) those of serine, threonine, or hydroxyproline. Such as a free hydroxyl group, (e) an aromatic residue such as phenylalanine, tyrosine, or tryptophan, or (f) an amide group of glutamine. Suitable methods are described, for example, in PCT patent application WO 87/05330 (published September 11, 1987) and Aplin et al., CRC Crit. Rev. Biochem., Pages 259-306 (1981).

Covalent binding of agents such as polyethylene glycol (PEG) or human serum albumin to human DNase I variants reduces the immunogenicity and / or toxicity of the variant, as has been observed with other proteins, and / or Or extend its half-life. Abuchowski et al. Biol. Chem. 252 : 3582-3586 (1977); Poznansky et al., FEBS Letters 239 : 18-22 (1988); Goodson et al., Biotechnology 8 : 343-346 (1990); Katre, J. et al. Immunol. 144 : 209-213 (1990); Harris, Polyethylene Glycol Chemistry (Plenum Press, 1992). In addition, modification of native human DNase I or a variant thereof by these agents at or adjacent to (ie within about 5 amino acid residues of) amino acid residues that affect actin binding, Actin-resistant mutants can be obtained.

  In a further embodiment, the human DNase I actin-resistant mutant is a mutation in the Asn residue that occurs at position 74 of the native human DNase I amino acid sequence to reduce or prevent deamidation of the DNase I mutant. (Eg, N74D, N74K, or N74S mutation). Frenz et al., PCT patent application WO93 / 25670 (published December 23, 1993). As another example, a human DNase I actin-resistant mutant is an amino acid sequence that reduces the mutant's sensitivity to degradation by proteases (eg, neutrophil esterases) that may be present in sputum and other biological materials. Mutations or other covalent modifications can be included.

  The DNA-hydrolyzing activity and actin-binding affinity of human DNase I variants as described above are readily determined using assays and methods known in the art and described herein. Any such mutant having DNA-hydrolyzing activity (as defined above) and reduced binding affinity for actin is an actin-resistant mutant within the scope of the present invention.

  The human DNase I actin-resistant mutants of the present invention are used to reduce the viscoelasticity of DNA-containing materials such as sputum, mucus, or other secretions. Such mutants may be associated with lung disease with abnormal viscosity or dense secretion and acute or chronic lung disease including infectious pneumonia, bronchitis or tracheobronchitis, bronchiectasis, cystic fibrosis, asthma, tuberculosis and fungal infections. It is particularly useful in the treatment of patients with In such treatment, a solution of the actin-resistant variant or a micronized dry preparation is routinely infused into the patient's respiratory tract (eg, bronchi) or lung, eg, by aerosol treatment.

  Actin-resistant mutants are diseases such as empyema, meningitis, abscess, peritonitis, sinusitis, otitis, periodontitis, pericarditis, pancreatitis, cholelithiasis, endocarditis and septic arthritis It is also useful for additional treatment of abscesses or severe narrow-area infections and for local treatment of various inflammation and infection lesions such as skin and / or mucous membranes, surgical injuries, ulcerative lesions and burned lesions. Actin-resistant mutants can improve the efficiency of antibodies used in the treatment of such infections (eg, gentamicin activity is significantly reduced by reversible binding to intact DNA).

  Natural human DNase I and its actin-resistant mutants may also be useful in the treatment of systemic lupus erythematosus (SLE), a life-threatening autoimmune disease characterized by the production of various autoantibodies. DNA is a major antigen component of immune complications. In this case, human DNase I (natural or variant) can be administered systemically, for example, by intravenous, subcutaneous, intrathecal or intramuscular administration to a diseased patient.

  Natural human DNase I and its actin-resistant mutants also help patients with cystic fibrosis, chronic bronchitis, asthma, pneumonia or other pulmonary diseases, or their breathing is aided by a ventilator or other mechanical device It may also be useful to prevent new outbreaks and / or exacerbations of respiratory infections, such as may occur in patients who are affected, or other patients at risk of developing respiratory infections, such as post-surgical patients.

  Actin-resistant mutants can be formulated according to known methods to prepare therapeutically useful compositions. A preferred therapeutic composition is a solution of an actin-resistant mutant in a buffered or unbuffered aqueous solution, preferably an isotonic salt solution such as 150 mM sodium chloride containing 1.0 mM calcium chloride at pH 7. These solutions are particularly suitable for use with commercially available nebulizers, including jet nebulizers and ultrasonic nebulizers, which are useful for direct administration to the affected patient's respiratory tract or lungs.

  In another embodiment, the therapeutic composition is substantially a solution of the actin-resistant mutant solution described in co-pending US patent application Ser. No. 08 / 206,020 (filed Mar. 4, 1994). It consists of a dry powder of actin-resistant mutants preferably prepared by spray-drying.

  In a further embodiment, the therapeutic composition consists of cells that actively produce an actin-resistant mutant of human DNase I. Such cells can be introduced directly into the patient's tissue or can be encapsulated within a porous membrane which can then be implanted into the patient, in each case increased concentrations of DNA-hydrolysate. It provides for the delivery of actin-resistant mutants to areas within a patient's body in need of degradation activity. For example, a patient's own cells can be transformed in vivo or ex vivo with DNA encoding an actin-resistant mutant of human DNase I, thus producing DNase I directly in the patient. used.

  A therapeutically effective amount of an actin-resistant human DNase I variant will depend, for example, on the amount of DNA and actin in the substance to be treated, the subject to be treated, the route of administration, and the condition of the patient. Thus, it may be necessary for the therapist to titer the dose and modify the route of administration necessary to obtain the optimal therapeutic effect. In view of its reduced binding affinity for actin in the presence of actin for native human DNase I and consequently increased DNA-hydrolysis activity, the amount of actin-resistant mutant required to achieve a therapeutic effect is: It will be lower than the amount of natural human DNase I needed to achieve the same effect under the same conditions. In general, a therapeutically effective amount of an actin resistant variant will be a dose of about 0.1 μg to about 5 mg variant per kg patient body weight administered within a pharmaceutical composition described herein. .

  Actin-resistant DNase I variants may optionally be antibiotics, bronchodilators, anti-inflammatory agents, mucolytic agents (eg n-acetyl-cysteine), actin-binding or actin-cleaving proteins (eg gelsolin; Matsudaria et al. , Cell 54: 139-140 (1988); Stossel et al., PCT patent application WO94 / 22465 (published Oct. 13, 1994), protease inhibitors, or gene therapy products (eg, cystic fibrosis transmembrane conductance modulators) (CFTR) gene, Riordan et al., Science 245: 1066-1073 (1989)) can also be combined with or administered with one or more other pharmacological agents used to treat the above listed diseases.

  The following examples are provided for illustration only and are not intended to limit the invention in any way. All patents and documents cited herein are expressly regarded as part of this specification.

  Example 1 Mutagenesis of human DNase I

E. coli strain CJ236 (BioRad Laboratories, Richmond, Calif., USA) was transformed with plasmid pRK.DNase 3. using the method of Chung et al. (Nuc. Acids Res. 16 : 3580 (1988)). The plasmid pRK.DNase 3. used to make the present invention is a PCT patent application WO 90/07572 (1990) except that the nucleic acid sequence encoding human DNase I is that shown in FIG. Transformed cells were placed on LB agar plates containing 50 μg / ml carbenicillin and grown overnight at 37 ° C. 50 μg / ml carbenicillin and 10 μl VCSM13 helper phage 2YT broth (5 ml) containing (Stratagene, La Jolla, Calif. USA) with individual colonies from agar plates. And seeds, stirring was grown overnight at 37 ° C.. Isolated single-stranded DNA from this culture and used as template for subsequent mutagenesis.

Site-directed mutagenesis was achieved using synthetic oligonucleotides according to the method of Kunkel et al. (Meth. Enzymol. 154 : 367-382 (1987). The mutagenic oligonucleotide is 5 'to the mispaired codon. 21-mer or 24-mer with 9 or 12 exact base pairings and 9 exact base pairs 3 'to the mispaired codon. Was subjected to dideoxy sequencing (Sanger et al., Proc. Nat. Acad. Sci. USA 74 : 5463-5467 (1977)). E. coli strain XL1 Blue MRF ′ (Stratagene) was transformed and after plating and single colony isolation, individual colonies were used to inoculate 0.5 liter LB broth containing 50 μg / ml carbenicillin. While Following overnight growth at 37 ° C., cells were harvested by centrifugation and the mutant DNA (in the expression vector) was purified using a Qiagen chip-500 column (Qiagen Inc., Chatsworth, Calif., USA).

  Figures 2-6 show the different human DNase I variants that were generated. In the figures and throughout the specification, the representation of amino acid substitution mutations present in DNase I is omitted by the first alphabetic character, the number and the second alphabetic character. The first alphabetic letter is a single letter omission of an amino acid residue in natural (wild type) human mature DNase I, the number indicates the position of that residue in natural human mature DNase I (numbering shown in FIG. 1), And the second alphabetic letter is a single letter omission of the amino acid residue at that position in mutant DNase I. For example, in a DNase I mutant with a D53R mutation, the aspartic acid (D) residue at position 53 in native human mature DNase I is replaced by an asparagine (R) residue. Multiple mutations in a single variant are similarly displayed, with each different mutation present in the variant separated by a colon (:). For example, the designation D53R: Y65A indicates that the variant has a D53R mutation and a Y65A mutation.

  Example 2 Expression of human DNase mutant

Human embryonic kidney 293 cells (ATCC CRL 1573, American Type Culture Collection, Rockville, MD, USA) were grown in serum containing 150 mm plastic petri dishes. Using the calcium phosphate method (Gorman et al., DNA and Protein Eng. Tech. 2 : 3-10 (1990)), log phase cells were treated with 22.5 μg of purified mutant DNA (prepared as described above) and 17 μg adenoviral DNA. Over-co-transfected. Sixteen hours after transfection, the cells were washed with 15 ml phosphate buffered saline and the medium was changed to serum-free medium. The first time, 24 harvests or 72 hours after serum-free medium change, and finally 96 hours later, two harvests of cell culture medium were taken from each plate. A total of approximately 50 ml of cell culture supernatant containing the DNase I mutant was thus obtained. The pool of culture supernatant collected from each plate was concentrated 5 to 50 fold with a Centriprep 10 concentrator and the concentrate was assayed to measure various biochemical and biological activities of the DNase I mutant.

  293 cells were transformed into plasmid pRK. A concentrate containing native human DNase was prepared by the same procedure as described above except that it was transiently transfected with DNase.3.

  Example 3 Biochemical and biological activity of human DNase I mutant

  I. Relative specific activity

The relative specific activity of the DNase I variant was assessed by comparing the activity of the variant with that of native human DNase I in two different assays. In particular, the relative specific activity of the mutant is the concentration of the mutant measured in methyl green activity (expressed in μg / ml) (Sinicropi et al., Anal. Biochem. 222 : 351--358 (1994); Kurmick, Arch. Biochem 29:. 41-53 (1950) ) and is defined as divided by the concentration of the variant as determined by DN DNase I ELISA assay (described below) (μg / ml). Standard curves were determined using Pulmozyme® human DNase I in both the methyl green activity assay and the DNase I ELISA assay. The relative specific activities of native human DNase I and variants are shown in FIGS. 2A-D.

The methyl green activity assay (Sinicropi et al., Anal. Biochem. 222 : 351-358 (1994); Kurnick, Arch. B1ochem. 29 : 41-53 (1950)) utilizes a methyl green dye, which is approximately 10 in DNA. Intercalation for each base results in a green substrate. As the DNA is cleaved by DNase I, the methyl green dye is released and oxidized to a colorless form. Thus, the loss of green color is proportional to the amount of DNase I added to the assay sample. The amount of DNase I present in the assay is then quantified by comparison with a standard curve prepared by assaying known amounts of DNase I.

  The DNase I ELISA assay involves coating a microtiter plate with a goat anti-DNase I polyclonal antibody, adding the sample to be assayed, and a rabbit anti-DNase I polyclonal antibody conjugated to horseradish peroxidase (HRP). Detecting any of the resulting bound DNase I. When HRP substrate and color developer are added, color generation is proportional to the amount of DNase I present in the sample. The amount of DNase I present in the assay is then quantified by comparison with a standard curve prepared by assaying known amounts of DNase I.

  In both assays, multiple dilutions of the sample were assayed, the values falling within the central range of the standard curve were averaged, and the standard deviation was calculated.

  DNase I concentrations measured by DNase I ELISA were also used to normalize DNase I concentrations in other assays that characterize DNase I variants (eg, assay for inhibition by actin described below).

  II. Actin inhibition of DNase I hydrolysis activity

G-actin (Kabsh et al., Ann. Rev. Biophys. Biomol. Struct. 21 : 49-76 (1992))) is actin (commercially available (Sigma, St. Louis, Mo., USA)) or Pardee et al., Meth. Enzymol 85 : 164-181 (1982) 1 mg / ml solution at 4 ° C. with 5 mM HEPES, pH 7.2, 0.2 mM CaCl ₂ , 0.5 mM ATP, 0.5 mM β-mercapto Prepared by dialyzing overnight against ethanol. After 5 minutes centrifugation at 13,000 × g, the amount of G-actin is quantified by measuring the absorbance at 290 nm; the 1 mg / ml solution has an absorbance of 0.66 OD. Preliminary experiments under the same conditions using the amount of G-actin preparation required to inhibit the DNA-hydrolyzing activity of native human DNase I substantially but not completely (> 50% inhibition) in each assay Measured with

Sensitivity to actin inhibition is determined by two different assays, the methyl green assay described above and an increase in light absorption assay based on an increase in absorbance at 260 nm upon denaturation and depolymerization of DNA (Kunitz, J. Gen. Physiol. 33 : 349-362). . (1950); Kunitz, J.Gen.Physiol 33: 363-377 (1950)) in any crab was evaluated by measuring the DNA- hydrolytic activity of the mutant in the presence and absence of actin . The percent inhibition selected in these assays is shown in FIGS.

In a light absorption increase assay, concentrated culture supernatant (prepared as above, containing DNase I variant) was added to buffer before addition to a cuvette containing 40 μg DNA in a total assay volume of 1.0 ml. 1 hour at room temperature with or without 2- to 3-fold molar excess of actin in A (25 mM HEPES, pH 7.5 CaCl ₂ , 4 mM MgCl ₂ , 4 mM MgCl ₂ , 0.1% BSA) Incubated. The final concentration of DNase I variant in the assay was approximately 26 nM as determined by DNase I ELISA. The rate of DNA hydrolysis by the DNase I mutant in the presence and absence of actin was measured. The percent activity shown in FIGS. 3 and 4 determines the ratio of the DNA hydrolyzing activity of human DNase I (natural or mutant) in the presence of actin to the DNA-hydrolyzing activity in the absence of actin, Calculated by multiplying.

In the methyl green assay, the concentrated culture supernatant (prepared as described above and containing the DNase I mutant) was added to buffer B (25 mM HEPES, pH 7.5, 4 mM CaCl ₂ , 4 mM MgCl ₂ , 0.1). Incubate for 16 hours at 37 ° C. with or without a 1000-fold molar excess of actin in% BSA, 0.01% thimerosol, and 0.05% Tween 20). The concentration of active enzyme in each case was assessed by comparison with a standard curve of Pulmozyme®. The “percent activity” remaining of the mutant refers to the ratio of the activity in the presence of actin to the activity in the absence of actin multiplied by 100.

  As shown in FIGS. 3 and 4, the DNA-hydrolysis activity of natural human DNase is substantially reduced in the presence of actin. By comparison, various single and multiple residue variants of native human DNase are shown to have higher DNA-hydrolyzing activity in the presence of actin than native human DNase, as shown in FIG. Relatively resistant to inhibition.

  III. Actin binding ELISA

A microtiter-based assay was developed to measure the binding of native human DNase I and DNase I variants that immobilize actin. First, the wells of a Maxi Sorp plate (Nunc., Inc., Naperville, Illinois, USA) were washed with human GC globulin at a concentration of 10 μg / ml in 25 mM HEPES, 4 mM MgCl ₂ , 4 mM CaCl ₂ , pH 7.2. (Calbiochem., La Jolla, California, USA), actin binding protein (Goldschmidt-Clermont et al., Biochem. J. 228 : 471-477 (1985), McLeod et al., J. Biol. Chem. 264 : 1260-1267 ( 1989), Houmeida et al., Eur. J. Biochem. 203 : 499-503 (1992)) 100 μl per well was coated at 4 ° C. for 16-24 hours. After discarding the GC globulin, 200 μl of buffer C per well (buffer C is the same as buffer B with 0.5 mM adenosine triphosphate added; buffer C is all subsequent unless otherwise noted. Excess reactive sites were blocked by adding (used as assay diluent in the process) and incubating the plate on a shaker at room temperature for 1-2 hours. Each subsequent incubation step was performed on a Mini Orbital Shaker (Bello Biotechnology, Vineland, NJ, USA) for 1 hour at room temperature; between each step, the plate was emptied and a Microwasher II plate washer (Skatron A / S, Norway) was washed 6 times with phosphate buffered saline containing 0.05% Tween 20. Next, G-actin prepared as described above was diluted to 50 μg / ml in buffer C and 100 μl was added to each well; plates were incubated, washed, various dilutions of Pulmozyme® and native human DN Cell culture medium containing ase I or a variant thereof was added to the wells and the plates were incubated and washed. Finally, 100 μl of a 1 / 25,000 dilution of anti-human DNase I rabbit polyclonal antibody-horseradish peroxidase conjugate (original stock concentration was 465 μg / ml) was added to each well. After incubation and washing, color development was initiated by the addition of 100 μl color developing reagent (solubilized according to Sigma Fast manufacturer's recommendations. Phenylenediamine and urea / H ₂ O ₂ tablets), and 100 μl per well. It was stopped by the addition of 4.5N H ₂ SO ₄ . Absorbance at 492 nm was recorded and plotted against the concentration of DNase I originally added to the wells. Sigmoidal curves were obtained for mutants bound to native human DNase I and actin; these curves were nonlinear regression analysis (Marquardt, J. Soc. Indust. Appl. Math. 11 : 431-441 (1963). The concentration of each DNase I (natural or mutant) required to give a half-maximal signal in the assay is calculated from the curve and referred to as the EC ₅₀ value. The molecular weight of native human DNase I and the variant was estimated to be 37,000 daltons.

The relative binding affinity of each human DNase I variant was calculated by dividing the EC ₅₀ value of the variant by the EC ₅₀ value of native human DNase I measured in an ELISA assay, and the results are shown in FIGS. 5A-D. Show. As an example, if if the relative binding assay of human DN DNase I variant was calculated to be 5, this value is 5 times larger than The EC ₅₀ values of The EC ₅₀ values natural human DN DNase for variants, Or in other words, the mutants are shown to be 5-fold less than the affinity of native human DNase I for actin in this ELISA assay.

  IV.痰 Compression assay

  Sputum from cystic fibrosis patients before and after incubation with native human DNase I and different DNase I mutants using the sputum compression assay (PCT application WO94 / 10567, published May 11, 1994) The relative viscoelasticity of (“CF 痰”) was measured. After mixing the CF sputum with the DNase I sample and incubating at room temperature for 20 minutes, the semi-solid solution was loaded onto the capillary, which was then centrifuged at 12,000 rpm for 20 minutes. Following swiftness, the pellet height was measured and compared to the solution + pellet height. These measurements are then used to calculate the percent compression of the heel, which correlates with the viscoelasticity of the heel.

［配列表］

The percent compression measured upon treatment of CF sputum with native human DNase I and human DNase I actin-resistant mutants is shown in FIG. These results indicate that the human DNase I actin-resistant mutant is more effective than native human DNase I in reducing the viscoelasticity of CF 痰 as measured by a compression assay.
Figures 2-6 show data for the following mutants:

[Sequence Listing]

1 shows the amino acid sequence of human mature DNase I (SEQ ID NO: 1). The numbers indicate the sequential position of amino acid residues within the sequence. The relative specific activities of native human DNase I and variants are shown. The relative specific activities of native human DNase I and variants are shown. The relative specific activities of native human DNase I and variants are shown. The relative specific activities of native human DNase I and variants are shown. In each of FIGS. 2A-D, error bars are standard deviations (n-weight ratio). The relative specific activity of Pulmozyme® human DNase I (Genentech, Inc., South San Francisco, California, USA) is defined as 1.0. The relative specific activity of native human DNase I is greater than that of Pulmozyme® due to the generation of PulmozymeR, a deamidated form of human DNase I with reduced DNA-hydrolysis activity (Frenz et al., December 23, 1993). PCT patent application WO93 / 25670) published on the day. 2 shows DNA-hydrolyzing activity of human DNase I activity and single-residue mutants of human DNase I in the presence of actin as measured by a light absorption increase assay. “Percent activity” is the percent DNA-hydrolysis activity of DNase I (natural or mutant) calculated as described in Example 3; the DNA-hydrolysis activity of DNase I in the absence of actin is Defined as 100 percent active. Error bars represent standard deviation. FIG. 6 shows the DNA-hydrolysis activity of natural human DNase I and multiple residue variants of human DNase in the presence of actin as measured by an increased light absorption assay or a methyl green assay. “Percent activity” is the percent DNA-hydrolysis activity of DNase I (natural or mutant) calculated as described in Example 3; the DNA-hydrolysis activity of DNase I in the absence of actin is Defined as 100 percent active. Error bars represent standard deviation. FIG. 5 shows the relative binding affinity of human DNase I variants for actin as measured by an actin binding ELISA assay (described in Example 3). FIG. 5 shows the relative binding affinity of human DNase I variants for actin as measured by an actin binding ELISA assay (described in Example 3). FIG. 5 shows the relative binding affinity of human DNase I variants for actin as measured by an actin binding ELISA assay (described in Example 3). FIG. 5 shows the relative binding affinity of human DNase I variants for actin as measured by an actin binding ELISA assay (described in Example 3). In each of FIGS. 5A-D, the EC ₅₀ value is the concentration of DNase I (natural or mutant) required to give half the maximum signal in the assay. Error bars represent standard deviation. EC ₅₀ values for Pulmozyme® and native human DNase I are 67 ± 23 pM (n = 31) and 87 ± 14 pM (n = 32), respectively. The relative binding affinity shown in the figures, a been The EC ₅₀ values measured for human DN DNase I variant divided by the native human DN DNase I has been The EC ₅₀ values determined for. Variants with EC ₅₀ values greater than those that could be measured in the assay are greater than a certain value (eg>10,>100,>300,>2000,>20000,> 35000) ratio (EC ₅₀ (DN Ase I variant) / EC ₅₀ (natural DNase I). 2 shows the mucolytic activity of native human DNase I and human DNase I variants in sputum samples from cystic fibrosis patients as measured by compression assay. Error bars represent the standard deviation of the mean. 2 shows a schematic representation of the actin binding ELISA assay described in Example 3.

Claims (7)

  Has at least 90% identity with the amino acid sequence of native human DNase I shown in SEQ ID NO: 1 and is located in one or more of the following positions: G49R, D53C, A114G, A114H, and S94N: Y96T A variant of human DNase I having increased DNA-hydrolyzing activity over native human DNase I, having at least one amino acid substitution for an amino acid.   2. The variant of claim 1 having at least 95% identity with the amino acid sequence of native human DNase I shown in SEQ ID NO: 1.   2. The variant of claim 1 having at least 98% identity with the amino acid sequence of native human DNase I shown in SEQ ID NO: 1.   An isolated nucleic acid encoding a variant of human DNase I according to claim 1.   A pharmaceutical composition comprising a variant of human DNase I according to claim 1 and optionally a pharmaceutically acceptable excipient.   The composition of claim 5, wherein the composition is in liquid form.   6. A composition according to claim 5, wherein the composition is in powder form.

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