Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN DNASE RESISTANT TO ACTIN INHIBITION
Field of the Invention
The present invention relates to newly identified human deoxyribonuclease
(DNase) protein, nucleic
acid encoding such protein, the use of such protein and nucleic acid, as well
as the production of such protein
and nucleic acid, for example, by recombinant DNA methods.
Background of the Invention
Deoxyribonuciease (DNase) is a phosphodiesterase capable of hydrolyzing
polydeoxyribonucleic acid,
= and is known to occur in several molecular forms. Based on their biochemical
properties and enzymatic
activities, DNase proteins have been classified as two types, DNase I and
DNase II. DNase 1 proteins have a
pH optimum near neutrality, an obligatory requirement for divalent cations,
and produce 5'-phosphate
nucleotides on hydrolysis of DNA. DNase II proteins exhibit an acid pH
optimum, can be activated by divalent
cations, and produce 3'-phosphate nucleotides on hydrolysis of DNA.
DNase from various species have been purified to a varying degree. For
example, various forms of
bovine DNase I have been purified and completely sequenced (Liao, et al., J.
Biol. Chem. 248:1489-1495
(1973); Oefiner, et al., J. Mol. Biol. 192:605-632 (1986); Lahm, et al., J.
Mol. Biol. ZU:645-667 (1991)), and
DNA encoding bovine DNase I has been cloned and expressed (Worrall, et al., J.
Biol. Chem 2_~5:2 1 889-2 1 895
(1990)). Porcine and orcine DNase I proteins also have been purified and
compietely sequenced (Paudel, et
al., J. Biol. Chem. 291:16006-16011 (1986); Paudel, et al., J. Biol. Chem.
26_1:16012-16017 (1986)).
DNA encoding a human DNase I has been isolated and sequenced and the DNA has
been expressed
in recombinant host cells, thereby enabling the production of human DNase I in
commercially useful quantities.
Shak, et al., Proc. Natl. Acad. Sci. 87:9188-9192 (1990). The term "human
DNase I" will be used hereafter
to refer to the mature polypeptide disclosed in Shak, et al.
DNA encoding other polypeptides having homology to human DNase I also have
been identified.
Rosen, et al., PCT Patent Publication No. WO 95/30428, published November 16,
1995; Parrish, et al., Hum.
Mol. Genet. 4:1557-1564 (1995).
DNase I has a number of known utilities and has been used for therapeutic
purposes. Its principal
therapeutic use has been to reduce the viscoelasticity of pulmonary secretions
(mucus) in such diseases as
pneumonia and cystic fibrosis (CF), thereby aiding in the clearing of
respiratory airways. See e.g., Lourenco,
et al., Arch. Intern. Med. IAZ:2299-2308 (1982); Shak, et al., Proc. Natl.
Acad. Sci. $7:9188-9192 (1990);
Hubbard, et al., New Engl. J. Med. 32&:812-815 (1992); Fuchs, et al., New
Engl. J. Med. 33:637-642 (1994);
Bryson, et al., Drugs 4$:894-906 (1994). Mucus also contributes to the
morbidity of chronic bronchitis,
asthmatic bronchitis, bronchiectasis, emphysema, acute and chronic sinusitis,
and even the common cold.
The puimonary secretions of persons having such diseases are complex
materials, that include mucus
glycoproteins, mucopolysaccharides, proteases, actin, and DNA. DNase I is
effective in reducing the
viscoelasticity of pulmonary secretions by hydrolyzing, or degrading, high-
molecular-weight DNA that is
present in such secretions. Shak, et al., Proc. Natl. Acad. Sci. $Z:9188-9192
(1990); Aitken, et al., J. Am.
Med. Assoc. ZE:1947-I951 (1992). The DNA-hydrolytic activity of DNase I in
pulmonary secretions may
be reduced, however, as a result of the interaction of the DNase I with actin.
Lazarides, et al., Proc. Natl. Acad.
Sci. 21:4742-4746 (1974); Mannherz, et al., Eur. J. Biochem. JQA:367-379
(1980). Accordingly, forms of
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DNase I that bind actin with lower affmity than human native DNase I, but that
still possess DNA-hydrolytic
activity should be useful therapeutic agents, especially in the treatment of
patients having pulmonary secretions
that comprise relatively large amounts of actin. Variants of human DNase I
having reduced affinity for actin
have been prepared synthetically and shown to be more potent than the native
enzyme in reducing the viscosity
of sputum of cystic fibrosis patients. Lazarus, et at., PCT Pulication
W096/26279, published 29 August 1996.
Summary of the Invention
The present invention provides a novel DNase, as well as analogs and variants
thereof, that have
DNA-hydrolytic activity but that are resistant to inhibition by actin. This
novel polypeptide, referred to as LS-
DNase, is of human origin.
The invention also provides nucteic acids encoding LS-DNase, recombinant
vectors comprising such
nucleic acids, recombinant host cells transformed with those nucleic acids or
vectors, and processes for
producing LS-DNase by means of recombinant DNA technology. The invention
includes the use of such
nucleic acids and vectors for j0 Vlvo or r~ vivo gene therapy.
The invention also provides pharmaceutical compositions comprising LS-DNase,
optionally together
with a pharmaceuticaliv acceptable excipient, as well as substantially
purified antibodies that are capable of
binding to LS-DNase.
The invention also provides methods for reducing the viscoelasticity or
viscous consistency of DNA-
containing material in a patient, comprising administering a therapeutically
effective dose of LS-DNase to the
patient. The invention is particularly directed to a method of treating a
patient having a disease such as cystic
fibrosis, chronic bronchitis, pneumonia, bronchiectasis, emphysema, asthma, or
systemic lupus erythematosus,
that comprises administering a therapeutically effective amount of LS-DNase to
the patient. The invention also
is directed to the use of LS-DNase in i,n vitro diagnostic assays of a viscous
material (e.g., sputum) from a
patient.
These and other aspects of the invention will be apparent to the ordinary
skilled artisan upon
consideration of the following detailed description.
Brief Description of the Fi ures
Figure 1 shows the nucleotide sequence (SEQ. ID. NO: 1) and deduced amino acid
sequence (SEQ.
ID. NO: 2) of LS-DNase. The predicted leader (signal) amino acid sequence is
underlined and the start of the
mature protein is indicated by the arrowhead.
Figure 2 shows an alignment of the amino acid sequences of human LS-DNase
(SEQ. ID. NO: 3) and
human DNase I (SEQ. ID. NO: 4). Identical amino acid residues are boxed,
conservative amino acid
substitutions are indicated by a dot ('), and conserved cysteine residues are
indicated by arrowheads. Two
potential glycosylations sites in human DNase I are indicated by asterisks ().
Amino acid residues of human
DNase I that are involved in actin binding are shaded. Conserved catalytic
residues are in inverted text (white
on black). =
Figure 3 shows the nucleotide sequence (SEQ. ID. NO: 11) of murine LS-DNase.
The ATG start
codon for the predicted protein is indicated by the arrowhead, and the
nucleotide sequence encoding the
predicted leader (signal) amino acid sequence of the protein is underlined.
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Detailed Description
The various aspects of the present invention are accomplished by first
providing isolated DNA
comprising the nucleotide coding sequence for LS-DNase. By providing the full
nucleotide coding sequence
for LS-DNase, the invention enables the production of LS-DNase by means of
recombinant DNA technology,
thereby making available for the first time sufficient quantities of
substantially pure LS-DNase protein for
diagnostic and therapeutic uses.
As used herein, the term "LS-DNase" refers to the polypeptide having the amino
acid sequence of the
mature protein set forth in Figure 1, as well as modified and variant fotms
thereof as described herein. The
term "human LS-DNase" refers to the poiypeptide having the amino acid sequence
of the mature protein set
forth in Figure 1.
Modified and variant forms of LS-DNase are produced jn vitro by means of
chemical or enzymatic
treatment or 2n viv by means of recombinant DNA technology. Such polypeptides
differ from human LS-
DNase, for example, by virtue of one or more amino acid substitutions,
insertions, and/or deletions, or in the
extent or pattern of glycosylation, but substantially retain a biological
activity of LS-DNase. Preferably, the
modified and variant forms of LS-DNase have DNA-hydrolytic activity that is
substantially the same as that
of human LS-DNase.
A "variant" or "amino acid sequence variant" of LS-DNase is a polypeptide that
comprises an amino
acid sequence different from that of human LS-DNase. Generally, a variant will
possess at least 80% sequence
identity (homology), preferably at least 90% sequence identity, more
preferably at least 95% sequence identity,
and most preferably at least 98% sequence identity with human LS-DNase.
Percentage sequence identity is
determined, for example, by the Fitch, et al., Proc. Natl. Acad. Sci. USA
84:1382-1386 (1983), version of the
algorithm described by Needleman, et al., J. Mol. Biol. 4~:443-453 (1970),
after aligning the sequences to
provide for maximum homology. Such variants include naturaily occurring
allelic forms of human LS-DNase
that are of human origin as well as natuarlly occurring homologs of human LS-
DNase that are found in other
animal species.
"DNA-hydrolytic activity" refers to the enzymatic activity of a DNase in
hydrolyzing (cleaving)
substrate DNA to yield 5'-phosphorylated oligonucleotide end products. DNA-
hydrolvtic activity is readily
determined by any of several different methods known in the art, including
analytical polyacrylamide and
agarose gel electrophoresis, hyperchromicity assay (Kunitz, J. Gen. Physiol.
22:349-362 (1950); Kunitz, J.
Gen. Physiol. 22:363-377 (1950)), or methyl green assay (Kurnick, Arch.
Biochem. 29:41-53 (1950);
Sinicropi, et al., Anal. Biochem. 22.1:351-358 (1994)).
For convenience, substitutions, insertions, and/or deletions in the amino acid
sequence of human LS-
DNase are usually made by introducing mutations into the corresponding
nucleotide sequence of the DNA
encoding human LS-DNase, for example by site-directed mutagenesis. Expression
of the mutated DNA then
= 35 results in production of the variant LS-DNase, having the desired amino
acid sequence.
Whereas any technique known in the art can be used to perform site-directed
mutagenesis, e.g. as
disclosed in Sambrook, et al., Molecular CloninQ: A Laboratorv Manual, Second
Edition (Cold Spring Harbor
Laboratory Press, New York (1989)), oligonucleotide-directed mutagenesis is
the preferred method for
preparing the LS-DNase variants of this invention. This method, which is well
known in the art (Zoller, et al.,
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WO 97/28266 PCT/US97/01506
Meth. Enzymol. 100:4668-500 (1983); Zoller, et al., Meth. Enzymol. 154:329-350
(1987); Carter. Meth.
Enzymol. 154:382-403 (1987); Kunkel, et al., Meth. Enzymol. 154:367-382
(1987); Horwitz, et al., Meth.
Enzymol. 1$:599-61 1(1990)), is particularly suitable for making substitution
variants, although it may also
be used to conveniently prepare deletion and insertion variants, as well as
variants having multiple substitution.
insertion, and/or deletion mutations.
Briefly, in carrying out site-directed mutagenesis of DNA encoding human LS-
DNase (or a variant
thereof), the DNA is altered by first hybridizing an oligonucleotide encoding
the desired mutation to a single
strand of the DNA. After hybridization, a DNA polymerase is used to synthesize
an entire second strand, using
the hybridized oligonucleotide as a primer, and using the single strand of the
DNA as a template. Thus, the
oligonucleotide encoding the desired mutation is incorporated in the resulting
double-stranded DNA.
Oligonucleotides may be prepared by anv suitable method, such as by
purification of a naturally
occurring DNA or by jU vitro synthesis. For example, oligonucleotides are
readily synthesized using various
techniques in organic chemistry, such as described by Narang, et al., Meth.
Enzymol. 6$:90-98 (1979); Brown,
et al., Meth. Enzymol. $:109-151 (1979); Caruthers, et al., Meth. Enzymol. ,j
5A:287-313 (1985). The general
approach to selecting a suitable oligonucleotide for use in site-directed
mutagenesis is well known. Typically,
the oligonucleotide will contain 10-25 or more nucleotides, and will include
at least 5 nucleotides on either side
of the sequence encoding the desired mutation so as to ensure that the
oligonucleotide will hybridize
preferentially at the desired location to the single-stranded DNA template
molecule.
"Polymerase chain reaction," or "PCR," generally refers to a method for
amplification of a desired
nucleotide sequence jg vi , as described, for example, in U.S. Pat. No.
4,683,195. In general, the PCR
method involves repeated cycles of primer extension synthesis, using
oligonucleotide primers capable of
hybridizing preferentially to a template nucleic acid.
PCR mutagenesis (Higuchi, in PCR Protocols, pp.177-183 (Academic Press, 1990);
Vallette, et al.,
Nuc. Acids Res. JZ:723-733 (1989)) is also suitable for making the variants of
LS-DNase. Briefly, when small
amounts of template DNA are used as starting material in a PCR, primers that
differ slightly in sequence from
the corresponding region in the template DNA can be used to generate
relatively large quantities of a specific
DNA fragment that differs from the template sequence only at the positions
where the primers differ from the
template. For introduction of a mutation into a plasmid DNA, for example, the
sequence of one of the primers
includes the desired mutation and is designed to hybridize to one strand of
the plasmid DNA at the position
of the mutation; the sequence of the other primer must be identical to a
nucleotide sequence within the opposite
strand of the plasmid DNA, but this sequence can be located anywhere along the
plasmid DNA. It is preferred,
however, that the sequence of the second primer is located within 200
nucleotides from that of the first, such
that in the end the entire amplified region of DNA bounded by the primers can
be easily sequenced. PCR
amplification using a primer pair like the one just described results in a
population of DNA fragments that
differ at the position of the mutation specified by the primer, and possibly
at other positions, as template
copying is somewhat error-prone. Wagner, et al., in PCR To2ics, pp.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(s). This
product DNA is used to replace the corresponding region
in the plasmid that served as PCR template using standard recombinant DNA
methods. Mutations at separate
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WO 97/28266 PCT/US97/01506
positions can be introduced simultaneously by either using a mutant second
primer. or performing a second
PCR with different mutant primers and ligating the two resulting PCR fragments
simultaneously to the plasmid
fragment in a three (or more)-part ligation.
Another method for preparing variants, cassette mutagenesis, is based on the
technique described by
Wells et al., Gene, 24:315-323 (1985). The starting material is the plasmid
(or other vector) comprising the
DNA sequence to be mutated. The codon(s) in the starting DNA to be mutated are
identified. There must be
a unique restriction endonuctease site on each side of the identified mutation
site(s). If no such restriction sites
exist, they may be generated using the above-described oligonucleotide-
mediated mutagenesis method to
introduce them at appropriate locations in the DNA. The plasmid DNA is cut at
these sites to linearize it. A
double-stranded oligonucleotide encoding the sequence of the DNA between the
restriction sites but containing
the desired mutation(s) is synthesized using standard procedures, wherein the
two strands of the oligonucleotide
are synthesized separately and then hybridized together using standard
techniques. This double-stranded
oligonucleotide is referred to as the cassette. This cassette is designed to
have 5' and 3' ends that are
compatible with the ends of the linearized plasmid, such that it can be
directly ligated to the plasmid. The
resulting plasmid contains the mutated DNA sequence.
The presence of mutation(s) in a DNA is determined by methods well known in
the art, including
restriction mapping and/or DNA sequencing. A preferred method for DNA
sequencing is the dideoxy chain
termination method of Sanger, et al., Proc. Natl. Acad. Sci. USA 72.:3918-3921
(1979).
DNA encoding LS-DNase is inserted into a replicable vector for further cloning
or expression.
"Vectors" are plasmids and other DNAs that are capable of replicating within a
host cell, and as such, are useful
for performing two functions in conjunction with compatible host cells (a
vector-host system). One function
is to facilitate the cloning of nucleic acid that encodes LS-DNase, i.e., to
produce usable quantities of the
nucleic acid. The other function is to direct the expression of LS-DNase. One
or both of these functions are
performed by the vector in the particular host cell used for cloning or
expression. The vectors will contain
different components depending upon the function they are to perform.
The LS-DNase of the present invention may be in the form of a preprotein
wherein the DNase includes
a leader or signal sequence, or may be in the fotm of a mature protein which
lacks a leader or signal sequence.
The LS-DNase also may be in the form of a fusion protein wherein additional
amino acid residues are
covalently joined to the amino- or carboxy-terminus of the preprotein or
mature form of the DNase.
To produce LS-DNase, an expression vector will comprise DNA encoding LS-DNase,
as described
above, operably linked to a promoter and a ribosome binding site. The LS-DNase
then is expressed directly
in recombinant cell culture, or as a fusion with a heterologous polypeptide,
preferably a signal sequence or
other polypeptide having a specific cleavage site at the junction between the
heterologous polypeptide and the
LS-DNase amino acid sequence.
"Operably linked" refers to the covalent joining of two or more DNA sequences,
by means of
etazymatic ligation or otherwise, in a configuration relative to one another
such that the normal function of the
sequences can be performed. For example, DNA for a presequence or secretory
leader is operably linked to
DNA for a polypeptide if it is expressed as a preprotein that participates in
the secretion of the polypeptide:
a promoter or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence;
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or a ribosome binding site is operably linked to a coding sequence if it is
positioned so as to facilitate
translation. Generally, "operably linked" means that the DNA sequences being
linked are contiguous and, in
the case of a secretory leader, contiguous and in reading phase. Linking is
accomplished by ligation at
convenient restriction sites. If such sites do not exist, then synthetic
oligonucleotide adaptors or linkers are
used, in conjunction with standard recombinant DNA methods.
Prokaryotes (e.g., F,. ggU, strains of Bacillus, Pseudomonas, and other
bacteria) are the preferred host
cells for the initial cloning steps of this invention. They are particularly
useful for rapid production of large
=
amounts of DNA, for production of single-stranded DNA templates used for site-
directed mutagenesis, and for
DNA sequencing of the variants generated. Prokaryotic host cells also may be
used for expression of DNA
encoding LS-DNase. Polypeptides that are produced in prokaryotic cells
typically will be non-glycosylated.
In addition, LS-DNase may be expressed in eukaryotic host cells, including
eukaryotic microbes (e.g.,
yeast) or cells derived from an animal or other multicellular organism (e.g.,
Chinese hamster ovary cel[s, and
other mammalian cells), or in live animals (e.g., cows, goats, sheep). Insect
cells also may be used.
Cloning and expression methodologies are well known in the art. Examples of
prokaryotic and
eukaryotic host cells, and starting expression vectors, suitable for use in
producing LS-DNase are, for example,
those disclosed in Shak, PCT Patent Publication No. WO 90/07572, published
July 12, 1990. To obtain
expression of LS-DNase, an expression vector of the invention is introduced
into host cells by transformation
or transfection, and the resulting recombinant host cells are cultured in
conventional nutrient media, modified
as appropriate for inducing promoters, selecting recombinant cells, or
amplifying LS-DNase DNA. The culture
conditions, such as temperature, pH, and the like, are those previously used
with the host cell, and as such will
be apparent to the ordinarily skilled artisan.
"Transformation" and "transfection" are used interchangeably to refer to the
process of introducing
DNA into a cell. Following transformation or transfection, the DNA may
integrate into the host cell genome,
or may exist as an extrachromosomal element. If prokaryotic cells or cells
that contain substantial cell wall
constructions are used as hosts, the preferred methods of transfection of the
cells with DNA is the calcium
treatment method described by Cohen et al., Proc. Natl. Acad. Sci. U:2110-2114
(1972) or the polyethylene
- glycol method of Chung et al., Nuc. Acids. Res. _1~k:3580 (1988). If yeast
are used as the host, transfection is
generally accomplished using polyethylene glycol, as taught by Hinnen, Proc.
Natt. Acad. Sci. U.S.A., 25:
1929-1933 (1978). If mammalian cells are used as host cells, transfection
generally is carried out 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). However, other known methods for introducing
DNA into prokaryotic and
eukaryotic cells, such as nuclear injection, electroporation, or protoplast
fusion also are suitable for use in this
invention.
Particularly useful in this invention are expression vectors that provide for
the transient expression
in mammalian cells of DNA encoding LS-DNase. In general, transient expression
involves the use of an =
expression vector that is able to efficiently replicate in a host cell, such
that the host cell accumulates many
copies of the expression vector and, in turn, synthesizes high levels of a
desired polypeptide encoded by the
expression vector. Transient expression systems, comprising a suitable
expression vector and a host cell, allow
for the convenient positive identification of polypeptides encoded by cloned
DNAs, as well as for the rapid
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screening of such polvpeptides for desired biological or physiological
properties. Wong, et al., Science
=:810-815 (1985); Lee, et al., Proc. Nat Acad. Sci. USA U:4360-4364 (1985);
Yang, et al., Cell 47:3-10
(1986). Thus, transient expression systems are conveniently used for
expressing the DNA encoding amino acid
sequence variants of LS-DNase, in conjunction with assays to identify those
variants that have such useful
properties as increased half-life or decreased immunogenicity in viv ,
increased DNA hydrolytic activity, or
=
increased resistance to inhibition by actin. The inhibition of DNase activity
by actin is readily determined using
assays and methods known in the art and as described herein.
= LS-DNase preferably is 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 cells are grown. In
that case, it may be desirable to
grow the cells in a serum free culture medium, since the absence of serum
proteins and other serum components
in the medium may facilitate purification of the variant. If it is not
secreted, then the LS-DNase is recovered
from lysates of the host cells. When the LS-DNase is expressed in a host cell
other than one of human origin,
the variant will be completely free of proteins of human origin. In any event,
it will be necessary to purify the
LS-DNase from recombinant cell proteins in order to obtain substantially
homogeneous preparations of the LS-
DNase. For therapeutic uses, the purified LS-DNase preferably will be greater
than 99% pure (i.e., any other
proteins will comprise less than 1% of the total protein in the purified
composition).
It is further contemplated that LS-DNase may be produced by a method involving
homologous
recombination and amplification, for example, as described in PCT Patent
Publication No. WO 91/06667,
published May 16, 1991. Briefly, this method involves transforming cells
containing an endogenous gene
encoding LS-DNase with a homologous DNA, which homologous DNA comprises (1) an
amplifiable gene
(e.g., a gene encoding dihydrofolate reductase (DHFR)), and (2) at least one
flanking sequence, having a length
of at least about 150 base pairs, which is homologous with a nucleotide
sequence in the cell genome that is
within or in proximity to the gene encoding LS-DNase. The transformation is
carried out under conditions such
that the homologous DNA integrates into the cell genome by recombination.
Cells having integrated the
homologous DNA then are subjected to conditions which select for amplification
of the amplifiable gene,
whereby the LS-DNase gene amplified concomitantly. The resulting cells then
are screened for production of
desired amounts of LS-DNase. Flanking sequences that are in proximity to a
gene encoding LS-DNase are
readily identified, for example, by the method of genomic walking, using as a
starting point the nucleotide
sequence of LS-DNase shown in Figure 1. Spoerel, et al., Meth. Enzymol. 1-
~2:598-603 (1987).
Generally, purification of LS-DNase is accomplished by taking advantage of the
differential
physicochemical properties of the LS-DNase as compared to the contaminants
with which it may be associated.
For example, as a first step, the culture medium or host cell lysate is
centrifuged to remove particulate cell
debris. The LS-DNase thereafter is purified from contaminant soluble proteins
and polypeptides, for example,
by ammonium sulfate or ethanol precipitation, gel filtration (molecular
exclusion chromatography), ion-
exchange chromatography, hydrophobic chromatography, immunoaffinity
chromatography (e.g., using a
column comprising anti-LS-DNase antibodies coupied to Sepharose), tentacle
cation exchange chromatography
(Frenz, et al., U.S. Patent No. 5,279,823, issued January 18, 1994), reverse
phase HPLC, and/or gel
electrophoresis.
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In some host cells (especially bacterial host cells) the LS-DNase may be
expressed initially in an
insoluble, aggregated form (referred to in the art as "refractile bodies" or
"inclusion bodies") in which case it
will be necessary to solubilize and renature the LS-DNase in the course of its
purification. Methods for
solubilizing and renaturing recombinant protein refractile bodies are known in
the art (see e.g., Builder, et al.,
U.S. Patent No. 4,511,502, issued April 16, 1985).
In another embodiment of this invention, covalent modifications are made
directly to LS-DNase to
give it a desired property (for example, increased half-life or decreased
immunogenicity j0, vivo= increased
DNA hydrolytic activity, or increased resistance to inhibition by actin), and
may be made instead of or in
addition to the amino acid sequence substitution, insertion, and deletion
mutations described above.
Covalent modifications are introduced by reacting targeted amino acid residues
of LS-DNase with an
organic derivatizing agent that is capable of reacting with selected amino
acid side-chains or N- or C-terminal
residues. Suitable derivatizing agents and methods are well known in the art.
Covalent coupling of glycosides
to amino acid residues of the protein may be used to modify or increase the
number or profile of carbohydrate
substituents.
The covalent attachment of agents such as polyethylene glycol (PEG) or human
serum albumin to LS-
DNase may reduce immunogenicity and/or toxicity of the LS-DNase and/or prolong
its half-life, as has been
observed with other proteins. Abuchowski, et al., J. Biol. Chem. M:3582-3586
(1977); Poznansky, et al.,
FEBS Letters 2,39:18-22 (1988); Goodson, et al., Biotechnology $:343-346
(1990); Katre, J. Immunol.
144:209-213 (1990); Harris, Polyethylene G vcol Chemistrv (Plenum Press,
1992). In addition, modification
of LS-DNase by these agents at or adjacent to (i.e., within about five amino
acid residues of) an amino acid
residue that affects actin binding may produce a variant having increased
resistance to inhibition by actin. As
another example, the variant or modified form of LS-DNase may comprise an
amino acid sequence mutation
or other covalent modification that reduces the susceptibility of the variant
to degradation by proteases (e.g.,
neutrophil elastase) that may be present in sputum and other biological
materials, as compared to human LS-
DNase.
Antibodies to LS-DNase are produced by immunizing an animal with LS-DNase or a
fragment thereof,
optionally in conjunction with an immunogenic polypeptide, and thereafter
recovering antibodies from the
serum of the immunized animals. Alternatively, monoclonal antibodies are
prepared from cells of the
immunized animal in conventional fashion. The antibodies also can be made in
the form of chimeric (e.g_,
humanized) or single chain antibodies or Fab fragments, using methods well
known in the art. Preferably, the
antibodies will bind to LS-DNase but will not substantially bind to (i.e.,
cross react with) other DNase proteins
(such as human and bovine DNase I). The antibodies can be used in methods
relating to the localization and
activity of LS-DNase, for example, for detecting LS-DNase and measuring its
levels in tissues or clinical
samples. Immobilized anti-LS-DNase antibodies are particularly useful in the
detection of LS-DNase in
clinicat samples for diagnostic purposes, and in the purification of LS-DNase.
Purified LS-DNase is used to reduce the viscoelasticity of DNA-containing
material, such as sputum,
mucus, or other pulmonary secretions. LS-DNase is particularly useful for the
treatment of patients with
pulmonary disease who have abnormal viscous or inspissated secretions and
conditions such as acute or chronic
bronchial pulmonary disease, including infectious pneumonia, bronchitis or
tracheobronchitis, bronchiectasis,
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cystic fibrosis, asthma, tuberculosis, and fungal infections. For such
therapies, a solution or finely divided dry
preparation of the LS-DNase is instilled in conventional fashion into the
airways (e.g., bronchi) or lungs of a
patient, for example by aerosolization.
LS-DNase also is useful for adjunctive treatment of abscesses or severe closed-
space infections in
conditions such as empyema, meningitis, abscess, peritonitis, sinusitis,
otitis, periodontitis, pericarditis,
pancreatitis, cholelithiasis, endocarditis and septic arthritis, as well as in
topical treatments in a variety of
inflammatory and infected lesions such as infected lesions of the skin and/or
mucosal membranes, surgical
wounds, ulcerative lesions and bums. LS-DNase may improve the efficacy of
antibiotics used in the treatment
of such infections (e.g., gentamicin activity is markedly reduced by
reversible binding to intact DNA).
LS-DNase also is useful for preventing the new development and/or exacerbation
of respiratory
infections, such as may occur in patients having cystic fibrosis, chronic
bronchitis, asthma, pneumonia, or other
pulmonary disease, or patients whose breathing is assisted by ventilator or
other mechanical device, or other
patients at risk of developing respiratory infections, for example post-
surgical patients.
LS-DNase also is useful for the treatment for systemic tupus erythematosus
(SLE), a life-threatening
autoimmune disease characterized by the production of diverse autoantibodies.
DNA is a major antigenic
component of the immune complexes. In this instance, the LS-DNase may be given
systemically, for example
by intravenous, subcutaneous, intrathecal, or intramuscular administration to
the affected patient.
Finally, LS-DNase is useful for the treatment of other non-infected conditions
in which there is an
accumulation of cellular debris that includes cellular DNA, such as
pyelonephritis and tubulo-interstitial kidney
disease.
LS-DNase can be formulated according to known methods to prepare
therapeutically useful
compositions. Typically, the LS-DNase is formulated with a physiologically
acceptable excipient (or carrier)
for therapeutic use. Such excipients are used, for example, to provide liquid
formulations and sustained-release
formulations of,LS-DNase. A preferred therapeutic composition is a solution of
LS-DNase in a buffered or
unbuffered aqueous solution, and preferably is an isotonic salt solution such
as 150 mM sodium chloride
containing 1.0 mM calcium chloride at pH 7. These solutions are particularly
adaptable for use in
commercially-available nebulizers including jet nebulizers and ultrasonic
nebulizers useful for administration
directly into the airways or lungs of an affected patient. Another preferred
therapeutic composition is a dry
powder of LS-DNase, preferably prepared by spray-drying of a solution of the
LS-DNase, essentially as
described in PCT Publication W095/23613. In all cases, it is desirable that
the therapeutic compositions be
sterile. Preferably, the therapeutic compositions are disposed in a container
fabricated of plastic or other non-
glass material.
In a further embodiment, the therapeutic composition comprises cells actively
producing LS-DNase.
Such ceils may be directly introduced into the tissue of a patient, or may be
encapsulated within porous
membranes which are then implanted in a patient (see e.g., Aebischer, et al.,
U.S. Patent No. 4,892,538, issued
January 9, 1990; Aebischer, et al., U.S. Patent No. 5,283,187, issued February
1, 1994), in either case
providing for the delivery of the LS-DNase into areas within the body of the
patient in need of increased
concentrations of DNA-hydrolytic activity. For example, the patient's own
cells could be transformed, either
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in vivo or 1& v' with DNA encoding LS-DNase, and then used to produce the LS-
DNase directly within the
patient. This latter method is commonly referred to as gene therapy.
The therapeutically effective amount of LS-DNase will depend, for example,
upon the amount of DNA
and actin in the material to be treated, the therapeutic objectives, the route
of administration, and the condition
of the patient. Accordingly, it will be necessary for the therapist to titer
the dosage and modify the route of
administration as required to obtain the optimal therapeutic effect. In view
of its reduced inhibition by actin
and consequential increased DNA-hydrolytic activity in the presence of actin
relative to human DNase I, the
amount of LS-DNase required to achieve a therapeutic effect may be less than
the amount of human DNase
I necessary to achieve the same effect under the same conditions. Generally,
the therapeutically effective
amount of LS-DNase will be a dosage of from about 0.1 g to about 5 mg of the
variant per kilogram of body
weight of the patient, administered within pharmaceutical compositions, as
described herein.
LS-DNase optionally is combined with or administered in concert with one or
more other
pharmacologic agents used to treat the conditions listed above, such as
antibiotics, bronchodilators. anti-
inflammatory agents, mucolytics (e.g. n-acetyl-cysteine), actin binding or
actin severing proteins (e.g., gelsolin;
Matsudaira et al., Cell 5A:139-140 (1988); Stossel, et al., PCT Patent
Publication No. WO 94/22465.
published October 13, 1994; protease inhibitors; or gene therapy product
(e.g., comprising the cystic fibrosis
transmembrane conductance regulator (CFTR) gene); Riordan, et al., Science
W:1066-1073 (1989)).
This invention also provides methods for determining the presence of a nucleic
acid molecule
encoding LS-DNase in test samples prepared from cells, tissues, or biological
fluids, comprising contacting
the test sample with isolated DNA comprising all or a portion of the
nucleotide coding sequence for LS-DNase
and determining whether the isolated DNA hybridizes to a nucleic acid molecule
in the test sample. DNA
comprising all or a portion of the nucleotide coding sequence for LS-DNase is
also used in hybridization assays
to identify and to isolate nucleic acids sharing substantial sequence identity
to the coding sequence for LS-
DNase, such as nucleic acids that encode naturally-occurring allelic variants
of LS-DNase.
Also provided is a method for ampiifying a nucleic acid molecule encoding LS-
DNase that is present
in a test sample, comprising the use of an oligonucleotide having a portion of
the nucleotide coding sequence
for LS-DNase as a primer in a poiymerase chain reaction.
The following examples are offered by way of illustration oniy and are not
intended to limit the
invention in any manner. All patent and literature references cited herein are
expressly incorporated.
EXAMPLE I
Clonine LS-DNase cDNA
Full-length cDNA encoding LS-DNase was identified by screeniag a human liver
cDNA library (in
*
ll-UniZAP XR, Stvagene, La Jolla, CA) with a mixture of the following
oligonucleotide probes, each of which
had been end-labeled with T4 polynucleotide kinase and Y72P-adenosine
triphosphate to a high specif5c
radioactivity:
5'-ACTGTAGTTTAAATTCAACTGGAAAGTGGTCGCTGACATCCAGGG-3' (SEQ. ID. NO: 5)
5'-GATGTCATTGTGAAGGTCATCAAACGCTGTGACATCATACTCGTG-3' (SEQ. ID. NO: 6)
5'-GTGTTTTCCAGGGGAGCCCTTTGTGGTCTGGTTCCAATCTCCCCA-3' (SEQ. ID. NO: 7)
5'-CTGGAGGTCTCCCAGCACTGGCAGAGCAAGGACGTGATCCTGCTT-3' (SEQ. ID. NO: 8)
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5'-GCCCAGCATCATCGCGAAGTTCCTGGCTGGCTATCACCTCGCGCT-3' (SEQ. ID. NO: 9)
5'-CCAGTACAAGGAGATGTACCTCTTCGTTTACAGGAAAGACGCCGT-3' (SEQ. ID. NO: 10)
The fu-st three of the oligonucleotide probes listed above (SEQ. ID. NOS: 5-7)
comprise portions of the EST
sequences having accession codes T68985, T69063, HSAAACIFW, T73558, T61400,
T73653. and T61368
in the Genbank database. The other two oligonucleotide probes listed above
(SEQ. ID. NOS: 9-10) comprise
portions of the EST sequences having accession codes R78020 and H42990 in the
Genbank database.
Hybridization of the probes to the cDNA library was carried out under low
stringency conditions (in
20% vol/vol formamide, 5X SSPE, 5X Denhardt's solution, 0.1 % sodium dodecyl
sulfate (SDS), and 100 g/ml
sonicated salmon sperm DNA), at 42 C. Post hybridization washes were carried
out in 2X SSC, 0.1 % SDS,
at 42 C. IX SSPE is 150mM NaCl, lOmM sodium phosphate. imM EDTA, pH 7.4. iX
Denhardt's solution
*
is 0.02% Ficoll, 0.02% bovine serum albumin, and 0.02% polyvinyl-pyrrolidone.
i X SSC is 0.15 M NaCI,
0.015 M sodium citrate, pH 7Ø
Hybridization-positive clones were found only with the first three of the
oligonucleotide probes listed
above (SEQ. ID. NOS: 5-7). Those clones were converted into phagemid-based
sequences following standard
procedures (Stratagene, La Jolla, Califomia, USA) and were sequenced. The
largest inserted nucleotide
sequence found amongst the hybridization-positive clones was 1079 base-pairs
in length, including an open
reading frame of 915 base-pairs that encodes a predicted protein that is 305
amino acid residues in length. The
nucleotide sequence of the 1079 base-pair insert (SEQ. ID. NO: 1) and the
amino acid sequence of predicted
protein (SEQ. ID. NO: 2) are shown in Figure 1.
The predicted protein includes a signal sequence that is 20 amino acid
residues in length. Cleavage
of the signal sequence releases the mature protein (LS-DNase), which has a
predicted molecular weight of
33,400 Daltons and a predicted pI of 9.7. The amino acid sequence of LS-DNase
is 46% identical to the amino
acid sequence of human DNase I (Figure 2).
LS-DNase contains five cysteine residues, two of which (Cys- 174 and Cys-21 1)
coincide with a pair
of cysteine residues in human DNase I that are disulfide bonded, suggesting
that LS-DNase and human DNase
I may have similar tertiary structures. Amino acid residues known to be
important for the DNA-hydrolytic
activity of human DNase I are conserved in LS-DNase, including the active site
histidine residues His- 135 and
His-254. Conversely, several amino acid residues known to comprise the actin-
binding site of human DNase
I are not conserved in LS-DNase. In particular, Val-67 and Ala-114 of human
DNase I are replaced by Ile-69
and Phe-115, respectively, at the homologous positions in LS-DNase. An
analogous replacement of Vai-67
by lie occurs in rat DNase I, which has approximately 1000-fold lower affinity
for actin as compared to human
DNase I.
EXAMPLE 2
Expression of LS-DNase cDNA
The cDNA encoding LS-DNase was subcloned into a mammalian expression vector
pRK5 (Gorman,
et al., DNA and Protein Engineering Techniques ?.:1 (1990); European Patent
Publication EP 307,247,
pubiished March 15, 1989). The resulting recombinant vector is referred to as
pRK5/LS-DNase. Human
embryonic kidney 293 cells (American Type Culture Collection, CRL 1573) were
grown in serum-containing
Dulbecco Modified Eagle's medium (DMEM) to 70% confluency and then transiently
transfected with
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pRK5/LS-DNase, or as a control, pRK5 alone. 24 hours post-transfection, the
cells were washed with
phosphate buffered saline and transferred to serum-free medium containing
insulin. 72-96 hours later,
conditioned medium was collected from the cell cultures and concentrated
approximately 10-fold. Proteins
expressed in the cell cultures were analyzed by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE).
Cells transfected with pRK-5/LS-DNase were found to produce a unique, sharply
resolving protein
~
of about 32,000 - 34,000 Daltons, that was not produced in cells transfected
with pRK5 alone. The molecular
weight size of this protein is in good agreement with that predicted for LS-
DNase.
EXAMPLE 3
Biological Activitv of LS-DNase
Concentrated cell culture supernatants, prepared as described above, were
tested for DNase activity
in a hyperchromicity assay (Kunitz, J. Gen. Physiol. 12:349-362 (1950);
Kunitz, J. Gen. Physiol. 22:363-377
(1950)), and a methyl green assay (Kurnick, Arch. Biochem. 2,2:41-53 (1950);
Sinicropi, et aI., Anal. Biochem.
2=:351-358 (1994)). In both assays, DNase activity was detected in the
supematants from cells transfected
with pRK5/LS-DNase, but not in the supernatants from cells transfected with
pRK5 alone.
EXAMPLE 4
Resistance to Actin Inhibition
To determine whether the DNA-hydrolytic activity of LS-DNase is inhibited by
actin, a plasmid
nicking assay was used. This assay measures the conversion of supercoiled
double-stranded pBR322 plasmid
DNA to nicked, linear, and degraded forms. Specifically, various DNase samples
were added to 20 }tl of
solution containing 25 g/mi supercoiled double-stranded pBR322 DNA in 25mM
HEPES buffer, ImM
MgCI21 1mM CaCl2, 100 g/mi bovine serum albumin, and the samples were
incubated for 10 minutes at 21 C.
To determine inhibition by actin, the DNase samples were pre-incubated with
actin for 15 minutes at 21 C.
prior to being added to the solution of pBR322 DNA. Reactions were stopped by
the addition of EDTA to a
finat concentration of 10mM, together with xylene cyanol, bromphenol blue, and
glycerol. The integrity of the
25-- pBR322 DNA was analyzed by electrophoresis of the reaction mixtures on
0.8% weight/vol. agarose gels.
After electrophoresis, the gels were stained with a solution of ethidium
bromide and the DNA in the gels was
visuaiized by ultraviolet light.
As expected, human DNase I converted the starting plasmid DNA to degraded
forms, and the DNA-
hydrolytic activity of human DNase I was inhibited by added actin in a
concentration-dependent manner. LS-
DNase converted the starting plasmid DNA to nicked, linear, and degraded
forms, but the DNA-hydrolytic
activity of LS-DNase was not inhibited by concentrations of actin that fully
inhibited human DNase I.
EXAMPLE 5
Pattern of Expression of LS-DNase in Human Tissue
Northern blots of various human tissues were performed using a radiolabeled
probe comprising a
portion of the coding sequence of the cloned LS-DNase cDNA. Expression of LS-
DNase messenger RNA
(mRNA) was found to be highest in liver and spleen. LS-DNase mRNA either was
poorly expressed or not
expressed in other tissues examined. No LS-DNase mRNA was detectable in
pancreas tissue.
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Northern blots of various human tissues also were performed using a
radiolabeled probe comprising
a portion of the nucleotide coding sequence for human DNase I. In contrast to
LS-DNase mRNA. human
DNase I mRNA appeared to be exclusively expressed in pancreas tissue.
EXAMPLE 6
Cjoningof LS-DNase Variant
= A 649 base-pair EcoRI-Pstl fragment of the coding sequence of the cloned LS-
DNase cDNA was used
to screen a murine liver cDNA library (in X-gt10, Clontech, Palo Alto,
California. USA). From about two
million clones screened. more than 60 hybridization positive clones were
identified. Partiai sequencing of six
random positive clones showed that they all originated from the same gene. The
inserted nucleotide sequence
of one of those positive clones was completely sequenced. The insert was 1124
base-pairs in length, including
an open reading frame of 930 base-pairs that encodes a predicted protein,
referred to as murine LS-DNase, that
is 310 amino acid residues in length.
The nucleotide sequence of the 1124 base-pair insert (SEQ. ID. NO: 11) is
shown in Figure 3. The
open reading frame begins with the ATG codon at nucleotide 173 and continues
to the stop codon at nucleotide
1103. The first 75 nucleotides of the open reading frame (the first 25 amino
acid residues of the predicted
protein) encode a putative signal sequence. Accordingly, the predicted murine
mature LS-DNase protein is
285 amino acid residues in length, has a molecular weight of 33,100 Daltons
and a predicted pI of 9.4. The
amino acid sequence of the murine mature LS-DNase is 84% identical to the
amino acid sequence shown in
Figure 1 for human mature LS-DNase.
Northern blots of various mouse tissues were performed using a radiolabeled
probe comprising a
portion of the nucleotide coding sequence for murine LS-DNase. Expression of
murine LS-DNase messenger
RNA (mRNA) was found to be highest in liver and spleen. LS-DNase mRNA either
was poorly expressed or
not expressed in other tissues examined.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Genentech, Inc.
(ii) TITLE OF INVENTION: HUMAN DNASE
(iii) NUMBER OF SEQUENCES: 11
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Dennison Associates
(B) STREET: 133 Richmond Street West, Suite 301
(C) CITY: Toronto
(D) PROVINCE: Ontario
(E) COUNTRY: CANADA
(F) POSTAL CODE: M5H 2L7
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch, 1.44 Mb floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WinPatin (Genentech)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,242,562
(B) FILING DATE: February 3, 1997
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Dennison Associates
(B) REFERENCE/DOCKET NUMBER: JJ-10 189CA
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 368-8313
(B) TELEFAX: (416) 368-1645
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1079 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
GAATTCGGCA CGAGAGCACT CCAAGCACTG CTGTCTTCTC ACAGAGTCTT 50
GAAGCCAGAG CAGCGCCAGG ATGTCACGGG AGCTGGCCCC ACTGCTGCTT 100
CTCCTCCTCT CCATCCACAG CGCCCTGGCC ATGAGGATCT GCTCCTTCAA 150
CGTCAGGTCC TTTGGGGAAA GCAAGCAGGA AGACAAGAAT GCCATGGATG 200
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TCATTGTGAA GGTCATCAAA CGCTGTGACA TCATACTCGT GATGGAAATC 250
AAGGACAGCA ACAACAGGAT CTGCCCCATA CTGATGGAGA AGCTGAACAG 300
AAATTCAAGG AGAGGCATAA CGTACAACTA TGTGATTAGC TCTCGGCTTG 350
GAAGAAACAC ATATAAAGAA CAATATGCCT TTCTCTACAA GGAAAAGCTG 400
GTGTCTGTGA AGAGGAGTTA TCACTACCAT GACTATCAGG ATGGAGACGC 450
AGATGTGTTT TCCAGGGAGC CCTTTGTGGT CTGGTTCCAA TCTCCCCACA 500
CTGCTGTCAA AGACTTCGTG ATTATCCCCC TGCACACCAC CCCAGAGACA 550
TCCGTTAAGG AGATCGATGA GTTGGTTGAG GTCTACACGG ACGTGAAACA 600
CCGCTGGAAG GCGGAGAATT TCATTTTCAT GGGTGACTTC AATGCCGGCT 650
GCAGCTACGT CCCCAAGAAG GCCTGGAAGA ACATCCGCTT GAGGACTGAC 700
CCCAGGTTTG TTTGGCTGAT CGGGGACCAA GAGGACACCA CGGTGAAGAA 750
GAGCACCAAC TGTGCATATG ACAGGATTGT GCTTAGAGGA CAAGAAATCG 800
TCAGTTCTGT TGTTCCCAAG TCAAACAGTG TTTTTGACTT CCAGAAAGCT 850
TACAAGCTGA CTGAAGAGGA GGCCCTGGAT GTCAGCGACC ACTTTCCAGT 900
TGAATTTAAA CTACAGTCTT CAAGGGCCTT CACCAACAGC AAAAAATCTG 950
TCACTCTAAG GAAGAAAACA AAGAGCAAAC GCTCCTAGAC CCAAGGGTCT 1000
CATCTTATTA ACCATTTCTT GCCTCTAAAT AAAATGTCTC TAACAAAAAA 1050
AAAA-A-AAAAA AAAAAAAAAA AAACTCGAG 1079
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 305 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Arg Glu Leu Ala Pro Leu Leu Leu Leu Leu Leu Ser Ile
1 5 10 15
His Ser Ala Leu Ala Met Arg Ile Cys Ser Phe Asn Val Arg Ser
20 25 30
Phe Gly Glu Ser Lys Gln Glu Asp Lys Asn Ala Met Asp Val Ile
35 40 45
Val Lys Val Ile Lys Arg Cys Asp Ile Ile Leu Val Met Glu Ile
50 55 60
Lys Asp Ser Asn Asn Arg Ile Cys Pro Ile Leu Met Glu Lys Leu
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65 70 75
Asn Arg Asn Ser Arg Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser
80 85 90
Ser Arg Leu Gly Arg Asn Thr Tyr Lys Glu Gin Tyr Ala Phe Leu
95 100 105
Tyr Lys Glu Lys Leu Val Ser Val Lys Arg Ser Tyr His Tyr His
110 115 120
Asp Tyr Gln Asp Giy Asp Ala Asp Val Phe Ser Arg Glu Pro Phe
125 130 135
Val Val Trp Phe Gln Ser Pro His Thr Ala Val Lys Asp Phe Val
140 145 150
Ile Ile Pro Leu His Thr Thr Pro Glu Thr Ser Val Lys Glu Ile
155 160 165
Asp Glu Leu Val Glu Val Tyr Thr Asp Val Lys His Arg Trp Lys
170 175 180
Ala Glu Asn Phe Ile Phe Met Gly Asp Phe Asn Ala Gly Cys Ser
185 190 195
Tyr Val Pro Lys Lys Ala Trp Lys Asn Ile Arg Leu Arg Thr Asp
200 205 210
Pro Arg Phe Val Trp Leu Ile Gly Asp Gin Glu Asp Thr Thr Val
215 220 225
Lys Lys Ser Thr Asn Cys Ala Tyr Asp Arg Ile Vai Leu Arg Gly
230 235 240
Gln Glu Ile Val Ser Ser Val Val Pro Lys Ser Asn Ser Val Phe
245 250 255
Asp Phe Gin Lys Ala Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp
260 265 270
Val Ser Asp His Phe Pro Val Glu Phe Lys Leu Gln Ser Ser Arg
275 280 285
Ala Phe Thr Asn Ser Lys Lys Ser Val Thr Leu Arg Lys Lys Thr
290 295 300
Lys Ser Lys Arg Ser
305
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 285 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
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Met Arg Ile Cys Ser Phe Asn Val Arg Ser Phe Gly Glu Ser Lys
1 5 10 15
Gln Glu Asp Lys Asn Ala Met Asp Val Ile Val Lys Val Ile Lys
20 25 30
Arg Cys Asp Ile Ile Leu Val Met Glu Ile Lys Asp Ser Asn Asn
35 40 45
Arg Ile Cys Pro Ile Leu Met Glu Lys Leu Asn Arg Asn Ser Arg
50 55 60
Arg Gly Ile Thr Tyr Asn Tyr Val Ile Ser Ser Arg Leu Gly Arg
65 70 75
Asn Thr Tyr Lys Glu Gln Tyr Ala Phe Leu Tyr Lys Glu Lys Leu
s0 85 90
Vai Ser Val Lys Arg Ser Tyr His Tyr His Asp Tyr Gln Asp Gly
95 100 105
Asp Ala Asp Val Phe Ser Arg Glu Pro Phe Val Val Trp Phe Gln
110 115 120
Ser Pro His Thr Ala Val Lys Asp Phe Val Ile Ile Pro Leu His
125 130 135
Thr Thr Pro Glu Thr Ser Val Lys Glu Ile Asp Giu Leu Val Glu
140 145 150
Val Tyr Thr Asp Val Lys His Arg Trp Lys Ala Glu Asn Phe Ile
155 160 165
Phe Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val Pro Lys Lys
170 175 180
Ala Trp Lys Asn Ile Arg Leu Arg Thr Asp Pro Arg Phe Val Trp
185 190 195
Leu Ile Gly Asp Gln Glu Asp Thr Thr Val Lys Lys Ser Thr Asn
200 205 210
Cys Ala Tyr Asp Arg Ile Val Leu Arg Gly Gin Glu Ile Val Ser
215 220 225
Ser Val Val Pro Lys Ser Asn Ser Val Phe Asp Phe Gln Lys Ala
230 235 240
Tyr Lys Leu Thr Glu Glu Glu Ala Leu Asp Val Ser Asp His Phe
245 250 255
Pro Va1 Glu Phe Lys Leu Gln Ser Ser Arg Ala Phe Thr Asn Ser
260 265 270
Lys Lys Ser Val Thr Leu Arg Lys Lys Thr Lys Ser Lys Arg Ser
275 280 285
(2) INFORMATION FOR SEQ ID NO:4:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Leu Lys Ile Ala Ala Phe Asn Ile Gin Thr Phe Gly Glu Thr Lys
1 5 10 15
Met Ser Asn Ala Thr Leu Val Ser Tyr Ile Val Gln Ile Leu Ser
20 25 30
Arg Tyr Asp Ile Ala Leu Val Gln Glu Val Arg Asp Ser His Leu
35 40 45
Thr Ala Val Gly Lys Leu Leu Asp Asn Leu Asn Gln Asp Ala Pro
50 55 60
Asp Thr Tyr His Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser
65 70 75
Tyr Lys Glu Arg Tyr Leu Phe Val Tyr Arg Pro Asp Gln Val Ser
80 85 90
Ala Val Asp Ser Tyr Tyr Tyr Asp Asp Gly Cys Glu Pro Cys Gly
95 100 105
Asn Asp Thr Phe Asn Arg Glu Pro Ala Ile Val Arg Phe Phe Ser
110 115 120
Arg Phe Thr Glu Val Arg Glu Phe Ala Ile Val Pro Leu His Ala
125 130 135
Ala Pro Giy Asp Ala Val Ala Glu Ile Asp Ala Leu Tyr Asp Val
140 145 150
Tyr Leu Asp Val Gin Glu Lys Trp Gly Leu Glu Asp Val Met Leu
155 160 165
Met Gly Asp Phe Asn Ala Gly Cys Ser Tyr Val Arg Pro Ser Gin
170 175 180
Trp Ser Ser Ile Arg Leu Trp Thr Ser Pro Thr Phe Gln Trp Leu
185 190 195
Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Pro Thr His Cys Ala
200 205 210
Tyr Asp Arg Ile Val Val Ala Gly Met Leu Leu Arg Gly Ala Val
215 220 225
Val Pro Asp Ser Ala Leu Pro Phe Asn Phe Gln Ala Al.a Tyr Gly
230 235 240
Leu Ser Asp Gln Leu Ala G1n Ala Ile Ser Asp His Tyr Pro Val
245 250 255
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Glu Val Met Leu Lys
260
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQLTENCE DESCRIPTION: SEQ ID NO:5:
ACTGTAGTTT AAATTCAACT GGAAAGTGGT CGCTGACATC CAGGG 45
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GATGTCATTG TGAAGGTCAT CAAACGCTGT GACATCATAC TCGTG 45
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GTGTTTTCCA GGGGAGCCCT TTGTGGTCTG GTTCCAATCT CCCCA 45
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CTGGAGGTCT CCCAGCACTG GCAGAGCAAG GACGTGATCC TGCTT 45
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
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CA 02242562 1998-07-09
WO 97/28266 PCT/US97/01506
(A) LENGTH: 45 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GCCCAGCATC ATCGCGAAGT TCCTGGCTGG CTATCACCTC GCGCT 45
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CCAGTACAAG GAGATGTACC TCTTCGTTTA CAGGAAAGAC GCCGT 45
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1124 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GAATTCCGGC CCATTACCTT CATTTCCTTG GGGATTGAAA CGCGTGATGG 50
TGAGTTCCTC AGAGAAGTGA AAGTGACCTA GAGGGATCCA GTAATTCCTG 100
TTATCAGCCT GCTTTATAAG TCAGTGAGCC AGGCACTGTC TTCATCCAGC 150
CTGAAGTCCC AGGAGTGCAA AGATGTCCCT GCACCCAGCT TCCCCACGCC 200
TGGCCTCCCT GCTGCTCTTC ATCCTTGCCC TCCATGACAC CCTGGCCCTA 250
AGGCTCTGCT CCTTCAATGT GAGGTCCTTT GGAGCGAGCA AGAAGGAAAA 300
CCATGAAGCC ATGGATATCA TTGTGAAGAT CATCAAACGC TGTGACCTTA 350
TACTGTTGAT GGAAATCAAG GACAGCAGCA ACAACATCTG TCCCATGCTG 400
ATGGAGAAGC TGAATGGAAA TTCACGAAGA AGCACAACAT ACAACTATGT 450
GATTAGTTCT CGACTTGGAA GAAACACGTA CAAAGAGCAG TATGCCTTCG 500
TCTACAAGGA GAAGCTGGTG TCTGTGAAGA CAAAATACCA CTACCATGAC 550
TATCAGGATG GAGACACAGA CGTGTTTTCC AGGGAGCCCT TTGTGGTTTG 600
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CA 02242562 1998-07-09
WO 97/28266 PCTIUS97/01506
GTTCCATTCC CCCTTTACTG CTGTCAAGGA CTTCGTGATT GTCCCCTTGC 650
ACACAACTCC CGAGACCTCC GTTAAAGAGA TAGATGAGCT GGTCGATGTC 700
TACACGGATG TGAGAAGCCA GTGGAAGACA GAGAATTTCA TCTTCATGGG 750
TGATTTCAAC GCCGGCTGTA GCTATGTCCC CAAGAAGGCC TGGCAGAACA 800
TTCGTTTGAG GACGGACCCC AAGTTTGTTT GGCTGATTGG GGACCAAGAG 850
GACACTACGG TCAAGAAGAG TACCAGCTGT GCCTATGACA GGATTGTGCT 900
TTGTGGACAA GAGATAGTCA ACTCCGTGGT TCCCCGTTCC AGTGGCGTCT 950
TTGACTTTCA GAAAGCTTAT GACTTGTCTG AGGAGGAGGC CCTGGATGTC 1000
AGTGATCACT TTCCAGTTGA GTTTAAGCTA CAGTCTTCAA GGGCCTTCAC 1050
CAACAACAGA AAATCTGTTT CTCTCAAAAA GAGAAAAAAA GGCAATCGCT 1100
CCTAGGTATC ACGCTCCGGA ATTC 1124
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