Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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HUMAN ISRE-BINDING PROTEIN
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human ISRE-
binding
protein and to the use of these sequences in the diagnosis. treatment, and
prevention of cell
proiiferative and immune disorders.
BACKGROUND OF THE INVENTION
Proto-oncogenes are involved in the control of cell growth and division. When
10 inappropriately expressed, these genes may cause cancer. The mvc proto-
oncogene encodes a
member of the helix-loop-helix/leucine zipper superfamily of transcription
factors. The~c gene
is implicated in the control of normal cell proliferation, transformation. and
differentiation.
Transcription of mvc is activated by growth factors as an early-response gene,
and the myc gene
product (MYC) activates transcription of the growth factor delayed-response
genes. The delayed
15 response genes activated by MYC include cyclins A and E, and the human heat
shock protein 70.
The MYC protein also acts to repress transcription of several genes, including
genes encoding the
major histocompatibility (MHC) class 1 antigens and the neuronal cell adhesion
molecule (N
CAM).
The ~ gene family consists of at least seven closely related genes whose
members show
20 distinct tissue and temporal expression patterns. For example, the~c gene
product MYC is
expressed in a wide variety of adult tissues during proliferation and at all
stages of embryonic
development. On the other hand, expression of family members MYCN and MYCL is
restricted
to embryonic brain, kidney, and lung (Hesketh, R. (1995) In 'The Oncoeene
Factsbook, Academic
Press, San Diego CA, pp. 201-210).
25 Expression of myc in untransformed cells is growth factor dependent, ands
expression
is essential for progression through the cell cycle. Cells which cannot
express mvc will not divide,
even in the presence of growth factors. In addition, downregulation of mvc
expression is usually
associated with differentiation. Conversely, overexpression of myc leads to
proliferation that is
independent of growth factor stimulation. This proliferative behavior
eventually causes normal
30 cells to undergo programmed cell death. Genes known to cooperate with myc,
including ras and
raf, may be required to maintain cell viability. Unfortunately, their presence
may also lead to
transformation, rather than apoptosis, in cells overexpressing~c (Hesketh,
supra).
The expression of myc is controlled at both the transcriptional and
translational levels.
The myc gene consists of three exons which show 70-90% identity between
species. The 5' end of
35 exon 1, which is not part of the coding region for MYC protein, contains
two transcriptional
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activation sites, P 1 and P2. Two different proteins, p64 and p67, are
produced by the myc
transcript due to alternative translation initiation between a CUG start codon
near the 3' end of
exon I and an AUG start codon in exon 2 (Hesketh, su ra).
Several transcriptional regulatory elements have been mapped to the ~'
untranslated region
(UTR) of the ~ gene. These elements include AP-I and AP-2, the P2 promoter
elements
MEla2, E2F, and MElal, and the MYC-associated zinc finger protein (MAZ)
binding site. Two
interferon-stimulated response elements (ISREs), found upstream of several
interferon-dependent
genes, have also been mapped to the myc gene 5' UTR. One of the ISREs (irlA)
is located
upstream of P1, and the second ISRE (irlB) is located between P1 and P2. These
regulatory
elements provide both positive and negative regulation over transcription of
the~c gene
(Hesketh, s-upra; Stasiv, Y.Z. et al. ( 1994) Gene 145:267-272).
Factors shown to bind to regulatory elements in the myc ~' UTR include nuclear
factor 1
(NF1), TGF(3" E2F, and MAZ. Repression and activation of myc expression is
controlled by
interactions between these factors. For example, it has been suggested that
the protein factors
binding to the ISRE sequences in c-myc may play a role in the inhibition of
gene transcription
initiated in intron 1. Three binding proteins have been identified which show
affinity for the
ISREs in the mvc regulatory region. Two of these proteins bind to the ISREs in
the~c
regulatory region with low affinity, as well as to ISREs in the interferon-
responsive gene 9-16.
The third protein, IRLB, binds with higher affinity to irlB than to irlA, and
does not bind to the
ISRE in 9-16. The ISRE binding site for IRLB partially overlaps MElal. Melal
is the binding
site for E2F and MAZ, and is necessary for both transcriptional initiation and
a transcriptional
block. Thus, competition for binding between IRLB. E2F, and MAZ may regulate
the state of
transcription of the myc gene (Stasiv, su ra).
The discovery of new human ISRE-binding protein and the polynucleotides
encoding
them satisfies a need in the art by providing new compositions which are
useful in the diagnosis,
prevention, and treatment of cell proliferative and immune disorders.
SUMMARY OF THE INVENTION
The invention features substantially purified polypeptides, human ISRE-binding
protein,
referred to collectively as "HIBP" and individually as "HIBP-I" and "HIBP-2".
In one aspect, the
invention provides a substantially purified polypeptide comprising an amino
acid sequence
selected from the group consisting of SEQ ID NO:1-2. and fragments thereof.
The invention further provides a substantially purified variant having at
least 90% amino
acid identity to at least one of the amino acid sequences selected from the
group consisting of SEQ
ID NO:1-2, and fragments thereof. The invention also provides an isolated and
purified
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polynucleotide encoding the polypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-2, and fragments thereof. The invention also
includes an
isolated and purified polynucleotide variant having at least 70%
polynucleotide sequence identity
to the polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from
the group consisting of SEQ ID NO:1-2, and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide
which
hybridizes under stringent conditions to the polynucleotide encoding the
polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID NO:1-2.
and fragments
thereof. The invention also provides an isolated and purified poiynucleotide
having a sequence
10 which is complementary to the polynucleotide encoding the polypeptide
comprising the amino
acid sequence selected from the group consisting of SEQ ID NO:I-2, and
fragments thereof.
The invention also provides an isolated and purified polynucleotide comprising
a
polynucleotide sequence selected from the group consisting of SEQ ID N0:3-4,
and fragments
thereof. The invention further provides an isolated and purified
polynucleotide variant having at
I S least 70% polynucleotide sequence identity to the polynucleotide sequence
selected from the
group consisting of SEQ ID N0:3-4, and fragments thereof. 'The invention also
provides an
isolated and purified polynucleotide having a sequence which is complementary
to the
polynucleotide comprising a poiynucleotide sequence selected from the group
consisting of SEQ
ID N0:3-4, and fragments thereof.
20 The invention also provides a method for detecting a polynucleotide in a
sample
containing nucleic acids, the method comprising the steps of: (a) hybridizing
the complement of
the polynucleotide sequence to at least one of the polynucleotides of the
sample, thereby forming a
hybridization complex; and (b) detecting the hybridization complex, wherein
the presence of the
hybridization complex correlates with the presence of a polynucleotide in the
sample. In one
25 aspect, the method further comprises amplifying the polynucleotide prior to
hybridization.
The invention further provides an expression vector containing at least a
fragment of the
polynucleotide encoding the poiypeptide comprising an amino acid sequence
selected from the
group consisting of SEQ ID NO:1-2, and fragments thereof. In another aspect,
the expression
vector is contained within a host cell.
30 The invention also provides a method for producing a polypeptide, the
method comprising
the steps of: (a) culturing the host cell containing an expression vector
containing at least a
fragment of a polynucleotide under conditions suitable for the expression of
the polypeptide; and
(b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a
substantially
35 purified polypeptide having the amino acid sequence selected from the group
consisting of SEQ
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ID NO: l-2, and fragments hereof, in conjunction with a suitable
pharmaceutical carrier.
The invention further includes a purified antibody which binds to a
polypeptide selected
from the group consisting of SEQ ID NO:1-2, and fragments thereof. The
invention also provides
a purified agonist and a purified antagonist to the polypeptide.
5 The invention also provides a method for treating or preventing a disorder
associated with
decreased expression or activity of HIBP, the method comprising administering
to a subject in
need of such treatment an effective amount of a pharmaceutical composition
comprising a
substantially purified polypeptide having the amino acid sequence selected
from the group
consisting of SEQ ID NO:1-2, and fragments thereof, in conjunction with a
suitable
pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder
associated with
increased expression or activity of HIBP, the method comprising administering
to a subject in
need of such treatment an effective amount of an antagonist of a polypeptide
having an amino acid
sequence selected from the group consisting of SEQ ID NO:1-2. and fragments
thereof.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1 A-F show the amino acid sequence (SEQ ID NO:1 ) and nucleic acid
sequence
(SEQ ID N0:3) of H1BP-1. The aiignment was produced using MACDNASIS PRO
software
(Hitachi Software Engineering, S. San Francisco CA).
Figures 2A-D show the amino acid sequence (SEQ ID N0:2) and nucleic acid
sequence
(SEQ ID N0:4) of HIBP-2. The alignment was produced using MACDNASIS PRO
software.
Figures 3A and 3B show the amino acid sequence alignment beriveen HIBP-1
(2518547;
SEQ ID NO:1 ), HIBP-2 ( 1640136: SEQ ID N0:2), and human IRLB (GI 33969; SEQ
ID NO:S),
produced using the multisequence alignment program of LASERGENE software
(DNASTAR,
Madison WI).
DESCRIPTION OF THE INVENTION
Before the present proteins, nucleotide sequences, and methods are described,
it is
understood that this invention is not limited to the particular machines,
materials and methods
described, as these may vary. It is also to be understood that the terminology
used herein is for the
purpose of describing particular embodiments only, and is not intended to
limit the scope of the
present invention which will be limited only by the appended claims.
It must be nated that as used herein and in the appended claims, the singular
forms "a,"
"an," and "the" include plural reference unless the context clearly dictates
otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such host cells,
and a reference to "an
antibody" is a reference to one or more antibodies and equivalents thereof
known to those skilled
in the art, and so forth.
a
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Unless defined otherwise, all technical and scientific terms used herein have
the same
meanines as commonly understood by one of ordinary skill in the art to which
this invention
belongs. Although any machines, materials, and methods similar or equivalent
to those described
herein can be used to practice or test the present invention, the preferred
machines, materials and
methods are now described. All publications mentioned herein are cited for the
purpose of
describine and disclosing the cell lines, protocols, reagents and vectors
which are reported in the
publications and which might be used in connection with the invention. Nothing
herein is to be
construed as an admission that the invention is not entitled to antedate such
disclosure by virtue of
prior invention.
DEFINITIONS
"HIBP" refers to the amino acid sequences of substantially purified HIBP
obtained from
any species. particularly a mammalian species, including bovine, ovine.
porcine, murine, equine,
and preferably the human species, from any source, whether natural, synthetic,
semi-synthetic, or
recombinant.
15 The term "agonist" refers to a molecule which, when bound to HIBP,
increases or
prolongs the duration of the effect of HIBP. Agonists may include proteins,
nucleic acids,
carbohydrates, or any other molecules which bind to and modulate the effect of
HIBP.
An "allelic variant" is an alternative form of the gene encoding HIBP. Allelic
variants
may result from at least one mutation in the nucleic acid sequence and may
result in altered
20 mRNAs or in polypeptides whose structure or function may or may not be
altered. Any given
natural or recombinant gene may have none, one, or many allelic forms. Common
mutational
changes which give rise to allelic variants are generally ascribed to natural
deletions. additions, or
substitutions of nucleotides. Each of these types of changes may occur alone,
or in combination
with the others, one or more times in a gmen sequence.
25 "Altered" nucleic acid sequences encoding HIBP include those sequences with
deletions,
insertions. or substitutions of different nucleotides, resulting in a
polynucleotide the same as HIBP
or a polypeptide with at least one functional characteristic of HIBP. Included
within this definition
are polymorphisms which may or may not be readily detectable using a
particular oligonucleotide
probe of the polynucleotide encoding HIBP, and improper or unexpected
hybridization to allelic
30 variants, with a locus other than the normal chromosomal locus for the
polynucleotide sequence
encoding HIBP. The encoded protein may also be "altered," and may contain
deletions, insertions,
or substitutions of amino acid residues which produce a silent change and
result in a functionally
equivalent HIBP. Deliberate amino acid substitutions may be made on the basis
of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the
amphipathic nature of the
35 residues. as long as the biological or immunological activity of HIBP is
retained. For example,
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negatively charged amino acids may include aspartic acid and glutamic acid,
positively charged
amino acids may include lysine and arginine, and amino acids with uncharged
polar head groups
having similar hydrophilicity values may include lcucine, isoleucine. and
valine: glycine and
alanine: asparagine and glutamine; serine and threonine: and phenylalanine and
tyrosine.
5 The terms "amino acid" or "amino acid sequence" refer to an oligopeptide,
peptide,
polypeptide, or protein sequence. or a fragment of any of these, and to
naturally occurring or
synthetic molecules. In this context, "fragments." "immunogenic fragments," or
"antigenic
fragments" refer to fragments of HIBP which are preferably at least ~ to about
1 ~ amino acids in
length, most preferably at least 14 amino acids, and which retain some
biological activity or
10 immunological activity of HIBP. Where "amino acid sequence" is recited to
refer to an amino
acid sequence of a naturally occurring protein molecule, "amino acid sequence"
and like terms are
not meant to limit the amino acid sequence to the complete native amino acid
sequence associated
with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic
acid sequence.
15 Amplification is generally carried out using polymerase chain reaction
(PCR) technologies well
known in the art.
The term "antagonist" refers to a molecule which, when bound to HIBP,
decreases the
amount or the duration of the effect of the biological or immunological
activity of HIBP.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or
any other molecules
20 which decrease the effect of HIBP.
The term "antibody" refers to intact molecules as well as to fragments
thereof, such as
Fab, F(ab'),, and Fv fragments, which are capable of binding the epitopic
determinant. Antibodies
that bind HIBP polypeptides can be prepared using intact polypeptides or using
fragments
containing small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide
25 used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be
derived from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier protein if
desired. Commonly
used carriers that are chemically coupled to peptides include bovine serum
albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to
immunize the animal.
The term "antigenic determinant" refers to that fragment of a molecule (i.e.,
an epitope)
30 that makes contact with a particular antibody. When a protein or a fragment
of a protein is used to
immunize a host animal, numerous regions of the protein may induce the
production of antibodies
which bind specifically to antigenic determinants (given regions or three-
dimensional structures on
the protein). An antigenic determinant may compete with the intact antigen
(i.e., the immunogen
used to elicit the immune response) for binding to an antibody.
35 The term "antisense" refers to any composition containing a nucleic acid
sequence which
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is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules
may be produced by any method including synthesis or transcription. Once
introduced into a cell,
the complementary nucleotides combine with natural sequences produced by the
cell to form
duplexes and to block either transcription or translation. The designation
"negative" can refer to
the antisense strand, and the designation "positive" can refer to the sense
strand.
The term "biologically active." refers to a protein having structural.
regulatory, or
biochemical functions of a naturally occurring molecule. Likewise,
"immunologically active"
refers to the capability of the natural. recombinant, or synthetic HIBP, or of
any oligopeptide
thereof, to induce a specific immune response in appropriate animals or cells
and to bind with
l0 specific antibodies.
The terms "complementary" or "complementarily" refer to the natural binding of
polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds
to the
complementary sequence "3' T-C-A ~'." Complementarily between two single-
stranded molecules
may be "partial," such that only some of the nucleic acids bind, or it may be
"complete," such that
total complementarily exists between the single stranded molecules. The degree
of
complementarily between nucleic acid strands has significant effects on the
efficiency and strength
of the hybridization between the nucleic acid strands. This is of particular
importance in
amplification reactions, which depend upon binding between nucleic acids
strands, and in the
design and use of peptide nucleic acid (PNA) molecules.
A "composition comprising a given polynucleotide sequence" or a "composition
comprising a given amino acid sequence" refer broadly to any composition
containing the given
polynucleotide or amino acid sequence. The composition may comprise a dry
formulation or an
aqueous solution. Compositions comprising poiynucleotide sequences encoding
HIBP or
fragments of HIBP may be employed as hybridization probes. The probes may be
stored in freeze-
dried form and may be associated with a stabilizing agent such as a
carbohydrate. In
hybridizations, the probe may be deployed in an aqueous solution containing
salts (e.g., NaCI),
detergents (e.g., sodium dodecyl sulfate: SDS), and other components (e.g.,
Denhardt's solution,
dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been
resequenced to
resolve uncalled bases, extended using XL-PCR kit (Perkin-Elmer, Norwalk CT)
in the 5' and/or
the 3' direction, and resequenced, or which has been assembled from the
overlapping sequences of
more than one Incyte Clone using a computer program for fragment assembly,
such as the
GELVIEW Fragment Assembly system (GCG, Madison WI). Some sequences have been
both
extended and assembled to produce the consensus sequence.
The term "correlates with expression of a polynucieotide" indicates that the
detection of
7
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the presence of nucleic acids, the same or related to a nucleic acid sequence
encoding HIBP, by
northern analysis is indicative of the presence of nucleic acids encoding HIBP
in a sample, and
thereby correlates with expression of the transcript from the polynucleotide
encoding HIBP.
A "deletion" refers to a change in the amino acid or nucleotide sequence that
results in the
absence of one or more amino acid residues or nucleotides.
The term "derivative" refers to the chemical modification of a polypeptide
sequence, or a
polynucieotide sequence. Chemical modifications of a polynucleotide sequence
can include, for
example, replacement of hydrogen by an alkyl, acyl, or amino group. A
derivative polynucleotide
encodes a poiypeptide -which retains at least one biological or immunological
function of the
l0 natural molecule. A derivative polypeptide is one modified by
glycosylation, pegylation, or any
similar process that retains at least one biological or immunological function
of the polypeptide
from which it was derived.
The term "similarity" refers to a degree of complementarily. There may be
partial
similarity or complete similarity. The word "identity" may substitute for the
word "similarity." A
15 partially complementary sequence that at least partially inhibits an
identical sequence from
hybridizing to a target nucleic acid is referred to as "substantially
similar." The inhibition of
hybridization of the completely complementary sequence to the target sequence
may be examined
using a hybridization assay (Southern or northern blot. solution
hybridization, and the like) under
conditions of reduced stringency. A substantially similar sequence or
hybridization probe will
20 compete for and inhibit the binding of a completely similar (identical)
sequence to the target
sequence under conditions of reduced stringency. This is not to say that
conditions of reduced
stringency are such that non-specific binding is permitted, as reduced
stringency conditions
require that the binding of two sequences to one another be a specific (i.e.,
a selective) interaction.
The absence of non-specific binding may be tested by the use of a second
target sequence which
25 lacks even a partial degree of complementarily (e.g., less than about 30%
similarity or identity).
In the absence of non-specific binding, the substantially similar sequence or
probe will not
hybridize to the second non-complementary target sequence.
The phrases "percent identity" or "% identity" refer to the percentage of
sequence
similarity found in a comparison of two or more amino acid or nucleic acid
sequences. Percent
30 identity can be determined electronically, e.g., by using the MEGALIGN
program (DNASTAR)
which creates alignments between two or more sequences according to methods
selected by the
user, e.g., the clustal method. (See, e.g., Higgins, D.G. and P.M. Sharp (
1988) Gene 73:237-244.)
The clustal algorithm groups sequences into clusters by examining the
distances between all pairs.
The clusters are aligned pairwise and then in groups. The percentage
similarity between two
35 amino acid sequences. e.g.. sequence A and sequence B, is calculated by
dividing the length of
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sequence A, minus the number of gap residues in sequence A, minus the number
of gap residues
in sequence B, into the sum of the residue matches between sequence A and
sequence B, times
one hundred. Gaps of low or of no similarity between the two amino acid
sequences are not
included in determining percentage similarity. Percent identity between
nucleic acid sequences
5 can also be counted or calculated by other methods known in the art, e.g.,
the Jotun Hein method.
(See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.) Identity between
sequences can also
be determined by other methods known in the art, e.g., by varying
hybridization conditions.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may
contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of
the elements
required for stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino
acid
sequence in the non-antigen binding regions has been altered so that the
antibody more closely
resembles a human antibody, and still retains its original binding ability.
"Hybridization" refers to any process by which a strand of nucleic acid binds
with a
complementary strand through base pairing.
The term "hybridization complex"' refers to a complex formed between rivo
nucleic acid
sequences by virtue of the formation of hydrogen bonds between complementary
bases. A
hybridization complex may be formed in solution (e.g., Cot or Rat analysis) or
formed between one
nucleic acid sequence present in solution and another nucleic acid sequence
immobilized on a
20 solid support (e.g., paper, membranes, filters, chips, pins or glass
slides, or any other appropriate
substrate to which cells or their nucleic acids have been fixed).
The words "insertion" or "addition" refer to changes in an amino acid or
nucleotide
sequence resulting in the addition of one or more amino acid residues or
nucleotides, respectively,
to the sequence found in the naturally occurring molecule.
25 "Immune response'' can refer to conditions associated with inflammation,
trauma, immune
disorders, or infectious or genetic disease, etc. These conditions can be
characterized by
expression of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which
may affect cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on
a substrate.
30 The terms ''element" or "array element'' in a microarray context, refer to
hybridizable
polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of HIBP. For example,
modulation
may cause an increase or a decrease in protein activity. binding
characteristics. or any other
biological, functional, or immunological properties of HiBP.
35 The phrases "nucleic acid'' or "nucleic acid sequence." as used herein,
refer to a
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nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These
phrases also refer to
DNA or RNA of genomic or synthetic origin which may be single-stranded or
double-stranded
and may represent the sense or the antisense strand, to peptide nucleic acid
(PNA), or to any
DNA-like or RNA-like material. In this context, "fragments'' refers to those
nucleic acid
sequences which comprise a region of unique polynucleotide sequence that
specifically identifies
SEQ ID N0:3-4, for example, as distinct from any other sequence in the same
genome. For
example, a fragment of SEQ ID N0:3-4 is useful in hybridization and
amplification technologies
and in analogous methods that distinguish SEQ ID N0:3-4 from related
polynucleotide sequences.
A fragment of SEQ ID N0:3-4 is at least about I S-20 nucleotides in length.
The precise length of
l0 the fragment of SEQ ID N0:3-4 and the region of SEQ ID N0:3-4 to which the
fragment
corresponds are routinely determinable by one of ordinary skill in the art
based on the intended
purpose for the fragment. In some cases, a fragment. when translated, would
produce
polypeptides retaining some functional characteristic, e.g., antigenicity, or
structural domain
characteristic, e.g., ATP-binding site, of the full-length polypeptide.
The terms ''operably associated" or "operably linked" refer to functionally
related nucleic
acid sequences. A promoter is operably associated or operably linked with a
coding sequence if
the promoter controls the translation of the encoded polypeptide. While
operably associated or
operably linked nucleic acid sequences can be contiguous and in the same
reading frame, certain
genetic elements, e.g., repressor genes, are not contiguously linked to the
sequence encoding the
polypeptide but still bind to operator sequences that control expression of
the polypeptide.
The term "oligonucleotide" refers to a nucleic acid sequence of at least about
6
nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most
preferably about 20
to 25 nucleotides. which can be used in PCR amplification or in a
hybridization assay or
microarray. "Oligonucleotide" is substantially equivalent to the terms
"amplimer," "primer,"
''oligomer," and "probe," as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene
agent which
comprises an oligonucleotide of at least about 5 nucleotides in length linked
to a peptide backbone
of amino acid residues ending in lysine. The terminal lysine confers
solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop
transcript
elongation. and may be pegylated to extend their lifespan in the cell.
The term ''sample" is used in its broadest sense. A sample suspected of
containing nucleic
acids encoding HIBP, or fragments thereof, or HIBP itself, may comprise a
bodily fluid; an extract
from a cell, chromosome, organelle, or membrane isolated from a cell; a cell;
genomic DNA,
RNA, or cDNA, in solution or bound to a substrate: a tissue; a tissue print;
etc.
The terms "specific binding" or "specifically binding" refer to that
interaction between a
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protein or peptide and an agonist, an antibody, or an antagonist. The
interaction is dependent upon.
the presence of a particular structure of the protein, e.g., the antigenic
determinant or epitope,
recognized by the binding molecule. For example, if an antibody is specific
for epitope "A," the
presence of a polypeptide containing the epitope A, or the presence of free
unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the amount of
labeled A that binds
to the antibody.
The term "stringent conditions'' refers to conditions which permit
hybridization between
polynucleotides and the claimed polynucleotides. Stringent conditions can be
defined by salt
concentration. the concentration of organic solvent, e.g., formamide,
temperature, and other
conditions well known in the art. In particular, stringency can be increased
by reducing the
concentration of salt, increasing the concentration of formamide, or raising
the hybridization
temperature.
The term "substantially purified" refers to nucleic acid or amino acid
sequences that are
removed from their natural environment and are isolated or separated, and are
at least about 60%
free, preferably about 75% free, and most preferably about 90% free from other
components with
which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or
nucleotides by
different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including
membranes. filters,
chips, slides. wafers, fibers, magnetic or nonmagnetic beads, gels, tubing,
plates, polymers,
microparticles and capillaries. The substrate can have a variety of surface
forms, such as wells,
trenches, pins, channels and pores, to which polynucleotides or polypeptides
are bound.
"Transformation" describes a process by which exogenous DNA enters and changes
a
recipient cell. Transformation may occur under natural or artificial
conditions according to
various methods well known in the art, and may rely on any known method for
the insertion of
foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The
method for
transformation is selected based on the type of host cell being transformed
and may include, but is
not limited to, viral infection, electroporation, heat shock, iipofection, and
particle bombardment.
The term "transformed" cells includes stably transformed cells in which the
inserted DNA is
capable of replication either as an autonomously replicating plasmid or as
part of the host
chromosome. as well as transiently transformed cells which express the
inserted DNA or RNA for
limited periods of time.
A "variant" of HIBP polypeptides refers to an amino acid sequence that is
altered by one
or more amino acid residues. The variant may have "conservative" changes,
wherein a substituted
amino acid has similar structural or chemical properties (e.g., replacement of
leucine with
CA 02344657 2001-05-02
WO 00/17354 PCT/US99/21281
isoleucine). More rarely, a variant may have "nonconservative'~ changes (e.g.,
replacement of
glycine with tryptophan). Analogous minor variations may also include amino
acid deletions or
insertions. or both. Guidance in determining which amino acid residues may be
substituted,
inserted, or deleted without abolishing biological or immunological activity
may be found using
computer programs well known in the art, for example, LASERGENE sof=tware
(DNASTAR).
The term "variant," when used in the context of a polynucleotide sequence, may
encompass a polynucleotide sequence related to HIBP. This definition may also
include, for
example. ''allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice
variant may have significant identity to a reference molecule, but will
generally have a greater or
lesser number of polynucleotides due to alternate splicing of exons during
mRNA processing. The
corresponding polypeptide may possess additional functional domains or an
absence of domains.
Species variants are polynucleotide sequences that vary from one species to
another. The resulting
polypeptides generally will have significant amino acid identity relative to
each other. A
polymorphic variant is a variation in the polynucleotide sequence of a
particular gene between
individuals of a given species. Polymorphic variants also may encompass
"single nucleotide
polymorphisms" (SNPs) in which the polynucleotide sequence varies by one base.
The presence
of SNPs may be indicative of, for example, a certain population, a disease
state, or a propensity for
a disease state.
THE INVENTION
The invention is based on the discovery of new human ISRE-binding proteins
(HIBP), the
polynucleotides encoding HIBP, and the use of these compositions for the
diagnosis, treatment, or
prevention of cell proliferative and immune disorders.
Nucleic acids encoding the HIBP-1 of the present invention were identified in
Incyte
Clone 2518547 from the brain tumor cDNA library (BRAITUT21 ) using a computer
search for
nucleotide and/or amino acid sequence alignments. A consensus sequence, SEQ ID
N0:3, was
derived from the following overlapping and/or extended nucleic acid sequences:
Incyte Clones
2518547H1 (BRAITUT21), 1509622H1 and 150962276 (LUNGNOT14), 1562945F6
(SPLNNOT04), 1640136X 18C 1, l 640136X 13C 1, and 1640136X21 C 1 (UTRSNOT06),
and
143201481 (BEPINONO l ).
In one embodiment, the invention encompasses a polypeptide comprising the
amino acid
sequence of SEQ ID NO:1, as shown in Figures 1 A-F. HIBP-1 is 245 amino acids
in length and
has a potential casein kinase II phosphorylation site at residue S27; and two
potential protein
kinase C phosphorylation sites at residues SS and 7229. HIBP-I has a potential
signal peptide
from residue M 1 through 822, identified by SPScan. As shown in Figures 3A and
3B, HIBP-1
has chemical and structural similarity with human IRLB (GI 33969; SEQ ID
NO:S). In particular,
12
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- WO 00/17354 PCT/US99/21281
HIBP-1 and IRLB share 36% identity and have similar isoeiectric points (8.4
and 8.7,
respectively). In addition. the potential protein kinase C phosphorylation
site in HIBP-i at residue
7229 is conserved as a potential protein kinase A phosphorylation site in IRLB
at residue 5176. A
fragment of SEQ ID N0:3 from about nucleotide 797 to about nucleotide 856 is
useful in
hybridization or amplification technologies to identify SEQ ID N0:3 and to
distinguish between
SEQ ID N0:3 and a related sequence.
Northern analysis shows the expression of H1BP-I in various libraries, at
least 78% of
which are associated with cell proliferation and at least 28% of which are
associated with
inflammation and the immune response. Of particular note is the expression of
HIBP-I in
reproductive, hematopoietic/immune, and nervous tissues.
Nucleic acids encoding the HIBP-2 of the present invention were identified in
Incyte
Clone 1640136 from the myometrial tissue cDNA library (UTRSNOT06) using a
computer search
for nucleotide and/or amino acid sequence alignments. A consensus sequence.
SEQ ID N0:4, was
derived from the following overlapping and/or extended nucleic acid sequences:
Incyte Clones
1509622H1, 150962276, and 1509622F6 (LUNGNOT14), 494466H1 (HNT2NOT01),
1640136H1 and 1640136X17C1 (UTRSNOT06), 1562945 F6(SPLNNOT04),and 143201481
(BEPINONO 1 ).
In one embodiment, the invention encompasses a polypeptide comprising
the~amino acid
sequence of SEQ ID N0:2, as shown in Figures 2A-D. HIBP-2 is 220 amino acids
in length and
has a potential casein kinase II phosphorylation site at residue S27; and two
potential protein
kinase C phosphorylation sites at residues S5 and 7204. HIBP-2 has a potential
signal peptide
from residue Ml through 822, identified by SPScan. As shown in Figures 3A and
3B, HIBP-2
has chemical and structural similarity with HIBP-1 and is considered to be a
splice variant of
HIBP-1. In addition, as shown in Figures 3A and 3B, HIBP-2 has chemical and
structural
similarity with human IRLB (GI 33969; SEQ ID N0:5). In particular, HIBP-2 and
IRLB share
29% identity and have similar isoelectric points (8.73 and 8.70,
respectively). In addition, the
potential protein kinase C phosphorylation site in HIBP-2 at residue 7204 is
conserved as a
potential protein kinase A phosphorylation site in IRLB at residue S 176. A
fragment of SEQ ID
N0:4 from about nucleotide 513 to about nucleotide 575 is useful in
hybridization or amplification
technologies to identify SEQ ID N0:4 and to distinguish between SEQ ID N0:4
and a related
sequence.
Northern analysis shows the expression of HIBP-2 in various libraries, at
least $1 % of
which are associated with cell proliferation and at least 23% of which are
associated with
inflammation and the immune response. Of particular note is the expression of
HIBP-2 in
reproductive, hematopoietic/immune, and nervous tissues.
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WO 00/17354 PCT/US99/21281
The invention also encompasses HIBP variants. A preferred HIBP variant is one
which
has at least about 80%, more preferably at least about 90%, and most
preferably at least about 95%
amino acid sequence identity to the HIBP amino acid sequence, and which
contains at least one
functional or structural characteristic of HIBP.
The invention also encompasses polynucleotides which encode HIBP. In a
particular
embodiment, the invention encompasses a polynucleotide sequence comprising a
sequence
selected from the group consisting of SEQ ID N0:3-4, which encodes HIBP.
The invention also encompasses a variant of a polynucleotide sequence encoding
HIBP.
In particular, such a variant polynucleotide sequence will have at least about
70%. more preferably
at least about 85%, and most preferably at least about 95% polynucleotide
sequence identity to the
polynucleotide sequence encoding HIBP. A particular aspect of the invention
encompasses a
variant of a polynucleotide sequence comprising a sequence selected from the
group consisting of
SEQ ID N0:3-4, which has at least about 70%, more preferably at least about
85%, and most
preferably at least about 95% polynucleotide sequence identity to a nucleic
acid sequence selected
from the group consisting of SEQ ID N0:3-4. Any one of the polynucleotide
variants described
above can encode an amino acid sequence which contains at least one functional
or structural
characteristic of HIBP.
It will be appreciated by those skilled in the art that as a result of the
degeneracy of the
genetic code, a multitude of polynucleotide sequences encoding HIBP, some
bearing minimal
similarity to the polynucleotide sequences of any known and naturally
occurring gene, may be
produced. Thus, the invention contemplates each and every possible variation
of polynucleotide
sequence that could be made by selecting combinations based on possible codon
choices. These
combinations are made in accordance with the standard triplet genetic code as
applied to the
polynucleotide sequence of naturally occurring HIBP, and all such variations
are to be considered
as being specifically disclosed.
Although nucleotide sequences which encode HIBP and its variants are
preferably capable
of hybridizing to the nucleotide sequence of the naturally occurring HIBP
under appropriately
selected conditions of stringency, it may be advantageous to produce
nucleotide sequences
encoding HIBP or its derivatives possessing a substantially different codon
usage, e.g., inclusion
of non-naturally occurring codons. Codons may be selected to increase the rate
at which
expression of the peptide occurs in a particular prokaryotic or eukaryotic
host in accordance with
the frequency with which particular codons are utilized by the host. Other
reasons for
substantially altering the nucleotide sequence encoding HIBP and its
derivatives without altering
the encoded amino acid sequences include the production of RNA transcripts
having more
desirable properties. such as a greater half life. than transcripts produced
from the naturally
to
CA 02344657 2001-05-02
WO 00/17354 PCT/US99/21281
occurring sequence.
The invention also encompasses production of DNA sequences which encode HIBP
and
HIBP derivatives, or fragments thereof, entirely by synthetic chemistry. After
production, the
synthetic sequence may be inserted into any of the many available expression
vectors and cell
systems using reagents well known in the art. Moreover, synthetic chemistry
may be used to
introduce mutations into a sequence encoding HIBP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to the claimed polynucleotide sequences, and, in particular. to
those shown in SEQ ID
N0:3-4, and fragments thereof under various conditions of stringency. (See,
e.g., Wahl, G.M. and
l0 S.L. Berger ( 1987) Methods Enzymol. 152:399-407; Kimmel, A.R. ( 1987)
Methods Enzymol.
1 X2:507-51 1.) For example, stringent salt concentration will ordinarily be
less than about 750 mM
NaCI and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and
50 mM trisodium
citrate. and most preferably less than about 250 mM NaCI and 25 mM trisodium
citrate. Low
stringency hybridization can be obtained in the absence of organic solvent,
e.g., formamide, while
high stringency hybridization can be obtained in the presence of at least
about 35% formamide,
and most preferably at least about SO% formamide. Stringent temperature
conditions will
ordinarily include temperatures of at least about 30°C, more preferably
of at least about 37°C, and
most preferably of at least about 42°C. Varying additional parameters,
such as hybridization time,
the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the
inclusion or exclusion
of carrier DNA, are well known to those skilled in the art. Various levels of
stringency are
accomplished by combining these various conditions as needed. In a preferred
embodiment,
hybridization will occur at 30°C in 750 mM NaCI, 75 mM trisodium
citrate, and I% SDS. In a
more preferred embodiment, hybridization will occur at 37°C in 500 mM
NaCI, 50 mM trisodium
citrate. 1% SDS, 35% formamide, and 100 ~g/ml denatured salmon sperm DNA
(ssDNA). In a
most preferred embodiment, hybridization will occur at 42°C in 250 mM
NaCI, 25 mM trisodium
citrate, 1% SDS, 50 % formamide, and 200 ~g/ml ssDNA. Useful variations on
these conditions
will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash
stringency conditions can be defined by salt concentration and by temperature.
As above, wash
stringency can be increased by decreasing salt concentration or by increasing
temperature. For
example, stringent salt concentration for the wash steps will preferably be
less than about 30 mM
NaCI and 3 mM trisodium citrate, and most preferably less than about 1 ~ mM
NaCI and l.5 mM
trisodium citrate. Stringent temperature conditions for the wash steps will
ordinarily include
temperature of at least about 25°C, more preferably of at least about
42°C, and most preferably of
at least about 68°C. In a preferred embodiment. wash steps will occur
at 25°C in 30 mM NaCI, 3
!5
CA 02344657 2001-05-02
WO 00/17354 PCT/US99/21281
mM trisodium citrate. and 0.1 % SDS. In a more preferred embodiment, wash
steps will occur at
42°C in 1~ mM NaCI. 1.5 mM trisodium citrate, and 0.1% SUS. In a most
preferred embodiment,
wash steps will occur at 68°C in 15 mM NaCI, 1.5 mM trisodium citrate.
and 0.1% SDS.
Additional variations on these conditions will be readily apparent to those
skilled in the art.
Methods for DNA sequencing are well known in the art and may be used to
practice any
of the embodiments of the invention. The methods may employ such enzymes as
the Klenow
fragment of DNA polymerise 1. SEQUENASE (US Biochemical, Cleveland OH), Taq
polymerise
(Perkin-Elmer), thermosiable T'7 polymerise (Amersham Pharmacia Biotech,
Piscataway NJ), or
combinations of polymerises and proofreading exonucleases such as those found
in the
ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably, sequence
preparation is automated with machines such as the MICROLAB 2200 (Hamilton,
Reno NV),
Pettier Thermal Cycler 200 (PTC200; MJ Research, Watertown MA) and the ABI
CATALYST
800 (Perkin-Elmer). Sequencing is then carried out using either ABI 373 or 377
DNA sequencing
systems (Perkin-Elmer) or the MEGABACE 1000 DNA sequencing system (Molecular
Dynamics,
Sunnyvale CA). The resulting sequences are analyzed using a variety of
algorithms which are
well known in the art. (See, e.g., Ausubel, F.M. ( 1997) Short Protocols in
Molecular Bioloey,
John Wiley & Sons, New York NY, unit 7.7; Meyers, R.A. (1995) Molecular
Biolo~v and
Biotechnoloey, Wiley VCH, New York NY, pp. 856-853.)
The nucleic acid sequences encoding HIBP may be extended utilizing a partial
nucleotide
sequence and employing various PCR-based methods known in the art to detect
upstream
sequences, such as promoters and regulatory elements. For example, one method
which may be
employed. restriction-site PCR, uses universal and nested primers to amplify
unknown sequence
from genomic DNA within a cloning vector. (See, e.g.. Sarkar, G. ( 1993) PCR
Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend in divergent
directions to
amplify unknown sequence from a circularized template. The template is derived
from restriction
fragments comprising a known genomic locus and surrounding sequences. (See,
e.g., Triglia, T. et
al. (1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves
PCR
amplification of DNA fragments adjacent to known sequences in human and yeast
artificial
chromosome DNA. (See, e.g., Lagerstrom, M. et al. ( I991 ) PCR Methods Applic.
1:111-119.) In
this method. multiple restriction enzyme digestions and ligations may be used
to insert an
engineered double-stranded sequence into a region of unknown sequence before
performing PCR.
Other methods which may be used to retrieve unknown sequences are known in the
art. (See, e.g.,
Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060). Additionally, one
may use PCR,
nested primers. and PROMOTERFINDER libraries (Clontech, Palo Alto CA) to walk
genomic
DNA. This procedure avoids the need to screen libraries and is useful in
finding intron/exon
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WO 00/17354 PCT/US99/21281
junctions. For all PCR-based methods, primers may be designed using
commercially available
software, such as OLIGO 4.06 Primer Analysis software (National Biosciences,
Plymouth MN) or
another appropriate program, to be about 22 to 30 nucleotides in length, to
have a GC content of
about 50% or more, and to anneal to the template at temperatures of about
6$°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that
have been
size-selected to include larger cDNAs. In addition, random-primed libraries,
which often include
sequences containing the 5' regions of genes, are preferable for situations in
which an oligo d(T)
library does not yield a full-length cDNA. Genomic libraries may be useful for
extension of
sequence into S' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used
to
analyze the size or confirm the nucleotide sequence of sequencing or PCR
products. In particular,
capillary sequencing may employ flowable polymers for electrophoretic
separation. four different
nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled
device camera for
detection of the emitted wavelengths. Output/light intensity may be converted
to electrical signal
using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Perkin-
Elmer),
and the entire process from loading of samples to computer analysis and
electronic data display
may be computer controlled. Capillary electrophoresis is especially preferable
for sequencing
small DNA fragments which may be present in limited amounts in a particular
sample.
In another embodiment of the invention, polynucleotide sequences or fragments
thereof
which encode HIBP may be cloned in recombinant DNA molecules that direct
expression of
HIBP, or fragments or functional equivalents thereof, in appropriate host
cells. Due to the
inherent degeneracy of the genetic code. other DNA sequences which encode
substantially the
same or a functionally equivalent amino acid sequence may be produced and used
to express
HIBP.
The nucleotide sequences of the present invention can be engineered using
methods
generally known in the art in order to alter HIBP-encoding sequences for a
variety of purposes
including, but not limited to, modification of the cloning, processing, and/or
expression of the
gene product. DNA shuffling by random fragmentation and PCR reassembly of gene
fragments
and synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example,
oligonucleotide-mediated site-directed mutagenesis may be used to introduce
mutations that create
new restriction sites, alter glycosylation patterns, change codon preference,
produce splice
variants, and so forth.
In another embodiment. sequences encoding HIBP may be synthesized, in whole or
in
part, using chemical methods well known in the art. (See, e.g., Caruthers,
M.H. et al. (1980)
Nucleic Acids Symp. Ser. 7:215-223; Horn. T. et al. ( 1980) Nucleic Acids
Symp. Ser. 7:225-232.)
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Alternatively, HIBP itself or a fragment thereof may be synthesized using
chemical methods. For
example, peptide synthesis can be performed using various solid-phase
techniques. (See, e.g.,
Roberge, J.Y. et al. ( 1995) Science 269:202-204.) Automated synthesis may be
achieved using
the ABI 431 A Peptide Synthesizer (Perkin-Elmer). Additionally, the amino acid
sequence of
HIBP, or any part thereof. may be altered during direct synthesis and/or
combined with sequences
from other proteins, or any part thereof, to produce a variant polypeptide.
The peptide may be substantially purified by preparative high performance
liquid
chromatography. (See, e.g, Chiez, R.M. and F.Z. Regnier ( 1990) Methods
Enzymol. 182:392-
421.) The composition of the synthetic peptides may be confrmed by amino acid
analysis or by
sequencing. (See, e.g., Creighton, T. ( 1984) Proteins, Structures and
Molecular Properties, WH
Freeman, New York NY.)
In order to express a biologically active HIBP, the nucleotide sequences
encoding HIBP or
derivatives thereof may be inserted into an appropriate expression vector,
i.e., a vector which
contains the necessary elements for transcriptional and translational control
of the inserted coding
IS sequence in a suitable host. These elements include regulatory sequences,
such as enhancers,
constitutive and inducible promoters, and 5' and 3' untranslated regions in
the vector and in
polynucleotide sequences encoding HIBP. Such elements may vary in their
strength and
specificity. Specific initiation signals may also be used to achieve more
efficient translation of
sequences encoding HIBP. Such signals include the ATG initiation codon and
adjacent
sequences, e.g. the Kozak sequence. In cases where sequences encoding HIBP and
its initiation
codon and upstream regulatory sequences are inserted into the appropriate
expression vector, no
additional transcriptional or translational control signals may be needed.
However, in cases where
only coding sequence, or a fragment thereof, is inserted, exogenous
translational control signals
including an in-frame ATG initiation codon should be provided by the vector.
Exogenous
translational elements and initiation codons may be of various origins, both
natural and synthetic.
The efficiency of expression may be enhanced by the inclusion of enhancers
appropriate for the
particular host cell system used. (See, e.g., Scharf, D. et al. ( 1994)
Results Probl. Cell Differ.
20:125-162. )
Methods which are well known to those skilled in the art may be used to
construct
expression vectors containing sequences encoding HIBP and appropriate
transcriptional and
translational control elements. These methods include in vitro recombinant DNA
techniques,
synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook,
J. et al. (1989)
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview
NY, ch. 4, 8, and
16-17; Ausubel, F.M. et al. ( 1995) Current Protocols in Molecular Bioloay,
John Wiley & Sons,
New York NY. ch. 9. 13, and 16.)
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A variety of expression vector/host systems may be utilized to contain and
express
sequences encoding HIBP. These include. but are not limited to, microorganisms
such as bacteria
transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast
transformed with yeast expression vectors; insect cell systems infected with
viral expression
vectors (e.g., baculovirus); plain cell systems transformed with viral
expression vectors (e.g.,
cauliflower mosaic virus, CaMV, or tobacco mosaic virus,TMV) or with bacterial
expression
vectors (e.g., Ti or pBR322 plasmids); or animal cell systems. The invention
is not limited by the
host cell employed.
In bacterial systems, a number of cloning and expression vectors may be
selected
t 0 depending upon the use intended for polynucleotide sequences encoding
HIBP. For example,
routine cloning, subcloning, and propagation of polynucleotide sequences
encoding HIBP can be
achieved using a multifunctional E. coli vector such as PBLUESCRIPT
(Stratagene, La Jolla CA)
or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding HIBP
into the vector's
multiple cloning site disrupts the lacZ gene, allowing a colorimetric
screening procedure for
I S identification of transformed bacteria containing recombinant molecules.
In addition, these
vectors may be useful for in vitro transcription, dideoxy sequencing, single
strand rescue with
helper phage, and creation of nested deletions in the cloned sequence. (See,
e.g., Van Heeke, G.
and S.M. Schuster ( 1989) J. Biol. Chem. 264:5503-5509.) When large quantities
of HIBP are
needed, e.g. for the production of antibodies, vectors which direct high level
expression of H1BP
30 may be used. For example, vectors containing the strong, inducible TS or T7
bacteriophage
promoter may be used.
Yeast expression systems may be used for production of HIBP. A number of
vectors
containing constitutive or inducible promoters, such as alpha factor, alcohol
oxidase, and PGH,
may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In
addition, such vectors
25 direct either the secretion or intracellular retention of expressed
proteins and enable integration of
foreign sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, 1995, suara;
Bitter, G.A. et al. ( 1987) Methods Enzymol. 153:516-544; and Scorer, C.A. et
al. ( 1994)
Bio/Technology 12:181-184.)
Plant systems may also be used for expression of HIBP. Transcription of
sequences
30 encoding HIBP may be driven viral promoters, e.g., the 35S and 195
promoters of CaMV used
alone or in combination with the omega leader sequence from TMV (Takamatsu, N.
( 1987)
EMBO J. 6:307-311 ). Alternatively, plant promoters such as the small subunit
of RUBISCO or
heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO
J. 3:1671-1680;
Broglie, R. et al. ( 1984) Science 224:838-843; and Winter, J. et al. ( 1991 )
Results Probl. Cell
;; Differ. 17:85-105.) These constructs can be introduced into plant cells by
direct DNA
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WO 00/17354 PCT/US99/21281
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill
Yearbook of
Science and Technoloav ( 1992) McGraw Hill, New York NY, pp. 191-196.)
In mammalian cells. a number of viral-based expression systems may be
utilized. In cases
where an adenovirus is used as an expression vector, sequences encoding HIBP
may be ligated
into an adenovirus transcription/translation complex consisting of the late
promoter and tripartite
leader sequence. Insertion in a non-essential E 1 or E3 region of the viral
genome may be used to
obtain infective virus which expresses HIBP in host cells. (See, e.g., Logan,
J. and T. Shenk
(1984) Proc. Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription
enhancers, such as
the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in
mammalian host
cells. SV40 or EBV-based vectors may also be used for high-level protein
expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger
fragments
of DNA than can be contained in and expressed from a plasmid. HACs of about 6
kb to 10 Mb
are constructed and delivered via conventional delivery methods (liposomes.
polycationic amino
polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.J.
et al. ( 1997) Nat.
Genet.15:345-355.)
For long term production of recombinant proteins in mammalian systems, stable
expression of HIBP in cell lines is preferred. For example, sequences encoding
HIBP can be
transformed into cell lines using expression vectors which may contain viral
origins of replication
and/or endogenous expression elements and a selectable marker gene on the same
or on a separate
vector. Following the introduction of the vector, cells may be allowed to grow
for about I to 2
days in enriched media before being switched to selective media. The purpose
of the selectable
marker is to confer resistance to a selective agent, and its presence allows
growth and recovery of
cells which successfully express the introduced sequences. Resistant clones of
stably transformed
cells may be propagated using tissue culture techniques appropriate to the
cell type.
Any number of selection systems may be used to recover transformed cell lines.
These
include, but are not limited to, the herpes simplex virus thymidine kinase and
adenine
phosphoribosyltransferase genes, for use in tk or apr cells, respectively.
(See, e.g., Wigler, M. et
al. (1977) Cell I 1:223-232; Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite,
antibiotic, or herbicide resistance can be used as the basis for selection.
For example, dhfr confers
resistance to methotrexate; neo confers resistance to the aminoglycosides,
neomycin and G-418;
and als and par confer resistance to chlorsulfuron and phosphinotricin
acetyltransferase.
respectively. (See, e.g., Wigler, M, et al. ( I980) Proc. Natl. Acad. Sci. USA
77:3567-3570;
Colbere-Garapin, F. et al. ( 1981 ) J. Mol. Biol. I50: I-14.) Additional
selectable genes have been
described, e.g., trpB and hisD, which alter cellular requirements for
metabolites. (See, e.g.,
Hartman. S.C. and R.C. Mulligan ( 1988) Proc. Natl. Acad. Sci. USA 85:8047-
8051.) Visible
CA 02344657 2001-05-02
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markers. e.g., anthocyanins. green fluorescent proteins (GFP; Clontech),13
glucuronidase and its
substrate 13-glucuronide. or luciferase and its substrate luciferin may be
used. These markers can
be used not only to identify transformants, but also to quantify the amount of
transient or stable
protein expression attributable to a specific vector system. (See, e.g.,
Rhodes, C.A. ( 1995)
Methods Mol. Biol. 55:121-131.)
Although the presence/absence of marker gene expression suggests that the gene
of
interest is also present. the presence and expression of the gene may need to
be confirmed. For
example, if the sequence encoding HIBP is inserted within a marker gene
sequence, transformed
cells containing sequences encoding HIBP can be identified by the absence of
marker gene
function. Alternatively, a marker gene can be placed in tandem with a sequence
encoding HIBP
under the control of a single promoter. Expression of the marker gene in
response to induction or
selection usually indicates expression of the tandem gene as well.
In eeneral, host cells that contain the nucleic acid sequence encoding HIBP
and that
express HiBP may be identified by a variety of procedures known to those of
skill in the art.
IS These procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations, PCR
amplification, and protein bioassay or immunoassay techniques which include
membrane,
solution, or chip based technologies for the detection and/or quantification
of nucleic acid or
protein sequences.
Immunological methods for detecting and measuring the expression of HIBP using
either
specific polyclonal or monoclonal antibodies are known in the art. Examples of
such techniques
include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),
and
fluorescence activated cell sorting (FACS). A two-site, monoclonal-based
immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on HIBP is
preferred, but a
competitive binding assay may be employed. These and other assays are well
known in the art.
(See, e.g., Hampton, R. et al. ( 1990) Serological Methods. a Laboratory
Manual, APS Press, St.
Paul MN, Sect. IV; Coligan, J.E. et al. ( 1997) Current Protocols in
Immunolosy, Greene Pub.
Associates and Wiley-Interscience, New York NY; and Pound, J.D. ( 1998)
Immunochemical
Protocols, Humana Press, Totowa NJ.)
A wide variety of labels and conjugation techniques are known by those skilled
in the art
and may be used in various nucleic acid and amino acid assays. Means for
producing labeled
hybridization or PCR probes for detecting sequences related to polynucleotides
encoding HIBP
include oligolabeling, nick translation, end-labeling, or PCR amplification
using a labeled
nucleotide. Alternatively, the sequences encoding HIBP, or any fragments
thereof, may be cloned
into a vector for the production of an mRNA probe. Such vectors are known in
the art, are
commercially available, and may be used to synthesize RNA probes in vitro by
addition of an
21
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WO 00/17354 PCT/US99/21281
appropriate RNA polymerase such as T7. T3, or SP6 and labeled nucleotides.
These procedures
may be conducted using a variety of commercially available kits, such as those
provided by
Amersham Pharmacia Biotech, Promega (Madison WI), and LJS Biochemical.
Suitable reporter
molecules or labels which may be used for ease of detection include
radionuclides, enzymes,
fluorescent, chemiluminescent, or chromogenic agents. as well as substrates,
cofactors, inhibitors,
magnetic particles, and the like.
Host cells transformed with nucleotide sequences encoding HIBP may be cultured
under
conditions suitable for the expression and recovery of the protein from cell
culture. The protein
produced by a transformed cell rnay be secreted or retained intracellularly
depending on the
sequence and/or the vector used. As will be understood by those of skill in
the art, expression
vectors containing polynucleotides which encode HIBP may be designed to
contain signal
sequences which direct secretion of HIBP through a prokaryotic or eukaryotic
cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
inserted sequences or to process the expressed protein in the desired fashion.
Such modifications
of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation,
phosphorylation, iipidation, and acylation. Post-translational processing
which cleaves a "prepro"
form of the protein may also be used to specify protein targeting, folding,
and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are
available from
the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the
correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified. or recombinant
nucleic acid
sequences encoding HIBP may be ligated to a heterologous sequence resulting in
translation of a
fusion protein in any of the aforementioned host systems. For example, a
chimeric HIBP protein
containing a heterologous moiety that can be recognized by a commercially
available antibody
may facilitate the screening of peptide libraries for inhibitors of HIBP
activity. Heterologous
protein and peptide moieties may also facilitate purification of fusion
proteins using commercially
available affinity matrices. Such moieties include, but are not limited to,
glutathione S-transferase
(GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding
peptide (CBP), 6-
His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable
purification
of their cognate fusion proteins on immobilized glutathione, maltose,
phenylarsine oxide,
calmodulin, and metal-chelate resins, respectively. FLAG, c-nwc, and
hemagglutinin (HA) enable
immunoaffinity purification of fusion proteins using commercially available
monoclonal and
polyclonal antibodies that specifically recognize these epitope tags. A fusion
protein may also be
engineered to contain a proteolytic cleavaee site located between the HIBP
encoding sequence and
CA 02344657 2001-05-02
WO 00/17354 PCTNS99/21281
the heterologous protein sequence. so that HIBP may be cleaved away from the
heterologous
moiety following purification. Methods for fusion protein expression and
purification are
discussed in Ausubel ( 1995, suara, ch 10). A variety of commercially
available kits may also be
used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled HIBP may
be achieved
in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract
systems (Promega). These
systems couple transcription and translation of protein-coding sequences
operably associated with
the T7, T3, or SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid
precursor, preferably 'SS-methionine.
IO Fragments of HIBP may be produced not only by recombinant production, but
also by
direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton,
supra, pp. 55-60.)
Protein synthesis may be performed by manual techniques or by automation.
Automated synthesis
may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin-
Elmer). Various
fragments of HIBP may be synthesized separately and then combined to produce
the full length
IS molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and
motifs, exists
between regions of HIBP and human IRLB. In addition, the expression of HIBP is
closely
associated with reproductive, immune, and nervous tissues, cell proliferation,
inflammation, and
20 immune response. Therefore, HIBP appears associated with cell proliferative
and immune
disorders. In disorders associated with decreased expression or activity of
HIBP, it is desirable to
increase the expression or activity of HIBP. In disorders associated with
increased expression or
activity of HIBP, it is desirable to decrease the expression or activity of
HIBP.
Therefore, in one embodiment, HIBP or a fragment or derivative thereof may be
25 administered to a subject to treat or prevent a disorder associated with
decreased expression or
activity of HIBP. Examples of such disorders include, but are not limited to,
cell proliferative
disorders such as actinic keratosis, arteriosclerosis, atherosclerosis,
bursitis, cirrhosis, hepatitis,
mixed connective tissue disease (MCTD), myeiofibrosis, paroxysmal nocturnal
hemoglobinuria,
polycythemia vera, psoriasis, primary thrombocythemia; cancers including
adenocarcinoma,
30 leukemia, lymphoma, melanoma, myeloma, sarcoma. teratocarcinoma, and, in
particular, cancers
of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall
bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas,
parathyroid, penis,
prostate, salivary glands, skin, spleen, testis, thymus. thyroid, and uterus;
and immune disorders
such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory
35 distress syndrome, allergies, ankylosing spondylitis. amyloidosis. anemia,
asthma. atherosclerosis,
23
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WO 00/17354 PCT/US99/21281
autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis,
cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes
mellitus. emphysema,
episodic lymphopenia with lymphocytotoxins, erythroblastosis fetalis. erythema
nodosum,
atrophic gastritis, glomerulonephritis, Goodpasture~s syndrome, gout, Graves'
disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple
sclerosis,
myasthenia gravis. myocardial or pericardial inflammation, osteoarthritis,
osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome. rheumatoid
arthritis, sclerodetma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome.
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and
helminthic infections. and trauma.
In another embodiment, a vector capable of expressing HIBP or a fragment or
derivative
thereof may be administered to a subject to treat or prevent a disorder
including, but not limited to,
those described above.
(5 In a further embodiment, a pharmaceutical composition comprising a
substantially
purified HIBP in conjunction with a suitable pharmaceutical carrier may be
administered to a
subject to treat or prevent a disorder including, but not limited to, those
provided above.
In still another embodiment, an agonist which modulates the activity of HIBP
may be
administered to a subject to treat or prevent a disorder including, but not
limited to, those listed
above.
In a further embodiment, an antagonist of HIBP may be administered to a
subject to treat
or prevent a disorder associated with increased expression or activity of HIBP
including, but not
limited to, those listed above. In one aspect, an antibody which specifically
binds HIBP may be
used directly as an antagonist or indirectly as a targeting or delivery
mechanism for bringing a
pharmaceutical agent to cells or tissue which express HIBP.
In an additional embodiment, a vector expressing the complement of the
polynucleotide
encoding HIBP may be administered to a subject to treat or prevent a disorder
including, but not
limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists,
complementary sequences, or vectors of the invention may be administered in
combination with
other appropriate therapeutic agents. Selection of the appropriate agents for
use in combination
therapy may be made by one of ordinary skill in the art. according to
conventional pharmaceutical
principles. The combination of therapeutic agents may act synergistically to
effect the treatment
or prevention of the various disorders described above. Using this approach,
one may be able to
achieve therapeutic efficacy with lower dosages of each agent. thus reducing
the potential for
24
CA 02344657 2001-05-02
WO 00/17354 PCT/US99/21281
adverse side effects.
An antagonist of HIBP may be produced using methods which are generally known
in the
art. In particular, purified HIBP may be used to produce antibodies or to
screen libraries of
pharmaceutical agents to identify those which specifically bind HIBP.
Antibodies to HIBP may
also be generated using methods that are well known in the art. Such
antibodies may include, but
are not limited to, polyclonal, monoclonal. chimeric, and single chain
antibodies, Fab fragments,
and fragments produced by a Fab expression library. Neutralizing antibodies
(i.e., those which
inhibit dimer formation) are especially preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits,
rats, mice,
humans, and others may be immunized by injection with HIBP or with any
fragment or
oligopeptide thereof which has immunogenic properties. Depending on the host
species, various
adjuvants may be used to increase immunological response. Such adjuvants
include. but are not
limited to, Freund's, mineral gels such as aluminum hydroxide, and surface
active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH,
and dinitrophenol.
IS Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corvnebacterium parvum
are especially preferable.
It is preferred that the oligopeptides, peptides. or fragments used to induce
antibodies to
HIBP have an amino acid sequence consisting of at least about 5 amino acids,
and, more
preferably, of at least about 10 amino acids. It is also preferable that these
oligopeptides, peptides,
or fragments are identical to a portion of the amino acid sequence of the
natural protein and
contain the entire amino acid sequence of a small, naturally occurring
molecule. Short stretches of
HIBP amino acids may be fused with those of another protein, such as KLH, and
antibodies to the
chimeric molecule may be produced.
Monoclonal antibodies to HIBP may be prepared using any technique which
provides for
the production of antibody molecules by continuous cell lines in culture.
These include, but are
not limited to, the hybridoma technique, the human B-cell hybridoma technique,
and the EBV-
hybridoma technique. (See, e.g., Kohler, G. et al. ( 1975) Nature 256:495-497;
Kozbor, D. et al.
(1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl.
Acad. Sci. USA
80:2026-2030; and Cole, S.P. et al. (1984) Mol. Cell Biol. 62:109-120.)
In addition, techniques developed for the production of "chimeric antibodies,"
such as the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with appropriate
antigen specificity and biological activity. can be used. (See, e.g.,
Morrison, S.L. et al. ( 1984)
Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature
312:604-608; and
Takeda. S. et al. (1985) Nature 314:452-454.) Alternatively. techniques
described for the
production of single chain antibodies may be adapted, using methods known in
the art. to produce
7J
CA 02344657 2001-05-02
WO 00/17354 PCT/US99/21281
HIBP-specific single chain antibodies. Antibodies with related specificity,
but of distinct idiotypic.
composition. may be generated by chain shuffling from random combinatorial
imrnunoglobulin
libraries. (See, e.g., Burton, D.R. ( 1991 ) Proc. Natl. Acad. Sci. USA
88:10134-10137.)
Antibodies may also be produced by inducing in vivo production in the
lymphocyte
population or by screening immunogiobulin libraries or panels of highly
specific binding reagents
as disclosed in the literature. (See, e.g., Urlandi, R. et al. ( 1989) Proc.
Natl. Acad. Sci. USA
86:3833-3837; Winter, G. et al. ( 1991 ) Nature 349:293-299.)
Antibody fragments which contain specific binding sites for H1BP may also be
generated.
For example, such fragments include, but are not limited to, F(ab')2 fragments
produced by
pepsin digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide
bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may
be constructed to
allow rapid and easy identification of monoclonal Fab fragments with the
desired specificity.
(See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.)
Various immunoassays may be used for screening to identify antibodies having
the
desired specificity. Numerous protocols for competitive binding or
immunoradiometric assays
using either polyclonal or monoclonal antibodies with established
specificities are well known in
the art. Such immunoassays typically involve the measurement of complex
formation between
HIBP and its specific antibody. A two-site, monoclonal-based immunoassay
utilizing monoclonal
antibodies reactive to two non-interfering HIBP epitopes is preferred, but a
competitive binding
assay may also be employed (Pound, supra).
Various methods such as Scatchard analysis in conjunction with
radioimmunoassay
techniques may be used to assess the affinity of antibodies for HIBP. Affinity
is expressed as an
association constant, K~, which is detined as the molar concentration of HIBP-
antibody complex
divided by the molar concentrations of free antigen and free antibody under
equilibrium
conditions. The K~ determined for a preparation of polyclonal antibodies,
which are
heterogeneous in their affinities for multiple HIBP epitopes, represents the
average affinity, or
avidity, of the antibodies for HIBP. The Ka determined for a preparation of
monoclonal
antibodies, which are monospecific for a particular HIBP epitope, represents a
true measure of
affinity. High-affinity antibody preparations with Ka ranging from about 1 Oy
to 10'= L/mole are
preferred for use in immunoassays in which the HIBP-antibody complex must
withstand rigorous
manipulations. Low-affinity antibody preparations with K, ranging from about
106 to l0' L/moie
are preferred for use in immunopurification and similar procedures which
ultimately require
dissociation of HIBP, preferably in active form, from the antibody (Catty, D.
( 1988) Antibodies,
Volume t: A Practical Approach, IRL Press, Washington, DC; Liddell. J.E. and
Cryer, A. ( 1991 )
A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
26
CA 02344657 2001-05-02
WO 00/17354 PCT/US99/21281
The titer and avidity of polyclonal antibody preparations may be further
evaluated to
determine the quality and suitability of such preparations for certain
downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2 mg specific
antibody/ml,
preferably 5-10 mg specific antibody/ml, is preferred for use in procedures
requiring precipitation
of HIBP-antibody complexes. Procedures for evaluating antibody specificity,
titer, and avidity,
and guidelines for antibody quality and usage in various applications. are
generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.)
In another embodiment of the invention. the polynucleotides encoding HIBP, or
any
fragment or complement thereof, may be used for therapeutic purposes. In one
aspect, the
complement of the polynucleotide encoding HIBP may be used in situations in
which it would be
desirable to block the transcription of the mRNA. In particular, cells may be
transformed with
sequences complementary to polynucleotides encoding HIBP. Thus, complementary
molecules or
fragments may be used to modulate HIBP activity, or to achieve regulation of
gene function. Such
technology is now well known in the art, and sense or antisense
oligonucleotides or larger
fragments can be designed from various locations along the coding or control
regions of sequences
encoding HIBP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or
vaccinia viruses,
or from various bacterial plasmids, may be used for delivery of nucleotide
sequences to the
targeted organ, tissue, or cell population. Methods which are well known to
those skilled in the art
can be used to construct vectors to express nucleic acid sequences
complementary to the
polynucleotides encoding HIBP. (See, e.g., Sambrook, supra; Ausubel, 1995,
supra.)
Genes encoding HIBP can be turned off by transforming a cell or tissue with
expression
vectors which express high levels of a polynucleotide, or fragment thereof
encoding HIBP. Such
constructs may be used to introduce untranslatable sense or antisense
sequences into a cell. Even
in the absence of integration into the DNA, such vectors may continue to
transcribe RNA
molecules until they are disabled by endogenous nucleases. Transient
expression may last for a
month or more with a non-replicating vector, and may last even longer if
appropriate replication
elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by
designing
complementary sequences or antisense molecules (DNA, RNA, or PNA) to the
control, 5', or
regulatory regions of the gene encoding HIBP. Oligonucleotides derived from
the transcription
initiation site. e.g., between about positions -10 and +10 from the start
site, are preferred.
Similarly, inhibition can be achieved using triple helix base-pairing
methodology. Triple helix
pairing is useful because it causes inhibition of the ability of the double
helix to open sufficiently
for the binding of polymerases. transcription factors, or regulatory
molecules. Recent therapeutic
27
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WO 00/17354 PCT/US99/21281
advances using triplex DNA have been described in the literature. (See, e.g.,
Gee. J.E. et al.
( 1994) in Huber, B.E. and B.1. Carr, Molecular and Immunolo~ic Approaches,
Futura Publishing,
Mt. Kisco NY. pp. 163-177.) A complementary sequence or antisense molecule may
also be
designed to block translation of mRNA by preventing the transcript from
binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific
cleavage
of RNA. The mechanism of ribozyme action involves sequence-specific
hybridization of the
ribozyme molecule to complementary target RNA, followed by endonucleoiytic
cleavage. For
example, engineered hammerhead motif ribozyme molecules may specifically and
efficiently
catalyze endonucleolytic cleavage of sequences encoding HIBP.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the target molecule for ribozyme cleavage sites, including the
following sequences:
GUA, GUU. and GUC. Once identified. short RNA sequences of between 1 S and 20
ribonucleotides. corresponding to the region of the target gene containing the
cleavage site, may
be evaluated for secondary structural features which may render the
oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing
accessibility to hybridization
with complementary oligonucleotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be
prepared by any method known in the art for the synthesis of nucleic acid
molecules. These
include techniques for chemically synthesizing oligonucleotides such as solid
phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules may be
generated by in vitro
and in vivo transcription of DNA sequences encoding HIBP. Such DNA sequences
may be
incorporated into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or
SP6. Alternatively, these cDNA constructs that synthesize complementary RNA.
constitutively or
inducibly, can be introduced into cell lines, cells, or tissues.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the S' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in ail of these molecules by the inclusion of
nontraditional bases such as
inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and
similarly modified forms
of adenine, c«idine, guanine, thymine, and uridine which are not as easily
recognized by
endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available and
equally
suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors
may be introduced into
stem cells taken from the patient and clonally propagated for autologous
transplant back into that
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WO 00/17354 PCT/US99/21281
same patient. Delivery by transfection, by liposome injections. or by
polycationic amino polymers.
may be achieved using methods which are well known in the art. (See, e.g.,
Goldman, C.K. et al.
(1997) Nat. Biotechnol. 15:462-466.)
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as dogs, cats, cows,
horses, rabbits,
monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a
pharmaceutical or sterile composition, in conjunction with a pharmaceutically
acceptable carrier,
for any of the therapeutic effects discussed above. Such pharmaceutical
compositions may consist
of HIBP, antibodies to HIBP, and mimetics, agonists. antagonists, or
inhibitors of HIBP. The
compositions may be administered alone or in combination with at least one
other agent, such as a
stabilizing compound, which may be administered in any sterile. biocompatible
pharmaceutical
carrier including, but not limited to, saline, buffered saline. dextrose, and
water. The compositions
may be administered to a patient alone, or in combination with other agents,
drugs, or hormones.
IS The pharmaceutical compositions utilized in this invention may be
administered by any
number of routes including, but not limited to, oral, intravenous,
intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain
suitable pharmaceutically-acceptable carriers comprising excipients and
auxiliaries which
facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. Further details on techniques for formulation and
administration may be found
in the latest edition of Remin~ton's Pharmaceutical Sciences (Maack
Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using
pharmaceutically acceptable carriers well known in the art in dosages suitable
for oral
administration. Such carriers enable the pharmaceutical compositions to be
formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and
the like, for ingestion by
the patient.
Pharmaceutical preparations for oral use can be obtained through combining
active
compounds with solid excipient and processing the resultant mixture of
granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be
added, if desired. Suitable
excipients include carbohydrate or protein fillers, such as sugars, including
lactose, sucrose,
mannitol, and sorbitol; starch from corn. wheat, rice, potato, or other
plants; cellulose, such as
methyl cellulose, hydroxypropylmethyl-cellulose, or sodium
carboxymethylcellulose; gums,
including arabic and tragacanth: and proteins, such as gelatin and collagen.
If desired.
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CA 02344657 2001-05-02
WO 00/17354 PCT/US99/21281
disintegrating or solubilizing agents may be added, such as the cross-linked
polyvinyl pyrrolidone, .
agar, and alginic acid or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suitable coatings, such as
concentrated
sugar solutions, which may also contain gum arabic, talc,
polyvinylpyrrolidone, carbopoi gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable
organic solvents or
solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee
coatings for
product identification or to characterize the quantity of active compound,
i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules
made of
gelatin, as well as soft, sealed capsules made of gelatin and a coating, such
as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or
binders. such as lactose or
starches, lubricants, such as talc or magnesium stearate, and, optionally,
stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in suitable
liquids, such as fatty
oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be
formulated in
aqueous solutions, preferably in physiologically compatible buffers such as
Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous injection
suspensions may contain
substances which increase the viscosity of the suspension, such as sodium
carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the active
compounds may be
prepared as appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include
fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl
oleate, triglycerides, or
liposomes. Non-lipid polycationic amino polymers may also be used for
delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to increase the
solubility of the
compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular
barrier to be
permeated are used in the formulation. Such penetrants are generally known in
the art.
The pharmaceutical compositions of the present invention may be manufactured
in a
manner that is known in the art, e.g., by means of conventional mixing,
dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping, or
lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed
with many
acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic,
tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other protonic
solvents than are the
corresponding free base forms. In other cases, the preferred preparation may
be a lyophilized
powder which may contain any or all of the following: 1 mM to 50 mM histidine,
0.1 % to 2%
sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined
with buffer prior to
use.
JO
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After pharmaceutical compositions have been prepared, they can be placed in an
appropriate container and labeled for treatment of an indicated condition. For
administration of
HIBP, such labeling would include amount, frequency, and method of
administration.
Pharmaceutical compositions suitable for use in the invention include
compositions
wherein the active ingredients are contained in an effective amount to achieve
the intended
purpose. The determination of an effective dose is welf within the capability
of those skilled in the
art.
For any compound, the therapeutically effective dose can be estimated
initially either in
cell culture assays, e.g., of neoplastic cells or in animal models such as
mice. rats, rabbits, dogs, or
pigs. An animal model may also be used to determine the appropriate
concentration range and
route of administration. Such information can then be used to determine useful
doses and routes
for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example
HIBP or fragments thereof, antibodies of HIBP, and agonists, antagonists or
inhibitors of HIBP,
which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity
may be
determined by standard pharmaceutical procedures in cell cultures or with
experimental animals,
such as by calculating the EDS° (the dose therapeutically effective in
50% of the population) or
LDS° (the dose lethal to 50% of the population) statistics. The dose
ratio of toxic to therapeutic
effects is the therapeutic index, and it can be expressed as the
LDS°1ED5° ratio. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred. The data
obtained from cell
culture assays and animal studies are used to formulate a range of dosage for
human use. The
dosage contained in such compositions is preferably within a range of
circulating concentrations
that includes the EDS° with little or no toxicity. The dosage varies
within this range depending
upon the dosage form employed, the sensitivity of the patient, and the route
of administration.
The exact dosage will be determined by the practitioner, in light of factors
related to the
subject requiring treatment. Dosage and administration are adjusted to provide
sufficient levels of
the active moiety or to maintain the desired effect. Factors which may be
taken into account
include the severity of the disease state, the general health of the subject,
the age, weight, and
gender of the subject, time and frequency of administration, drug
combination(s), reaction
sensitivities, and response to therapy. Long-acting pharmaceutical
compositions may be
administered every 3 to 4 days, every week, or biweekly depending on the half
life and clearance
rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 ~g to 100.000 ~cg, up to a total
dose of
about 1 gram. depending upon the route of administration. Guidance as to
particular dosages and
3~ methods of delivery is provided in the literature and generally available
to practitioners in the art.
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Those skilled in the art will employ different formulations for nucleotides
than for proteins or their .
inhibitors. Similarly, delivery of polynucleotides or polypeptides will be
specific to particular
cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind HIBP may be used for
the
diagnosis of disorders characterized by expression of HIBP, or in assays to
monitor patients being
treated with HIBP or agonists, antagonists, or inhibitors of HIBP. Antibodies
useful for diagnostic
purposes may be prepared in the same manner as described above for
therapeutics. Diagnostic
assays for HIBP include methods which utilize the antibody and a label to
detect HIBP in human
body fluids or in extracts of cells or tissues. The antibodies may be used
with or without
modification, and may be labeled by covalent or non-covalent attachment of a
reporter molecule.
A wide variety of reporter molecules, several of which are described above,
are known in the art
and may be used.
A variety of protocols for measuring HIBP, including ELISAs, RIAs, and FACS,
are
known in the art and provide a basis for diagnosing altered or abnormal levels
of NIBP expression.
Normal or standard values for HIBP expression are established by combining
body fluids or cell
extracts taken from normal mammalian subjects, preferably human, with antibody
to HIBP under
conditions suitable for complex formation. The amount of standard complex
formation may be
quantitated by various methods, preferably by photometric means. Quantities of
HIBP expressed
in subject, control, and disease samples from biopsied tissues are compared
with the standard
values. Deviation between standard and subject values establishes the
parameters for diagnosing
disease.
In another embodiment of the invention, the polynucleotides encoding HIBP may
be used
for diagnostic purposes. The polynucleotides which may be used include
oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides
may be
used to detect and quantitate gene expression in biopsied tissues in which
expression of HIBP may
be correlated with disease. The diagnostic assay may be used to determine
absence, presence, and
excess expression of HIBP, and to monitor regulation of HIBP levels during
therapeutic
intervention.
In one aspect, hybridization with PCR probes which are capable of detecting
polynucleotide sequences, including genomic sequences, encoding HIBP or
closely related
molecules may be used to identify nucleic acid sequences which encode H1BP.
The specificity of
the probe, whether it is made from a highly specific region. e.g., the 5'
regulatory region, or from a
less specific region, e.g., a conserved motif, and the stringency of the
hybridization or
amplification (maximal, high, intermediate, or low), will determine whether
the probe identifies
32
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only naturally occurring sequences encoding HIBP, allelic variants. or related
sequences.
Probes may also be used for the detection of related sequences, and should
preferably
have at least 50% sequence identity to any of the HIBP encoding sequences. The
hybridization
probes of the subject invention may be DNA or RNA and may be derived from the
sequence of
SEQ 1D N0:3-4, or from genomic sequences including promoters, enhancers, and
introns of the
HIBP gene.
Means for producing specific hybridization probes for DNAs encoding HIBP
include the
cloning of polynucleotide sequences encoding HIBP or HIBP derivatives into
vectors for the
production of mRNA probes. Such vectors are known in the art. are commercially
available, and
may be used to synthesize RNA probes in vitro by means of the addition of the
appropriate RNA
polymerases and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a
variety of reporter groups, for example, by radionuclides such as''-P or'SS,
or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin coupling
systems. and the like.
Polynucleotide sequences encoding HIBP may be used for the diagnosis of
disorders
associated with expression of HIBP. Examples of such disorders include, but
are not limited to,
cell proliferative disorders such as actinic keratosis, arteriosclerosis,
atherosclerosis, bursitis,
cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis,
paroxysmal nocturnal
hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia; cancers
including
adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,
teratocarcinoma, and, in
particular. cancers of the adrenal gland, bladder, bone, bone marrow, brain,
breast, cervix, gall
bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,
ovary, pancreas,
parathyroid, penis, prostate, salivary glands, skin, spleen. testis, thymus,
thyroid, and uterus; and
immune disorders such as acquired immunodeficiency syndrome (AIDS), Addison's
disease, adult
respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis,
anemia, asthma,
atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,
bronchitis, cholecystitis,
contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,
diabetes mellitus,
emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis
fetalis, erythema
nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout,
Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple
sclerosis,
myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis,
osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome. rheumatoid
arthritis, scleroderma,
Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus,
systemic sclerosis,
thrombocvtopenic purpura, ulcerative colitis, uveitis, Werner syndrome,
complications of cancer,
hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,
parasitic, protozoal, and
helminthic infections, and trauma. The polynucleotide sequences encoding HIBP
may be used in
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Southern or northern analysis, dot blot, or other membrane-based technologies;
in PCR
technologies; in dipstick, pin, and multiformat ELISA-like assays; and in
microarrays utilizing
fluids or tissues from patients to detect altered HIBP expression. Such
qualitative or quantitative
methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding HIBP may be useful
in assays
that detect the presence of associated disorders, particularly those mentioned
above. The
nucleotide sequences encoding HIBP may be labeled by standard methods and
added to a fluid or
tissue sample from a patient under conditions suitable for the formation of
hybridization
complexes. After a suitable incubation period, the sample is washed and the
signal is quantitated
and compared with a standard value. If the amount of signal in the patient
sample is significantly
altered in comparison to a control sample then the presence of altered levels
of nucleotide
sequences encoding HIBP in the sample indicates the presence of the associated
disorder. Such
assays may also be used to evaluate the efficacy of a particular therapeutic
treatment regimen in
animal studies. in clinical trials, or to monitor the treatment of an
individual patient.
In order to provide a basis for the diagnosis of a disorder associated with
expression of
HIBP, a normal or standard profile for expression is established. This may be
accomplished by
combining body fluids or cell extracts taken from normal subjects, either
animal or human, with a
sequence, or a fragment thereof, encoding HIBP, under conditions suitable for
hybridization or
amplification. Standard hybridization may be quantified by comparing the
values obtained from
normal subjects with values from an experiment in which a known amount of a
substantially
purified polynucleotide is used. Standard values obtained in this manner may
be compared with
values obtained from samples from patients who are symptomatic for a disorder.
Deviation from
standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is
initiated,
hybridization assays may be repeated on a regular basis to determine if the
level of expression in
the patient begins to approximate that which is observed in the normal
subject. The results
obtained from successive assays may be used to show the efficacy of treatment
over a period
ranging from several days to months.
With respect to cancer, the presence of an abnormal amount of transcript
(either under- or
overexpressed) in biopsied tissue from an individual may indicate a
predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to the
appearance of actual clinical symptoms. A more definitive diagnosis of this
type may allow health
professionals to employ preventative measures or aggressive treatment earlier
thereby preventing
the development or further progression of the cancer.
Additional diagnostic uses for oli~onucleotides designed from the sequences
encoding
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WO 00/17354 PCT/US99/21281
HIBP may involve the use of PCR. These oligomers may be chemically
synthesized, generated
enzymatically, or produced in vitro. Oligomers will preferably contain a
fragment of a
polynucleotide encoding HIBP, or a fragment of a polynucleotide complementary
to the
polynucleotide encoding HIBP. and will be employed under optimized conditions
for
S identification of a specific gene or condition. Oligomers may also be
employed under less
stringent conditions for detection or quantitation of closely related DNA or
RNA sequences.
Methods which may also be used to quantitate the expression of HIBP include
radiolabeling or biotinylating nucleotides, coamplification of a control
nucleic acid, and
interpolating results from standard curves. (See, e.g., Melby, I'.C. et al.
(1993) J. Immunol.
Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236.) The
speed of
quantitation of multiple samples may be accelerated by running the assay in an
ELISA format
where the oligomer of interest is presented in various dilutions and a
spectrophotometric or
colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any
of the
polynucleotide sequences described herein may be used as targets in a
microarray. The
microarray can be used to monitor the expression level of large numbers of
genes simultaneously
and to identify genetic variants, mutations, and polymorphisms. This
information may be used to
determine gene function, to understand the genetic basis of a disorder, to
diagnose a disorder, and
to develop and monitor the activities of therapeutic agents.
Microarrays may be prepared. used, and analyzed using methods known in the
art. (See,
e.g., Brennan. T.M. et al. ( I 995) U.S. Patent No. 5,474,796: Schena, M. et
al. ( 1996) Proc. Natl.
Acad. Sci. USA 93:10614-10619; Baideschweiler et al. (1995) PCT application
W095/251 I 16;
Shalom D. et al. ( 1995) PCT application W095/35505; Heifer, R.A. et al. (
1997) Proc. Natl. Acad.
Sci. USA 94:2150-2155; and Heller, M.J. et al. ( 1997) U.S. Patent No.
5.605,662.)
In another embodiment of the invention, nucleic acid sequences encoding HIBP
may be
used to generate hybridization probes useful in mapping the naturally
occurring genomic
sequence. The sequences may be mapped to a particular chromosome, to a
specific region of a
chromosome. or to artificial chromosome constructions, e.g., human artificial
chromosomes
(HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial
P I constructions, or single chromosome cDNA libraries. (See, e.g.,
Harrington, J.J. et al. ( 1997)
Nat. Genet. 15:345-355; Price, C.M. (1993) Blood Rev. 7:127-134; and Trask,
B.J. (1991) Trends
Genet. 7: I 49- I 54. )
Fluorescent in situ hybridization (FISH) may be correlated with other physical
chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich,
et ai. ( 1995) in
Meyers, supra, pp. 965-968.) Examples of genetic map data can be found in
various scientific
CA 02344657 2001-05-02
WO 00/17354 PCT/US99/21281
journals or at the Online Mendelian Inheritance in Man (OMIM) site.
Correlation between the
location of the gene encoding HIBP on a physical chromosomal map and a
specific disorder. or a
predisposition to a specific disorder, may help define the region of DNA
associated with that
disorder. The nucleotide sequences of the invention may be used to detect
differences in gene
sequences among normal, carrier. and affected individuals.
In situ hybridization of chromosomal preparations and physical mapping
techniques, such
as linkage analysis using established chromosomal markers, may be used for
extending genetic
maps. Often the placement of a gene on the chromosome of another mammalian
species, such as
mouse, may reveal associated markers even if the number or arm of a particular
human
t0 chromosome is not known. New sequences can be assigned to chromosomal arms
by physical
mapping. This provides valuable information to investigators searching for
disease genes using
positional cloning or other gene discovery techniques. Once the disease or
syndrome has been
crudely localized by genetic linkage to a particular genomic region, e.g.,
ataxia-telangiectasia to
1 Iq22-23, any sequences mapping to that area may represent associated or
regulatory genes for
further investigation. (See, e.g., Gatti, R.A. et al. ( 1988) Nature 336:577-
580.) The nucleotide
sequence of the subject invention may also be used to detect differences in
the chromosomal
Location due to translocation, inversion, etc., among normal, carrier, or
affected individuals.
In another embodiment of the invention, HIBP, its catalytic or immunogenic
fragments, or
oligopeptides thereof can be used for screening libraries of compounds in any
of a variety of drug
screening techniques. The fragment employed in such screening may be free in
solution, affixed
to a solid support, borne on a cell surface, or located intracellularly. The
formation of binding
complexes between HIBP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of
compounds having suitable binding affinity to the protein of interest. (See,
e.g., Geysen, et al.
( 1984) PCT application W084/03564.) In this method, large numbers of
different small test
compounds are synthesized on a solid substrate. The test compounds are reacted
with HIBP, or
fragments thereof, and washed. Bound HIBP is then detected by methods well
known in the art.
Purified HIBP can also be coated directly onto plates for use in the
aforementioned drug screening
techniques. Alternatively, non-neutralizing antibodies can be used to capture
the peptide and
immobilize it on a solid support.
In another embodiment, one may use competitive drug screening assays in which
neutralizing antibodies capable of binding HIBP specifically compete with a
test compound for
binding HIBP. In this manner, antibodies can be used to detect the presence of
any peptide which
shares one or more antigenic determinants with HIBP.
In additional embodiments, the nucleotide sequences which encode HIBP may be
used in
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WO 00/17354 PCT/US99/Z1281
any molecular biology techniques that have yet to be developed, provided the
new techniques rely
on properties of nucleotide sequences that are currently known, including, but
not limited to, such
properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one skilled in the art can,
using the
preceding description, utilize the present invention to its fullest extent.
The following preferred
specific embodiments are, therefore, to be construed as merely illustrative,
and not limitative of
the remainder of the disclosure in any way whatsoever.
The disclosures of all patents, applications, and publications mentioned above
and below,
in particular U.S. Ser. No. 09/157>091, are hereby expressly incorporated by
reference.
EXAMPLES
I. cDNA Library Construction
BRAITUT21
The BRAITUT21 cDNA library was constructed using RNA isolated from brain tumor
tissue removed from the midline frontal lobe of a 61-year-old Caucasian female
during excision of
a cerebral meningeal lesion. Pathology indicated subfrontal meningothelial
meningioma with no
atypia. One ethmoid and mucosal tissue sample indicated meningioma. Patient
history included
cerebrovascular disease, atherosclerotic coronary artery disease, epistaxis,
hyperlipidemia,
depressive disorder, irritable bowel, and skin cancer. Family history included
cerebrovascular
disease, senile dementia, atherosclerotic coronary artery disease, congestive
heart failure, and
breast cancer.
The frozen tissue was homogenized and iysed in Trizol reagent (Life
Technologies), a
monophasic solution of phenol and guanidine isothiocyanate, using a Polytron
PT-3000
homogenizer (Brinkmann Instruments, Westbury NY). After a brief incubation on
ice, chloroform
was added ( 1:5 v/v), and the lysate was centrifuged. The upper chloroform
layer was removed,
and the RNA extracted with isopropanol, resuspended in water, and treated with
DNase for 25 min
at 37°C. Extraction and precipitation were repeated. Poly(A+) RNA was
isolated using the
OLIGOTEX kit (QIAGEN, Inc., Chatsworth, CA).
UTRSNOT06
The UTRSNOT06 cDNA library was constructed using RNA isolated from myometrial
tissue obtained from a 50-year-old Caucasian female during a vaginal
hysterectomy. Pathology
indicated residual atypical complex endometrial hyperplasia. Pathology for the
associated tissue
removed during dilation and curettage indicated fragments of atypical complex
hyperplasia and a
single microscopic focus suspicious for grade 1 adenocarcinoma. Patient
history included benign
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breast neoplasm, hypothyroid disease, polypectomy, and arthralgia. Family
history included
cerebrovascular disease. arteriosclerotic coronary artery disease.
hyperlipidemia, and chronic
hepatitis.
The frozen tissue was homogenized and lysed in guanidinium isothiocyanate
solution
using a Polytron PT-3000 homogenizer (Brinkmann Instruments, Westbury NY). The
lysate was
centrifuged over a 5.7 M CsCI cushion using a Beckman SW28 rotor in a Beckman
L8-70M
Ultracentrifuge (Beckman Instruments, Fullerton CA) for 18 hours at 25,000 rpm
at ambient
temperature. The RNA was extracted with acid phenol, precipitated using sodium
acetate and
ethanol, resuspended in RNAse-free water, and treated with DNase. The RNA was
extracted with
acid phenol and precipitated as before. Poly(A+) RNA was isolated using the
OLIGOTEX kit
(QIAGEN, Inc.).
BRAITUT21 and UTRSNOT06
Poly(A+) RNA was used for cDNA synthesis and library construction according to
the
recommended protocols in the SUPERSCRIPT plasmid system (Life Technologies).
cDNAs were
fractionated on a SEPHAROSE CL4B column (Pharmacia Amersham Biotech) and those
cDNAs
exceeding 400 by were ligated into pINCY (Incyte Pharmaceuticals, Inc., Palo
Alto CA) and
subsequently transformed into DHSa competent cells (Life Technologies).
II. Isolation of cDNA Clones
Plasmid DNA was released from the cells and purified using the REAL Prep 96
plasmid
kit (QIAGEN, lnc.). The recommended protocol was employed except for the
following changes:
1 ) the bacteria were cultured in 1 ml of sterile Terrific Broth (Life
Technologies) with carbenicillin
at 25 mg/L and glycerol at 0.4%; 2) after the cultures were incubated for 19
hours, the cells were
lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation.
the piasmid DNA
pellets were resuspended in 0.1 ml of distilled water. The DNA samples were
stored at 4°C.
III. Sequencing and Analysis
The cDNAs were prepared for sequencing using the ABI CATALYST 800 (Perkin-
Elmer)
or the HYDRA microdispenser (Robbins Scientific) or MICROLAB 2200 (Hamilton)
systems in
combination with the PTC-200 thermal cyclers (MJ Research). The cDNAs were
sequenced using
the ABI PRISM 373 or 377 sequencing systems (Perkin-Elmer) and standard ABI
protocols, base
calling software, and kits. In one alternative, cDNAs were sequenced using the
MEGABACE
1000 DNA sequencing system (Molecular Dynamics). In another alternative, the
cDNAs were
amplified and sequenced using the ABI PRISM BIGDYE Terminator cycle sequencing
ready
reaction kit (Perkin-Elmer). In yet another alternative, cDNAs were sequenced
using solutions
and dyes from Amersham Pharmacia Biotech. Reading frames for the ESTs were
determined
using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7). Some of
the cDNA
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sequences were selected for extension using the techniques disclosed in
Example V.
The polynucleotide sequences derived from cDNA, extension, and shotgun
sequencing
were assembled and analyzed using a combination of software programs which
utilize algorithms
well known to those skilled in the art. Sequences were analyzed using
MACDNASIS PRO
software (Hitachi Software Engineering) and LASERGENE software (DNASTAR).
The poiynucleotide sequences were validated by removing vector, linker, and
polyA
sequences and by masking ambiguous bases, using algorithms and programs based
on BLAST,
dynamic programming, and dinucieotide nearest neighbor analysis. The sequences
were then
queried against a selection of public databases such as GenBank primate,
rodent, mammalian,
10 vertebrate, and eukaryote databases, and BLOCKS to acquire annotation,
using programs based on
BLAST, FASTA, and BLIMPS. The sequences were assembled into full length
polynucleotide
sequences using programs based on Phred, Phrap, and Consed, and were screened
for open
reading frames using programs based on GeneMark. BLAST, and FASTA. The full
length
polynucleotide sequences were translated to derive the corresponding full
length amino acid
15 sequences, and these full length sequences were subsequently analyzed by
querying against
databases such as the GenBank databases (described above), SwissProt, BLOCKS,
PRINTS,
PFAM, and Prosite.
The programs described above for the assembly and analysis of full length
polynucleotide
and amino acid sequences were used to identify polynucleotide sequence
fragments from SEQ ID
20 N0:3-4. Fragments from about 20 to about 4000 nucleotides which are useful
in hybridization
and amplification technologies were described in the Invention section above.
IV. Northern Analysis
Northern analysis is a laboratory technique used to detect the presence of a
transcript of a
gene and involves the hybridization of a labeled nucleotide sequence to a
membrane on which
25 RNAs -C-from a particular cell type or tissue have been bound. (See, e.g.,
Sambrook, supra, ch. 7;
Ausubel, 1995, supra, ch. 4 and 16.)
Analogous computer techniques applying BLAST were used to search for identical
or
related molecules in nucleotide databases such as GenBank or LIFESEQ database
(Incyte
Pharmaceuticals). This analysis is much faster than multiple membrane-based
hybridizations. In
30 addition, the sensitivity of the computer search can be modified to
determine whether any
particular match is categorized as exact or similar. The basis of the search
is the product score,
which is defined as:
seauence identity x % maximum BLAST score
100
35 The product score takes into account both the degree of similarity between
two sequences and the
39
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WO 00/17354 PCT/US99/21281
length of the sequence match. For example, with a product score of 40, the
match will be exact
within a 1% to 2% error, and, with a product score of 70, the match will be
exact. Similar
molecules are usually identified by selecting those which show product scores
between 15 and 40,
although lower scores may identify related molecules.
The results of northern analyses are reported a percentage distribution of
libraries in which
the transcript encoding HIBP occurred. Analysis involved the categorization of
eDNA libraries by
organ/tissue and disease. The organ/tissue categories included cardiovascular,
dermatologic,
developmental, endocrine, gastrointestinal, hematopoietic/immune,
musculoskeletal, nervous,
reproductive. and urologic. The disease categories included cancer,
inflammation/trauma, fetal,
neurological. and pooled. For each category, the number of libraries
expressing the sequence of
interest was counted and divided by the total number of libraries across all
categories. Percentage
values of tissue-specific and disease expression are reported in the
description of the invention.
V. Extension of HIBP Encoding Polynucleotides
The full length nucleic acid sequence of SEQ ID N0:3-4 were produced by
extension of
IS an appropriate fragment of the full length molecule using oligonucleotide
primers designed from
this fragment. One primer was synthesized to initiate 5' extension of the
known fragment, and the
other primer. to initiate 3' extension of the known fragment. The initial
primers were designed
using OLIGO 4.06 software (National Biosciences), or another appropriate
program, to be about
22 to 30 nucleotides in length, to have a GC content of about 50% or more, and
to anneal to the
target sequence at temperatures of about 68 °C to about 72 °C.
Any stretch of nucleotides which
would result in hairpin structures and primer-primer dimerizations was
avoided.
Selected human cDNA libraries were used to extend the sequence. If more than
one
extension was necessary or desired. additional or nested sets of primers were
designed.
High fidelity amplification was obtained by PCR using methods well known in
the art.
PCR was performed in 96-well plates using the PTC-200 thermal cycier (MJ
Research, Inc.). The
reaction mix contained DNA template, 200 nmol of each primer, reaction buffer
containing MgZ',
(NH4),SOa, and ~3-mercaptoethanol, Taq DNA polymerase (Amersham Pharmacia
Biotech),
ELONGASE enzyme (Life Technologies), and Pfu DNA polymerase (Stratagene), with
the
following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3
min; Step 2: 94°C, 15 sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3,
and 4 repeated 20 times; Step 6:
68°C, 5 min: Step 7: storage at 4°C. In the alternative, the
parameters for primer pair T7 and SK+
were as follows: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step
3: 57°C, 1 min; Step 4: 68°C, 2
min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68°C, 5 min;
Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 pl
PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN; Molecular Probes.
Eugene OR)
CA 02344657 2001-05-02
WO 00/17354 PCT/US99/21281
dissolved in 1 X TE and 0.5 pl of undiluted PCR product into each well of an
opaque fluorimeter
plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The
plate was
scanned in a Fluoroskan (I (Labsystems Oy, Helsinki, Finland) to measure the
fluorescence of the
sample and to quantify the concentration of DNA. A S ~I to 10 ul aliquot of
the reaction mixture
was analyzed by electrophoresis on a I % agarose mini-gel to determine which
reactions were
successful in extending the sequence.
The extended nucleotides were desalted and concentrated. transferred to 384-
well plates,
digested with CviJI cholera virus endonuclease (Molecular Biology Research,
Madison WI), and
sonicated or sheared prior to religation into pUC 18 vector (Amersham
Pharmacia Biotech). For
shotgun sequencing, the digested nucleotides were separated on low
concentration (0.6 to 0.8%)
agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended
clones were religated using T4 ligase (New England Biolabs, Beverly MA) into
pUC 18 vector
(Amersham Pharmacia Biotech). treated with Pfu DNA polymerase (Stratagene) to
fill-in
restriction site overhangs, and transfected into competent E. coli cells.
Transformed cells were
IS selected on antibiotic-containing media, individual colonies were picked
and cultured overnight at
37°C in 384-well plates in LB/2x carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase
(Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the
following
parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3:
60°C, 1 min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, S min: Step
7: storage at 4°C. DNA was
quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples
with low
DNA recoveries were reamplified using the same conditions as described above.
Samples were
diluted with 20% dimethysulphoxide (i:2, v/v), and sequenced using DYENAMIC
energy transfer
sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or
the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequences of SEQ ID N0:3-4 are used to obtain
5'
regulatory sequences using the procedure above, oligonucleotides designed for
such extension,
and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes
Hybridization probes derived from SEQ ID N0:3-4 are employed to screen cDNAs,
genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting
of about 20
base pairs, is specifically described, essentially the same procedure is used
with larger nucleotide
fragments. Oligonucleotides are designed using state-of the-art software such
as OLIGO 4.06
software (National Biosciences) and labeled by combining 50 pmol of each
oligomer, 250 ~Ci of
[y-'=P] adenosine triphosphate (Amersham Pharmacia Biotech). and T4
polynucleotide kinase
4l
CA 02344657 2001-05-02
WO 00/17354 PCT/US99/21281
(DuPont NEN, Boston MA). The labeled oligonucleotides are substantially
purified using a
SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia
Biotech).
An aliquot containing 10' counts per minute of the labeled probe is used in a
typical membrane-
based hybridization analysis of human genomic DNA digested with one of the
following
endonucleases: Ase 1, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred
to nylon
membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is
carried out for 16
hours at 40°C. To remove nonspecific signals, blots are sequentially
washed at room temperature
under increasingly stringent conditions up to 0.1 x saline sodium citrate and
0.5% sodium dodecyl
10 sulfate. After XOMAT-AR film (Eastman Kodak, Rochester NY) is exposed to
the blots for
several hours, hybridization patterns are compared visually.
VII. Microarrays
A chemical coupling procedure and an ink jet device can be used to synthesize
array
elements on the surface of a substrate. (See, e.g., Baldeschweiler, supra.) An
array analogous to a
dot or slot blot may also be used to arrange and link elements to the surface
of a substrate using
thermal, UV, chemical, or mechanical bonding procedures. A typical array may
be produced by
hand or using available methods and machines and contain any appropriate
number of elements.
After hybridization, nonhybridized probes are removed and a scanner used to
determine the levels
and patterns of fluorescence. The degree of complementarity and the relative
abundance of each
probe which hybridizes to an element on the microarray may be assessed through
analysis of the
scanned images.
Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may
comprise the elements of the microarray. Fragments suitable for hybridization
can be selected
using software well known in the art such as LASERGENE software (DNASTAR).
Full-length
25 cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide
sequences of the
present invention, or selected at random from a cDNA library relevant to the
present invention, are
arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed
to the slide using, e.g.,
UV cross-linking followed by thermal and chemical treatments and subsequent
drying. (See, e.g.,
Schena, M. et al. ( 1995) Science 270:467-470; Shalom D. et al. ( 1996) Genome
Res. 6:639-645.)
30 Fluorescent probes are prepared and used for hybridization to the elements
on the substrate. The
substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides
Sequences complementary to the HIBP-encoding sequences, or any parts thereof,
are
used to detect, decrease, or inhibit expression of naturally occurring HIBP.
Although use of
35 oligonucleotides comprising from about 1 S to 30 base pairs is described,
essentially the same
42
CA 02344657 2001-05-02
WO 00!17354 PCT/US99/21281
procedure is used with smaller or with larger sequence fragments. Appropriate
oligonucleotides
are designed using OLIGO 4.06 software (National Biosciences) and the coding
sequence of
HIBP. To inhibit transcription, a complementary oligonucleotide is designed
from the most
unique ~' sequence and used to prevent promoter binding to the coding
sequence. To inhibit
translation, a complementary oligonucleotide is designed to prevent ribosomal
binding to the
HIBP-encoding transcript.
IX. Expression of HIBP
Expression and purification of HIBP is achieved using bacterial or virus-based
expression systems. For expression of HIBP in bacteria, cDNA is subcloned into
an appropriate
1 o vector containing an antibiotic resistance gene and an inducible promoter
that directs high levels
of cDNA transcription. Examples of such promoters include, but are not limited
to, the trp-lac
(tac) hybrid promoter and the TS or T7 bacteriophage promoter in conjunction
with the lac
operator reeuiatory element. Recombinant vectors are transformed into suitable
bacterial hosts,
e.g., BL21(DE3). Antibiotic resistant bacteria express HIBP upon induction
with isopropyl beta-
t5 D-thiogalactopyranoside (IPTG). Expression of HIBP in eukaryotic cells is
achieved by infecting
insect or mammalian cell lines with recombinant Autoeraphica californica
nuclear polyhedrosis
virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin
gene of
baculovirus is replaced with cDNA encoding HIBP by either homologous
recombination or
bacterial-mediated transposition involving transfer pfasmid intermediates.
Viral infectivity is
20 maintained and the strong polyhedrin promoter drives high levels of cDNA
transcription.
Recombinant baculovirus is used to infect Snodoptera fru~iperda (Sf~) insect
cells in most cases,
or human hepatocytes, in some cases. Infection of the latter requires
additional genetic
modifications to baculovirus. (See Engelhard, E.K. et al. (1994) Proc. Natl.
Acad. Sci. USA
91:3224-3227; Sandig, V. et al. (1996) Hum. Gene Ther. 7:1937-1945.)
25 In most expression systems, HIBP is synthesized as a fusion protein with,
e.g.,
glutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-
His, permitting rapid,
single-step, affinity-based purification of recombinant fusion protein from
crude cell lysates.
GST, a 26-kilodalton enzyme from Schistosoma jaaonicum, enables the
purification of fusion
proteins on immobilized glutathione under conditions that maintain protein
activity and
30 antigenicity (Amersham Pharmacia Biotech). Following purification, the GST
moiety can be
proteolytically cleaved from HIBP at specifically engineered sites. FLAG, an 8-
amino acid
peptide, enables immunoaffinity purification using commercially available
monoclonal and
polyclonal anti-FLAG antibodies (Eastman Kodak). 6-His, a stretch of six
consecutive histidine
residues, enables purification on metal-chelate resins (QIAGEN). Methods for
protein expression
35 and purification are discussed in Ausubel ( 1995, su ra, ch 10 and 16).
Purified HIBP obtained by
43
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these methods can be used directly in the following activity assay.
X. Demonstration of HIBP Activity
HIBP activity is measured by its ability to regulate transformation of NIH3T3
mouse
fibrobiast cells transfected with a genomic~c construct. Genomic DNA encoding
MYC and its
5' regulatory regions is subcloned into an appropriate eukaryotic vector. This
construct is
transfected into NIH3T3 cells using methods known in the art. Transfected
cells are treated with
HIBP, E2F, a combination of HIBP and E2F, or are left untreated. Cells are
assessed for the
following quantifiable properties characteristic of oncogenically transformed
cells: growth in
culture to high density associated with loss of contact inhibition, growth in
suspension or in soft
agar, lowered serum requirements, and ability to induce tumors when injected
into
immunodeficient mice. The activity of HIBP is proportional to the extent of
transformation of
NIH3T3 cells treated with HIBP and E2F relative to cells treated with E2F
alone.
XI. Functional Assays
HIBP function is assessed by expressing the sequences encoding HIBP at
physiologically
elevated levels in mammalian cell culture systems. cDNA is subcloned into a
mammalian
expression vector containing a strong promoter that drives high levels of cDNA
expression.
Vectors of choice include pCMV SPORT plasmid (Life Technologies) and pCR3.1
plasmid
(Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter.
S-IO,ug of
recombinant vector are transiently transfected into a human cell line,
preferably of endothelial or
hematopoietic origin, using either liposome formulations or electroporation. 1-
2 ~g of an
additional plasmid containing sequences encoding a marker protein are co-
transfected. Expression
of a marker protein provides a means to distinguish transfected cells from
nontransfected cells and
is a reliable predictor of cDNA expression from the recombinant vector. Marker
proteins of
choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a
CD64-GFP fusion
protein. Flow cytometry (FCM), an automated, laser optics-based technique, is
used to identify
transfected cells expressing GFP or CD64-GFP, and to evaluate properties, for
example, their
apoptotic state. FCM detects and quantifies the uptake of fluorescent
molecules that diagnose
events preceding or coincident with cell death. These events include changes
in nuclear DNA
content as measured by staining of DNA with propidium iodide: changes in cell
size and
granularity as measured by forward light scatter and 90 degree side light
scatter; down-regulation
of DNA synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in
expression of cell surface and intracellular proteins as measured by
reactivity with specific
antibodies; and alterations in plasma membrane composition as measured by the
binding of
fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow
cytometry are
discussed in Ormerod, M.G. ( 1994) Flow Cvtometrv, Oxford, New York NY.
44
CA 02344657 2001-05-02
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The influence of HIBP on gene expression can be assessed using highly purified
populations of cells transfected with sequences encoding HIBP and either CD64
or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of transfected cells and bind
to conserved
regions of human immunoglobulin G (IgG). Transfected cells are efficiently
separated from
nontransfected cells using magnetic beads coated with either human IgG or
antibody against CD64
(DYNAL, Lake Success NY). mRNA can be purified from the cells using methods
well known
by those of skill in the art. Expression of mRNA encoding HIBP and other genes
of interest can
be analyzed by northern analysis or microarray techniques.
XII. Production of HH3P Specific Antibodies
HIBP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g.,
Harrington, M.G. ( 1990) Methods Enzymol. 182:488-495), or other purification
techniques, is
used to immunize rabbits and to produce antibodies using standard protocols.
Alternatively, the HIBP amino acid sequence is analyzed using LASERGENE
software
(DNASTAR) to determine regions of high immunogenicity, and a corresponding
oligopeptide is
synthesized and used to raise antibodies by means known to those of skill in
the art. Methods for
selection of appropriate epitopes, such as those near the C-terminus or in
hydrophilic regions are
well described in the art. (See, e.g., Ausubel, 1995, s_u_pra, ch. l l.)
Typically, oligopeptides I S residues in length are synthesized using an ABI
431A
Peptide Synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH
(Sigma-Aldrich,
St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to
increase immunogenicity. (See, e.g., Ausubel, 1995, s. unra.) Rabbits are
immunized with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide activity by, for example, binding the peptide to plastic, blocking
with 1 % BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
XIII. Purification of Naturally Occurring HIBP Using Specific Antibodies
Naturally occurring or recombinant HIBP is substantially purified by
immunoaffinity
chromatography using antibodies specific for HIBP. An immunoaffinity column is
constructed by
covalently coupling anti-HIBP antibody to an activated chromatographic resin,
such as
CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the
resin is
blocked and washed according to the manufacturer's instructions.
Media containing HIBP are passed over the immunoaffinity column, and the
column is
washed under conditions that allow the preferential absorbance of HIBP (e.g.,
high ionic strength
buffers in the presence of detergent). The column is eluted under conditions
that disrupt
antibody/HIBP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration
of a chaotrope,
such as urea or thiocyanate ion), and HIBP is collected.
CA 02344657 2001-05-02
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- XIV. Identification of Molecules Which Interact with HIBP
HIBP, or biologically active fragments thereof, are labeled with'Z'I Bolton-
Hunter
reagent. (See, e.g., Bolton, A.E. and W.M. Hunter ( 1973) Biochem. J. 133:529-
539.) Candidate
molecules previously arrayed in the wells of a mufti-well plate are incubated
with the labeled
HIBP, washed, and any wells with labeled HIBP complex are assayed. Data
obtained using
different concentrations of HIBP are used to calculate values for the number,
affinity, and
association of HIBP with the candidate molecules.
Various modifications and variations of the described methods and systems of
the
invention will be apparent to those skilled in the art without departing from
the scope and spirit of
the invention. Although the invention has been described in connection with
specific preferred
embodiments, it should be understood that the invention as claimed should not
be unduly limited
to such specific embodiments. Indeed, various modifications of the described
modes for carrying
out the invention which are obvious to those skilled in molecular biology or
related fields are
intended to be within the scope of the following claims.
46
CA 02344657 2001-05-02
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SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
HILLMAN, Jennifer L.
LAL, Preeti
CORLEY, Neil C.
GUEGLER, Karl J.
PATTERSON, Chandra
<120> HUMAN ISRE-BINDING PROTEIN
<130> PF-0459-1 PCT
<140> To Be Assigned
<141> Herewith
<1S0> 09/157,091
c151> 1998-09-18
<160> 5
<170> PERL Program
<210> 1
c211> 245
<212> PRT
<213> Homo sapiens
c220>
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Met Gly Gly Ala Ser Arg Arg Val Glu Ser Gly Ala Trp Ala Tyr
1 5 10 15
Leu Ser Pro Leu Val Leu Arg Lys Glu Leu G1u Ser Leu Val Glu
20 25 30
Asn Glu Gly Ser Glu Val Leu Ala Leu Pro Glu Leu Pro Ser Ala
35 40 45
His Pro Ile Ile Phe Trp Asn Leu Leu Trp Tyr Phe Gln Arg Leu
50 5S 60
Arg Leu Pro Ser Ile Leu Pro Gly Leu Val Leu Ala Ser Cys Asp
65 70 75
Gly Pro Ser His Ser Gln Ala Pro Ser Pro Trp Leu Thr Pro Asp
80 85 90
Pro Ala Ser Val Gln Val Arg Leu Leu Trp Asp Val Leu Thr Pro
95 100 105
Asp Pro Asn Ser Cys Pro Pro Leu Tyr Val Leu Trp Arg Val His
110 115 120
Ser Gln Ile Pro Gln Arg Val Val Trp Pro Gly Pro Val Pro Ala
125 130 135
Ser Leu Ser Leu Ala Leu Leu Glu Ser Val Leu Arg His Val Gly
140 145 150
Leu Asn Glu Val His Lys Ala Val Gly Leu Leu Leu Glu Thr Leu
155 160 165
1/4
CA 02344657 2001-05-02
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Gly Pro Pro Pro Thr Gly Leu His Leu Gln Arg Gly Ile Tyr Arg
170 175 180
Glu Ile Leu Phe Leu Thr Met Ala Ala Leu Gly Lys Asp His Val
185 190 195
Asp Ile Val Ala Phe Asp Lys Lys Tyr Lys Ser Ala Phe Asn Lys
200 205 210
Leu Ala Ser Ser Met Gly Lys Glu Glu Leu Arg His Arg Arg Ala
215 220 225
Gln Met Pro Thr Pro Lys Ala Ile Asp Cys Arg Lys Cys Phe Gly
230 235 240
Ala Pro Pro Glu Cys
245
<210> 2
<211> 220
<212> PRT
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 1640136CD1
<400> 2
Met Gly Gly Ala Ser Arg Arg Val Glu Ser Gly Ala Trp Ala Tyr
1 5 10 15
Leu Ser Pro Leu Val Leu Arg Lys Glu Leu Glu Ser Leu Val Glu
20 25 30
Asn Glu Gly Ser Glu Val Leu Ala Leu Pro Glu Leu Pro Ser Ala
35 40 45
His Pro Ile Ile Phe Trp Asn Leu Leu Trp Tyr Phe Gln Arg Leu
50 55 60
Arg Leu Pro Ser Ile Leu Pro Gly Leu Val Leu Ala Ser Cys Asp
65 70 75
Gly Pro Ser His Ser Gln Ala Pro Ser Pro Trp Leu Thr Pro Asp
80 85 90
Pro Ala Ser Val Gln Ala Arg Ser Pro Gln Arg Val Val Trp Pro
95 100 105
Gly Pro Val Pro Ala Ser Leu Ser Leu Ala Leu Leu Glu Ser Val
110 115 120
Leu Arg His Val Gly Leu Asn Glu Val His Lys Ala Val Gly Leu
125 130 135
Leu Leu Glu Thr Leu Gly Pro Pro Pro Thr Gly Leu His Leu Gln
140 145 150
Arg Gly Ile Tyr Arg Glu Ile Leu Phe Leu Thr Met Ala Ala Leu
155 160 165
Gly Lys Asp His Val Asp Ile Val Ala Phe Asp Lys Lys Tyr Lys
170 175 180
Ser Ala Phe Asn Lys Leu Ala Ser Ser Met Gly Lys Glu Glu Leu
185 190 195
Arg His Arg Arg Ala Gln Met Pro Thr Pro Lys Ala Ile Asp Cys
200 205 210
Arg Lys Cys Phe Gly Ala Pro Pro Glu Cys
215 220
<210> 3
2/4
CA 02344657 2001-05-02
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<211> 2033
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte ID No: 2518547CB1
<400> 3
cggctcgagg ccgcagcccc atggacagtc ttctgcaccc ccgggagcgc cctggatcca 60
ctgcctccga gagctcagcc tctctgggca gtgagtggga cctctcagaa tcttctctca 120
gcaacctgag tcttcgccgt tcctcagagc gcctcagtga cacccctgga tccttccagt 180
caccttccct ggaaattctg ctgtccagct gctccctgtg ccgtgcctgt gattcgctgg 240
tgtatgatga ggaaatcatg gctggctggg cacctgatga ctctaacctc aacacaacct 300
gccccttctg cgcctgcccc tttgtgcccc tgctcagtgt ccagaccctt gattcccggc 360
ccagtgtccc cagccccaaa tctgctggtg ccagtggcag caaagatgct cctgtccctg 420
gtggtcctgg ccctgtgctc agtgaccgaa ggctctgcct tgctctggat gagcccagct 480
ctgcaacggg cacatggggg gagcctcccg gcgggttgag agtggggcat gggcatacct 540
gagccccctg gtgctgcgta aggagctgga gtcgctggta gagaacgagg gcagtgaggt 600
gctggcgttg cctgaactgc cctctgccca ccccatcatc ttctggaacc ttttgtggta 660
tttccaacgg ctacgcctgc ccagtattct accaggcctg gtgctggcct cctgtgatgg 720
gccttcgcac tcccaggccc catctccttg gctaacccct gatccagcct ctgttcaggt 780
acggctgctg tgggatgtac tgacccctga ccccaatagc tgcccacctc tctatgtgct 840
ctggagggtc cacagccaga tcccccagcg ggtggtatgg ccaggccctg tacctgcatc 900
ccttagtttg gcactgttgg agtcagtgct gcgccatgtt ggactcaatg aagtgcacaa 960
ggctgtgggg ctcctgctgg aaactctagg gcccccaccc actggcctgc acctgcagag 1020
gggaatctac cgtgagatat tattcctgac aatggctgct ctgggcaagg accacgtgga 1080
catagtggcc ttcgataaga agtacaagtc tgcctttaac aagctggcca gcagcatggg 1140
caaggaggag ctgaggcacc ggcgggcgca gatgcccact cccaaggcca ttgactgccg 1200
aaaatgtttt ggagcacctc cagaatgcta gagaccttaa gcttccctct ccagcctagg 1260
gtggggaagt gaggaagaag ggattctaga gttaaactgc ctccctgttg ccttcatgga 1320
gttgggaaca ggctgggaag gatgcccagt caaaggctcc aagcgaggac aacaggaaga 1380
gggatccact gttaccaaaa gtcctgattc ccccatcacc aacctaccca gtttgttcgt 1440
gctgatgttg ggggagatct ggggggagtt ggtacagctc tgttcttccc ttgtcctata 1500
ccgggaactc ccctccaggg tacccacaga tctgcattgc cctggtcatt ttagaagttt 1560
ttgttttaaa aaacaactgg aaagatgcag agctactgag cctttgccct gaatgggagg 1620
tagggatgtc attctccacc aataatggtc cctcttccct gacgttgctg aaggagccca 1680
aggctctcca tgcctttcta cctaagtgtt tgtattttat tttaaattat ttattctgga 1740
gccacagccc ccttgcttat gaggttctta tggagagtga gaaagggaag ggaaataggg 1800
caccatggtc cggtggtttg tagttccttc aaagtcaggc actgggagct agaggagtct 1860
caagctcccc ttaggaagaa ctggtgcccc ctccagtcct aatttttctt gcctgccccg 1920
ccttggggaa tgcctcaccc acccaggtcc tgacctgtgc aataaggatt gttccctgcg 1980
aagttttgtt ggatgtaaat atagtaaaag ctgcttctgt ctttttcaaa aaa 2033
<210> 4
<211> 1320
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<223> Incyte ID No: 1640136CB1
<400> 4
ttgtgcccct gctcagtgtc cagacccttg attcccggcc caggtgccca aggcagggca 60
agtgctgtgg gtgggaatag ggaggtgaag gtccaaaagc tgacccctgc tgctatcccc 120
3/4
CA 02344657 2001-05-02
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ctgcagtgtc cccagcccca aatctgctgg tgccagtggc agcaaagatg ctcctgtccc 180
tggtggtcct ggccctgtgc tcagtgaccg aagctctgcc ttgctctgga tgagcccagc 240
tctgcaacgg gcacatgggg ggagcctccc ggcgggttga gagtggggca tgggcatacc 300
tgagccccct ggtgctgcgt aaggagctgg agtcgctggt agagaacgag ggcagtgagg 360
tgctggcgtt gcctgaactg ccctctgccc accccatcat cttctggaac cttttgtggt 420
atttccaacg gctacgcctg cccagtattc taccaggcct ggtgctggcc tcctgtgatg 480
ggccttcgca ctcccaggcc ccatctcctt ggctaacccc tgatccagcc tctgttcagg 540
ccagatcccc ccagcgggtg gtatggccag gccctgtacc tgcatccctt agtttggcac 600
tgttggagtc agtgctgcgc catgttggac tcaatgaagt gcacaaggct gtggggctcc 660
tgctggaaac tctagggccc ccacccactg gcctgcacct gcagagggga atctaccgtg 720
agatattatt cctgacaatg gctgctctgg gcaaggacca cgtggacata gtggccttcg 780
ataagaagta caagtctgcc tttaacaagc tggccagcag catgggcaag gaggagctga 840
ggcaccggcg ggcgcagatg cccactccca aggccattga ctgccgaaaa tgttttggag 900
cacctccaga atgctagaga ccttaagctt ccctctccag cctagggtgg ggaagtgagg 960
aagaagggat tctagagtta aactgcttcc ctgttgcctt catggagttg ggaacaggct 1020
gggaaggatg cccagtcaaa ggctccaagc gaggacaaca ggaaagagga tccactgtta 1080
cccgaagtcc tgattccccc attcaccacc tacccaattt gttccgtgcc gaatttttgg 1140
gggaaatttt gggggggaat ttggtaaaag cccctggttc ttcccccttg gtcctaataa 1200
ccgggggaac ctccccctcc agggggtaac cccaaaaagt tctggaaatt tgcccccggg 1260
gccaattttt aaaaaaattt ttgtggtttt taaaaacaca ccactctgga aaagttgggg 1320
<210> 5
<211> 191
<212> PRT
<213> Homo Sapiens
<300> misc_feature
<308> GenBank ID No: g33969
<400> 5
Trp Asn Leu Val Trp Tyr Phe Arg Arg Leu Asp Leu Pro Ser Asn
1 5 10 15
Leu Pro Gly Leu Ile Leu Ser Ser Glu His Cys Asn Lys Tyr Ser
20 25 30
Lys Ile Pro Arg His Cys Met Ser Glu Asp Ser Lys Tyr Val Leu
35 40 45
Ile Gln Met Leu Trp Asp Asn Met Lys Leu His Gln Asp Pro Gly
50 55 60
Gln Pro Leu Tyr Ile Leu Trp Asn Ala His Thr Gln Lys Tyr Pro
65 70 75
Met Val His Leu Leu Gln Lys Ser Asp Asn Ser Phe Asn Gln Glu
80 85 90
Leu Leu Lys Ser Met Val Lys Ser Ile Lys Met Asn Asp Val Tyr
95 100 105
Gly Pro Met Ser Gln Ile Leu Glu Thr Leu Asn Lys Cys Pro His
110 115 120
Phe Lys Arg Gln Arg Ser Leu Tyr Arg Glu Ile Leu Phe Leu Ser
125 130 135
Leu Val Ala Leu Gly Arg Glu Asn Ile Asp Ile Asp Ala Phe Asp
140 145 150
Lys Glu Tyr Lys Met Ala Tyr Asp Arg Leu Thr Pro Ser Gln Val
155 160 165
Lys Ser Thr His Asn Cys Asp Arg Pro Pro Ser Thr Gly Val Met
170 175 180
Glu Cys Arg Lys Thr Phe Gly Glu Pro Tyr Leu
185 190
4/4
