Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Human Brainiac-5
Field of the Invention
The present invention relates to a novel human gene encoding a polypeptide
related to the Notch family. More specifically, isolated nucleic acid
molecules are
provided encoding a human polypeptide named Brainiac-5. Brainiac-5
polypeptides
are also provided, as are vectors, host cells and recombinant methods for
producing the
same. Also provided are diagnostic methods for detecting disorders related to
the
immune and nervous systems, and therapeutic methods for treating and/or
preventing
such disorders.
The invention further relates to screening methods for identifying agonists
and
antagonists of Brainiac-5 activity.
Background of the Invention
Control of cell division is a basic aspect of multicellular existence that
depends
upon a programmed series of events. One factor in cellular proliferation and
its
control is the presence of various polypeptide growth factors. Growth factors
are
essential components of growth media for in vitro cell culture and are
involved in cell
survival in vivo. A partial list of growth factors identified to date include
platelet-derived growth factor (PDGF; implicated in the repair of the vascular
system
in vivo); epidermal growth factor (EGF; which acts as a mitogen for cells of
ectodermal and mesodermal origin); transforming growth factor (TGF)-alpha
(which
acts as a mitogen similarly to EGF, with the exception that it enables normal
cells to
grow in soft-agar); transforming growth factor (TGF)-beta (a mitogen for some
cells
and a growth inhibitor for others); and nerve growth factor (NGF; which is
involved in
the development and maintenance of sympathetic and embryonic neurons).
(Watson,
et al., Molecular Biology of the Gene, p. 975; Benjamin/Cummings ( 1987).)
It is clear that particular cell types require particular growth factors for
normal
growth and maintenance. Peptide growth factors are produced and secreted from
a
variety of tissues. The target cells are typically located near the site of
release of the
growth factor (paracrine response). In addition to growth promoting and
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differentiation-inducing activities, growth factors elicit a wide variety of
effects on
their target cells and are involved in processes such as inflammation, immune
reactions, and wound repair. (See, Pimentel, E. Handbook of Growth Facrors,
Volume
l: General Basics (CRC Press 1994).)
The Notch family of transmembrane receptor proteins have been demonstrated
to mediate cell fate decisions, and mutations in mammalian Notch genes have
been
implicated in leukemia, cervical cancer, colon cancer, breast cancer, stroke,
and
dementia. In Drosophila, three genes, fringe, Serrate, and Delta, are involved
in the
cellular interactions leading to Notch activation. Delta and Serrate encode
transmembrane ligands for Notch, whereas frznge encodes a pioneer protein.
Human
homologs of Notch, Delta, Senate (termed "Jagged"), and Fringe (termed
"Radical
Fringe" and "Lunatic Fringe") have been cloned. Expression studies in mouse
embryos support a conserved role for mammalian Fringe family members in
participation in the Notch signaling pathway.
Myocardial hypertrophy refers to a focal or general enlargement of the heart.
Normal hypertrophy is a compensatory action which functions to maintain the
pumping action of the heart. Abnormal hypertrophy occurs in a number of
situations
including hypertension, myocardial infarction, valve disease, and
cardiomyopathy.
(Simpson, P.C. Heart Failure 5:113 (1989).) The effects of peptide growth
factors on
cardiac myocytes are reflected in differentiated patterns of gene expression.
For
example, stimulation of the alpha-adrenergic receptor induces hypertrophy of
cultured
cardiac myocytes and produces specific changes in gene expression at the level
of
transcription. (Simpson, P. C. "Cardiac Myocyte Hypertrophy," Molecular
Biology of
the Cardiovascular System, Roberts, R. et al., ed.: 125-133 ( 1990).) In
cardiac
myocytes, the growth factors TGF-beta 1 and basic FGF concomitantly elicit
complex
and heterogeneous responses: selective inhibition of certain adult
transcripts,
concurrent with the upregulation of "fetal" contractile protein genes.
(Schneider, et
al., "Oncogenes and Myogenesis," Molecular Biology of the Cardiovascular
System,
Roberts, R. et al., ed.: 63-71 (1990).)
Monitoring of growth factor gene expression in myocytes and other cells of the
heart, including connective tissue, would be useful in detecting and studying
abnormal
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hypertrophy both in vitro and in vivo. Organ and clonal cell systems have been
developed to analyze cardiomyogenic differentiation. (See, for example, Bader,
D. et
al., Molecular Biology of the Cardiovascular System, Roberts, R. et al., ed.:
41-49
(1990).) Differentiation in these systems can be monitored by in vitro
analysis of
cardiac myogenesis and monoclanal antibodies that have been raised against
muscle-specific protein.
Additionally, polypeptide growth factors are very important cell culture
reagents for stimulating cellular growth and aiding survival of the cells in
vitro.
Homology with other members of the Fringe family and indication that mammalian
Fringe family members play an evolutionarily conserved role in the Notch
signaling
pathway suggests that these polypeptides, as well as Brainiac-5 polypeptides,
have
uses which include the treatment or prevention of disorders of cell fate or
differentiation (e.g., cancerous conditions, such as, leukemia, cervical
cancer, colon
cancer, breast cancer), treatment or prevention of disorders of the nervous
system, and
stimulation of tissue repair and regeneration.
The search continues to exist for polypeptides that stimulate and/or inhibit
growth of particular cells for both in vitro and in vivo uses. In addition,
the search
continues for novel tissue specific markers that can be employed qualitatively
to help
identify a particular cell or tissue type and employed qualitatively to assess
whether
cells, tissues or organs are abnormal in their expression of a particular
polypeptide.
,summary of the Invention
The present invention provides isolated nucleic acid molecules comprising a
polynucleotide encoding at least a portion of the Brainiac-5 polypeptide
having the
complete amino acid sequence shown in SEQ ID N0:2 or the complete amino acid
sequence encoded by the cDNA clone deposited as plasmid DNA as ATCC Deposit
Number 203572 on January 1 I, 1999. The nucleotide sequence determined by
sequencing the deposited Brainiac-5 clone, which is shown in Figures IA and IB
(SEQ ID NO:1 ), contains an open reading frame encoding an apparently
incomplete
polypeptide of 278 amino acid residues, beginning with an initial valine codon
at
nucleotide positions I-3, and a predicted molecular weight of about 30,475
Daltons.
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Nucleic acid molecules of the invention include those encoding the complete
amino
acid sequence shown in SEQ ID N0:2 excepting an N-terminal methionine residue,
or
the complete amino acid sequence encoded by the cDNA clone in ATCC Deposit
Number 203572 excepting an N-terminal methionine, which molecules also can
S encode additional amino acids fused to the N-terminus and/or C-terminus of
the
Brainiac-5 amino acid sequence.
Thus, one embodiment of the invention provides an isolated nucleic acid
molecule comprising, or alternatively consisting of, a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) a nucleotide
sequence
encoding the Brainiac-5 polypeptide having the complete amino acid sequence in
SEQ
ID N0:2 (i.e., positions 1 to 278 of SEQ ID N0:2); (b) a nucleotide sequence
encoding the Brainiac-5 polypeptide having the complete amino acid sequence
encoded by the cDNA clone contained in ATCC Deposit No. 203572; (c) a
nucleotide
sequence encoding the mature Brainiac-5 polypeptide having the amino acid
sequence
encoded by the cDNA clone contained in ATCC Deposit No. 203572; and (d) a
nucleotide sequence complementary to any of the nucleotide sequences in (a),
(b) or
(c), above.
Further embodiments of the invention include isolated nucleic acid molecules
that comprise a polynucleotide having (i.e., comprising, or alternatively
consisting of)
a nucleotide sequence at least 90% identical, and more preferably at least
92%, 95%,
96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a),
(b), (c) or
(d), above, or a polynucleotide which hybridizes under stringent hybridization
conditions to a polynucleotide in (a), (b), (c) or (d), above. This
polynucleotide which
hybridizes does not hybridize under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence consisting of only A residues or
of only
T residues.
An additional nucleic acid embodiment of the invention relates to an isolated
nucleic acid molecule comprising, or alternatively consisting of, a
polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion of a
Brainiac-5
polypeptide having an amino acid sequence in (a), (b) or (c), above. A further
nucleic
acid embodiment of the invention relates to an isolated nucleic acid molecule
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comprising, or alternatively consisting of, a polynucleotide which encodes the
amino
acid sequence of a Brainiac-S polypeptide having an amino acid sequence which
contains at least one conservative amino acid substitution, but not more than
50
conservative amino acid substitutions, even more preferably, not more than 40
conservative amino acid substitutions, still more preferably, not more than 30
conservative amino acid substitutions, and still even more preferably, not
more than 20
conservative amino acid substitutions. Of course, in order of ever-increasing
preference, it is highly preferable for a polynucleotide which encodes the
amino acid
sequence of a Brainiac-S polypeptide to have an amino acid sequence which
contains
not more than 10, 9, $, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid
substitutions.
The present invention also relates to recombinant vectors, which include the
isolated nucleic acid molecules of the present invention, and to host cells
containing
the recombinant vectors, as well as to methods of making such vectors and host
cells
and for using them for production of Brainiac-5 polypeptides or peptides by
recombinant techniques.
In accordance with a further embodiment of the present invention, there is
provided a process for producing such polypeptide by recombinant techniques
comprising culturing recombinant prokaryotic and/or eukaryotic host cells,
containing
a human Brainiac-5 nucleic acid sequence, under conditions promoting
expression of
said polypeptide and subsequent recovery of said polypeptide.
The invention also provides an isolated Brainiac-5 polypeptide comprising, or
alternatively consisting of, an amino acid sequence selected from the group
consisting
of: (a) the amino acid sequence of the Brainiac-5 polypeptide having the
complete
amino acid sequence shown in SEQ ID N0:2 (i.e., positions 1-278 of SEQ ID
N0:2);
(b) the complete amino acid sequence encoded by the cDNA clone contained in
the
ATCC Deposit No. 203572; (c) the complete amino acid sequence of the predicted
mature Brainiac-5 polypeptide encoded by the cDNA clone contained in the ATCC
Deposit No. 203572.
The polypeptides of the present invention also include polypeptides having
(i.e., comprising, or alternatively consisting of) an amino acid sequence at
least 80%
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identical, more preferably at least 90% identical, and still more preferably
92%, 95%,
96%, 97%, 98% or 99% identical to those described in (a), (b) or (c), above.
An additional embodiment of the invention relates to a peptide or polypeptide
which comprises, or alternatively consists of, the amino acid sequence of an
epitope-bearing portion of a Brainiac-5 polypeptide having (i.e., comprising,
or
alternatively consisting of) an amino acid sequence described in (a), (b) or
(c), above.
Peptides or polypeptides having the amino acid sequence of an epitope-bearing
portion
of a Brainiac-5 polypeptide of the invention include portions of such
polypeptides with
at least six or seven, preferably at least nine, and more preferably at least
about 30
amino acids to about 50 amino acids, although epitope-bearing polypeptides of
any
length up to and including the entire amino acid sequence of a polypeptide of
the
invention described above also are included in the invention.
A further embodiment of the invention relates to a peptide or polypeptide
which comprises, or alternatively consists of, the amino acid sequence of a
Brainiac-5
polypeptide having an amino acid sequence which contains at least one
conservative
amino acid substitution, but not more than 50 conservative amino acid
substitutions,
even more preferably, not more than 40 conservative amino acid substitutions,
still
more preferably, not more than 30 conservative amino acid substitutions, and
still even
more preferably, not more than 20 conservative amino acid substitutions. Of
course,
in order of ever-increasing preference, it is highly preferable for a peptide
or
polypeptide to have an amino acid sequence which comprises the amino acid
sequence
of a Brainiac-5 polypeptide, which contains at least one, but not more than
10, 9, 8, 7,
6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
In another embodiment, the invention provides an isolated antibody that binds
specifically to a Brainiac-5 polypeptide having an amino acid sequence
described in
(a), (b) or (c), above. The invention further provides methods for isolating
antibodies
that bind specifically to a Brainiac-5 polypeptide having an amino acid
sequence as
described herein. Such antibodies are useful diagnostically or therapeutically
as
described below.
The invention also provides for pharmaceutical compositions comprising
Brainiac-5 polypeptides, particularly human Brainiac-5 polypeptides, which may
be
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employed, for instance, to treat, prevent, and/or diagnose immune and/or
nervous
system diseases and disorders. Methods of treating individuals in need of
Brainiac-5
polypeptides are also provided.
The invention further provides compositions comprising a Brainiac-5
polynucleotide or polypeptide for administration to cells in vitro, to cells
ex vivo and to
cells in vivo, or to a multicellular organism. In certain particularly
preferred
embodiments of this aspect of the invention, the compositions comprise a
Brainiac-5
polynucleotide for expression of a Brainiac-5 polypeptide in a host organism
for
treatment, prevention, and/or diagnosis of disease. Particularly preferred in
this regard
is expression in a human patient for treatment, prevention, and/or diagnosis
of a
dysfunction associated with aberrant endogenous activity of a Brainiac-5
polynucleotide and/or polypeptide.
The present invention also provides a screening method for identifying
compounds capable of enhancing or inhibiting a biological activity of the
Brainiac-5
polypeptide, which involves contacting a receptor whose activity is inhibited
or
enhanced by the Brainiac-5 polypeptide with the candidate compound in the
presence
of a Brainiac-5 polypeptide, assaying cell division activity of the receptor
in the
presence of the candidate compound and of Brainiac-5 polypeptide, and
comparing the
receptor activity to a standard level of activity, the standard being assayed
when
contact is made between the receptor and in the presence of the Brainiac-5
polypeptide
and the absence of the candidate compound In this assay, an increase in
receptor
activity over the standard indicates that the candidate compound is an agonist
of
Brainiac-5 activity and a decrease in receptor activity compared to the
standard
indicates that the compound is an antagonist of Brainiac-S activity.
In another embodiment, a screening assay for agonists and antagonists is
provided which involves determining the effect a candidate compound has on
Brainiac-5 binding to another member of the Notch family (e.g., fringe,
Serrate, Delta,
Jagged, Radical Fringe, Lunatic Fringe, and Maniac Fringe). In particular, the
method involves contacting the Notch family member with a Brainiac-5
polypeptide
and a candidate compound and determining whether Brainiac-5 polypeptide
binding to
the Notch family member is increased or decreased due to the presence of the
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candidate compound. In this assay, an increase in binding of Brainiac-5 over
the
standard binding indicates that the candidate compound is an agonist of
Brainiac-5
binding activity and a decrease in Brainiac-S binding compared to the standard
indicates that the compound is an antagonist of Brainiac-5 binding activity.
The
antagonists may be employed to treat and/or prevent, diseases, disorders or
conditions
including, but not limited to, septic shock, inflammation, cerebral malaria,
activation
of the HIV virus, graft-host rejection, bone resorption, rheumatoid arthritis,
cachexia
(wasting or malnutrition), immune system function, lymphoma, and autoimrnune
disorders.
In yet another embodiment, the Brainiac-5 polypeptide(s) may bind to a cell
surface polypeptide which also function as a viral receptor or coreceptor.
Thus,
Brainiac-S, or agonists or antagonists thereof, may be used to regulate viral
infectivity
at the level of viral binding or interaction with the Brainiac-5 receptor or
coreceptor or
during the process of viral internalization or entry into the cell.
It has been discovered that Brainiac-5 is expressed not only in ovarian tumor,
but also (using BLAST analysis of the HGS EST database) in bone marrow stromal
cells and synovial sarcoma. Therefore, nucleic acids of the invention are
useful as
hybridization probes for differential identification of the tissues) or cell
types)
present in a biological sample. Similarly, polypeptides and antibodies
directed to
those polypeptides are useful to provide immunological probes for differential
identification of the tissues) or cell type(s). In addition, for a number of
disorders of
the above tissues or cells, particularly of the immune system, significantly
higher or
lower levels of Brainiac-S gene expression may be detected in certain tissues
(e.g.,
cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial
fluid or spinal fluid) taken from an individual having such a disorder,
relative to a
"standard" Brainiac-5 gene expression level, i.e., the Brainiac-5 expression
level in
healthy tissue from an individual not having the immune system disorder.
Thus, the invention provides a diagnostic method useful during diagnosis of
such a disorder, which involves: (a) assaying Brainiac-5 gene expression level
in cells
or body fluid of an individual; (b) comparing the Brainiac-5 gene expression
level with
a standard Brainiac-5 gene expression level, whereby an increase or decrease
in the
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assayed Brainiac-5 gene expression level compared to the standard expression
level is
indicative of disorder in the immune system.
Another embodiment of the invention is related to a method for treating or
diagnosing an individual in need of an increased level of Brainiac-5 activity
in the
body comprising administering to such an individual a composition comprising a
therapeutically effective amount of an isolated Brainiac-5 polypeptide of the
invention
or an agonist thereof.
A further embodiment of the invention is related to a method for treating or
diagnosing an individual in need of a decreased level of Brainiac-5 activity
in the body
comprising, administering to such an individual a composition comprising a
therapeutically effective amount of a Brainiac-5 antagonist. Preferred
antagonists for
use in the present invention are Brainiac-5-specific antibodies.
Brief Description of the Figures
The following drawings are illustrative of embodiments of the invention and
are not meant to limit the scope of the invention as encompassed by the
claims.
Figures lA and 1B show the nucleotide sequence (SEQ ID NO: l) and
deduced amino acid sequence (SEQ ID N0:2) of Brainiac-5. A single potential
asparagine-linked glycosylation site is marked in the amino acid sequence of
Brainiac-5. The potential site of glycosylation begins at asparagine-79 in
Figures lA
and 1B (SEQ ID N0:2). The potential glycosylation site is marked with a bold
pound
symbol (#) above the nucleotide sequence coupled with a bolded one letter
abbreviation for the asparagine (N) in the amino acid sequence in Figures lA
and 1B.
Regions of high identity between Brainiac-5 and the closely related Drosophila
Brainiac and human UDP-galactose-2-acetamido-2-deoxy-D-glucose-3-beta-
galactosyltransferase (an alignment of these sequences is presented in Figures
2A, 2B,
i
and 2C) are underlined in Figures lA and 1B. These regions are not limiting
and are
labeled as Conserved Domain (CD)-I, CD-II, CD-III, CD-IV, CD-V, CD-VI, CD-VII,
CD-VIII, CD-IX, CD-X, and CD-XI in Figures lA and 1B.
Figures 2A, 2B, and 2C show the regions of identity between the Brainiac-5
amino acid sequence and the translation product of the Drosophila melanogaster
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mRNA for Brainiac (SEQ ID N0:3; GenBank Accession No. U41449), and the human
UDP-galactose-2-acetamido-2-deoxy-D-glucose-3-beta-galactosyltransferase (SEQ
ID
N0:4; GenBank Accession No. Y15014), as determined by the computer program
MegAlign (DNA*STAR nucleotide and amino acid sequence analysis package) using
the default parameters.
Figure 3 shows an analysis of the Brainiac-5 amino acid sequence. Alpha,
beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic
regions;
flexible regions; antigenic index and surface probability are shown. In the
"Antigenic
Index or Jameson-Wolf" graph, the positive peaks indicate locations of the
highly
antigenic regions of the Brainiac-S polypeptide, i.e., regions from which
epitope-
bearing peptides of the invention can be obtained.
The data presented in Figure 3 are also represented in tabular form in Table
I.
The columns in Table I are labeled with the headings "Res", "Position", and
Roman
Numerals I-XIV. The column headings refer to the following features of the
amino
acid sequence presented in Figure 3 and Table I: "Res": amino acid residue of
SEQ ID
N0:2 and Figures lA and 1B; "Position": position of the corresponding residue
within
SEQ ID N0:2 and Figures lA and 1B; I: Alpha, Regions - Gamier-Robson; II:
Alpha,
Regions - Chou-Fasman; III: Beta, Regions - Garnier-Robson; IV: Beta, Regions -
Chou-Fasman; V: Turn, Regions - Garnier-Robson; VI: Turn, Regions - Chou-
Fasman; VII: Coil, Regions - Gamier-Robson; VIII: Hydrophilicity Plot - Kyte-
Doolittle; IX: Hydrophobicity Plot - Hopp-Woods; X: Alpha, Amphipathic Regions
-
Eisenberg; XI: Beta, Amphipathic Regions - Eisenberg; XII: Flexible Regions -
Karplus-Schulz; XIII: Antigenic Index - Jameson-Wolf; and XIV: Surface
Probability
Plot - Emini.
Detailed Description
The present invention provides isolated nucleic acid molecules comprising, or
alternatively consisting of, a polynucleotide encoding a Brainiac-5
polypeptide having
the amino acid sequence shown in SEQ ID NO:2, which was determined by
sequencing a cloned cDNA. The nucleotide sequence shown in Figures lA and 1B
(SEQ ID NO:1 ) was obtained by sequencing the HOGCC45 cDNA clone, which was
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deposited on January 1 l, 1999 at the American Type Culture Collection, 10801
University Boulevard, Manassas, Virginia 20110-2209, and given ATCC accession
number 203572. The deposited clone is contained in the pCMVSPORT 2.0 plasmid
(Life Technologies, Inc., Gaithersburg, MD).
The Brainiac-5 polypeptides of the present invention share sequence homology
with the translation products of the Drosophila melanogaster mRNA which
encodes
Brainiac (Figure 2; SEQ ID N0:3) human mRNA which encodes UDP-galactose-2-
acetamido-2-deoxy-D-glucose-3-beta-galactosyltransferase (Figure 2; SEQ ID
N0:4).
Drosophila Brainiac is thought to be an important neurogenic secreted molecule
that is
believed to play a role in the differentiation of embryonic cells into
neurons. Thus, it
is contemplated that the Brainiac-5 polynucleotides and polypeptides of the
invention
exert an effect on the differentiation of cells in the early stages of cell
and tissue
development, and rnay serve to aid in the differentiation of embryonic cells
into
dendritic or other immune system cells or neurons or other cells of the
nervous system.
Nucleic Acid Molecules
Unless otherwise indicated, all nucleotide sequences determined by sequencing
a DNA molecule herein were determined using an automated DNA sequencer (such
as
the Model 373 from Applied Biosystems, Inc., Foster City, CA), and all amino
acid
sequences of polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above. Therefore, as
is
known in the art for any DNA sequence determined by this automated approach,
any
nucleotide sequence determined herein may contain some errors. Nucleotide
sequences determined by automation are typically at least about 90°70
identical, more
typically at least about 95% t:o at least about 99.9% identical to the actual
nucleotide
sequence of the sequenced DNA molecule. The actual sequence can be more
precisely determined by other approaches including manual DNA sequencing
methods
well known in the art. As is also known in the art, a single insertion or
deletion in a
determined nucleotide sequence compared to the actual sequence will cause a
frame
shift in translation of the nucleotide sequence such that the predicted amino
acid
sequence encoded by a determined nucleotide sequence will be completely
different
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from the amino acid sequence actually encoded by the sequenced DNA molecule,
beginning at the point of such an insertion or deletion.
By "nucleotide sequence" of a nucleic acid molecule or polynucleotide is
intended, for a DNA molecule or polynucleotide, a sequence of
deoxyribonucleotides,
and for an RNA molecule or polynucleotide, the corresponding sequence of
ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T)
in the
specified deoxyribonucleotide sequence is replaced by the ribonucleotide
uridine (U).
Using the information provided herein, such as the nucleotide sequence in
Figures lA and 1B (SEQ ID NO:1), a nucleic acid molecule of the present
invention
encoding a Brainiac-5 polypeptide may be obtained using standard cloning and
screening procedures, such as those for cloning cDNA using mRNA as starting
material. Illustrative of the invention, the nucleic acid molecule described
in Figures
lA and 1B (SEQ ID NO: l) was discovered in a cDNA library derived from ovarian
tumor cells. Additional clones of the same gene were also identified in cDNA
libraries
from the following tissues: bone marrow stromal cells and synovial sarcoma
cells.
The determined nucleotide sequence of the Brainiac-5 cDNA of Figures 1 A
and 1B (SEQ ID NO:1) contains a partial open reading frame encoding a
polypeptide
of 278 amino acid residues, initiating with a valine codon at nucleotide
positions 1-3
of the nucleotide sequence in Figures 1 A and 1 B (SEQ ID NO:1 ), and a
deduced
molecular weight of about 30,475 Daltons. The amino acid sequence of the
Brainiac-5
polypeptide shown in SEQ ID N0:2 is about 37.7% identical to Drosophila
melanogaster mRNA for Brainiac (Figure 2), which can be accessed as GenBank
Accession No. U41449.
As one of ordinary skill would appreciate, due to the possibilities of
sequencing errors discussed above, the actual complete Brainiac-S polypeptides
encoded by the respective deposited cDNA clones, which comprises about 278
amino
acids, may be somewhat longer or shorter. More generally, the actual open
reading
frames comprising Brainiac-5 may be anywhere in the range of ~20 amino acids,
more
likely in the range of ~10 amino acids, of that predicted from the valine
codon at the
N-terminus shown in Figures 1 A and 1 B (SEQ ID NO: l ). It will further be
appreciated that, depending on the analytical criteria used for identifying
various
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functional domains, the exact "address" of a signal sequence or other
particular
domains of the Brainiac-5 polypeptides may differ slightly from the predicted
positions herein.
As indicated, nucleic acid molecules of the present invention may be in the
form of RNA, such as mRNA, or in the form of DNA, including, for instance,
cDNA
and genomic DNA obtained by cloning or produced synthetically. The DNA may be
double-stranded or single-stranded. Single-stranded DNA or RNA may be the
coding
strand, also known as the sense strand, or it may be the non-coding strand,
also
referred to as the anti-sense strand.
In specific embodiments, the polynucleotides of the invention are less than
100,000 kb, 50,000 kb, 10,000 kb, 1,000 kb, 500 kb, 400 kb, 350 kb, 300 kb,
250 kb,
200 kb, 175 kb, 150 kb, 125 kb, 100 kb, 75 kb, 50 kb, 40 kb, 30 kb, 25 kb, 20
kb, 15
kb, 10 kb, 7.5 kb, or 5 kb in length.
In further embodiments, polynucleotides of the invention comprise at least 15,
at least 30, at least 50, at least 100, or at least 250, at least 500, or at
least 1000
contiguous nucleotides of Brainiac-5 coding sequence, but consist of less than
or equal
to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25
kb, 20 kb,
15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5' or 3' coding
nucleotide set
forth in Figures lA and 1B (SEQ ID NO:1). In further embodiments,
polynucleotides
of the invention comprise at least 15, at least 30, at least 50, at least 100,
or at least
250, at least 500, or at least 1000 contiguous nucleotides of Brainiac-5
coding
sequence, but do not comprise all or a portion of any Brainiac-5 intron. In
another
embodiment, the nucleic acid comprising Brainiac-5 coding sequence does not
contain
coding sequences of a genomic flanking gene (i.e., 5' or 3' to the Brainiac-5
gene in
the genome). In other embodiments, the polynucleotides of the invention do not
contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15,
10, 5, 4,
3, 2, or 1 genomic flanking gene(s).
By "isolated" nucleic acid molecules) is intended a nucleic acid molecule,
DNA or RNA, which has been removed from its native environment. For example,
recombinant DNA molecules contained in a vector are considered isolated for
the
purposes of the present invention. Further examples of isolated DNA molecules
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include recombinant DNA molecules maintained in heterologous host cells or
purified
(partially or substantially) DNA molecules in solution. Isolated RNA molecules
include in vivo or in vitro RNA transcripts of the DNA molecules of the
present
invention. Isolated nucleic acid molecules according to the present invention
further
include such molecules produced synthetically. In another embodiment, an
"isolated"
nucleic acid molecule does not encompass a chromosome isolated or removed from
a
cell or a cell lysate (e.g., a "chromosome spread", as in a karyotype). In yet
another
embodiment, an "isolated" nucleic acid molecule does not encompass a eDNA or
genomic library which contains a sequence which encodes Brainiac-5. In further
embodiments, an "isolated" nucleic acid molecule does not encompass any
collection
of vectors which contain exceptionally large DNA, RNA, or cDNA inserts with
respect to the Brainiac-5 sequence disclosed herein (for example, cDNA or
genomic
libraries, or YAC or BAC artificial chromosomes, and the like) which contain a
sequence encoding Brainiac-5.
Isolated nucleic acid molecules of the present invention include DNA
molecules comprising, or alternatively consisting of, an open reading frame
(ORF), or
comprising a partial ORF, initiating with a valine codon at positions 1-3 of
the
nucleotide sequence shown in Figures lA and 1B (SEQ ID NO:1).
In addition, isolated nucleic acid molecules of the invention include DNA
molecules which comprise, or alternatively consist of, a sequence
substantially
different from those described above but which, due to the degeneracy of the
genetic
code, still encode Brainiac-5 polypeptides of the invention. In specific
embodiments,
Brainiac-5 variants in which 5-10, 1-5, or 1-2 amino acids are substituted,
deleted, or
added in any combination are preferred. Of course, the genetic code and
species-specific codon preferences are well known in the art. Thus, it would
be
routine for one skilled in the art to generate the degenerate variants
described above,
for instance, to optimize codon expression for a particular host (e.g., change
codons in
the human mRNA to those preferred by a bacterial host such as E. coli).
In another embodiment, the invention provides isolated nucleic acid molecules
encoding the Brainiac-S polypeptide having (i.e., comprising, or alternatively
consisting of) an amino acid sequence encoded by the cDNA clone contained in
the
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pIasmid deposited as ATCC Deposit No. 203572 on January 11, 1999. Preferably,
this
nucleic acid molecule will encode the mature polypeptide encoded by the
above-described deposited cDNA clone.
The invention further provides an isolated nucleic acid molecule having (i.e.,
S comprising, or alternatively consisting of) the nucleotide sequence shown in
Figures
lA and 1B (SEQ ID NO:1) or the nucleotide sequence of the. Brainiac-5 cDNA
contained in the above-described deposited clone, or a nucleic acid molecule
having
(i.e., comprising, or alternatively consisting of) a sequence complementary to
one of
the above sequences. Such isolated molecules, particularly DNA molecules, are
useful,
for example, as probes for gene mapping, by in situ hybridization with
chromosomes,
and for detecting expression of the Brainiac-S gene in human tissue, for
instance, by
Northern blot analysis.
The present invention is further directed to nucleic acid molecules encoding
portions of the nucleotide sequences described herein as well as to fragments
of the
isolated nucleic acid molecules described herein. In particular, the invention
provides
a polynucleotide having (i.e., comprising, or alternatively consisting of) a
nucleotide
sequence representing the portion of SEQ ID NO:1 which consists of positions 1-
977
of SEQ ID NO:1. Further, the invention includes a polynucleotide comprising,
or
alternatively consisting of, any portion of at least about 30 nucleotides,
preferably at
least about 50 nucleotides, of SEQ ID NO:1 from positions 1-977 of SEQ ID
NO:1,
excluding the sequences of the following related cDNA clones, and any
subfragments
therein: HOGCC45RA (SEQ ID NO:S); HTEDM56R (SEQ ID N0:6); HSSET36R
(SEQ ID N0:7); HSOBD70R (SEQ ID N0:8); :H 18701 (SEQ ID N0:9); and (SEQ ID
NO:10).
Further, the invention includes a polynucleotide comprising, or alternatively
consisting of, any portion of at least about 25 nucleotides, preferably at
least about 30
nucleotides, more preferably at least about 40 nucleotides, and even more
preferably at
least about SO nucleotides, of SEQ ID NO:1 from residue 1-600. More
preferably, the
invention includes a polynucleotide comprising, or alternatively consisting
of,
nucleotides 1-600; 25-600; 50-600; 75-600; 100-600; 125-600; 150-600; 175-600;
200-600; 225-600; 250-600; 275-600; 300-600; 325-600; 350-600; 375-600; 400-
600;
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425-600; 450-600; 475-600; 500-600; 525-600; 550-600; 575-600; 1-575; 25-575;
50-575; 75-575; 100-575; 125-575; 150-575; 175-575; 200-575; 225-575; 250-575;
275-575; 300-575; 325-57:5; 350-575; 375-575; 400-575; 425-575; 450-575; 475-
575;
S00-575; 525-575; 550-575; 1-550; 25-550; 50-550; 75-550; 100-550; 12S-550;
150-550; 175-550; 200-550; 225-550; 250-SSO; 275-550; 300-550; 325-550; 350-
SSO;
375-550; 400-550; 425-550; 450-550; 475-SSO; S00-550; 525-SSO; 1-525; 25-525;
50-525; 75-525; 100-525; 125-525; 150-525; 175-525; 200-525; 225-525; 250-525;
275-525; 300-525; 325-525; 350-525; 375-525; 400-525; 425-525; 450-525; 475-
525;
S00-525; 1-500; 25-500; 50-500; 75-500; 100-500; 125-500; 150-500; 175-500;
200-500; 225-500; 2S0-500; 275-500; 300-500; 325-500; 350-500; 375-500; 400-
500;
425-500; 450-500; 475-500; 1-475; 25-475; 50-475; 75-475; 100-475; 125-475;
150-475; 17S-475; 200-475; 225-475; 250-475; 275-475; 300-475; 325-475; 350-
475;
375-475; 400-475; 425-475; 450-475; 1-450; 25-450; 50-450; 75-450; 100-450;
125-450; 150-450; 175-450; 200-450; 225-450; 250-450; 275-450; 300-450; 325-
450;
350-450; 375-450; 400-450; 425-450; I-425; 25-425; 50-425; 75-425; 100-425;
125-425; 1 SO-425; I75-425; 200-425; 225-425; 250-4.25; 275-425; 300-425; 325-
425;
350-425; 375-425; 400-425; 1-400; 25-400; 50-400; 75-400; 100-400; 125-400;
150-400; 175-400; 200-400; 225-400; 250-400; 275-400; 300-400; 325-400; 350-
400;
375-400; 1-375; 25-375; SO-375; 75-375; 100-375; 125-375; 150-375; 175-375;
200-375; 225-375; 250-375; 275-375; 300-375; 325-375; 350-375; 1-350; 2S-350;
50-350; 75-350; 100-350; 125-350; 150-350; 175-350; 200-350; 225-350; 250-350;
275-350; 300-350; 325-350; 1-325; 25-325; SO-325; 75-325; 100-325; 125-325;
150-325; 175-325; 200-325; 225-325; 250-325; 275-325; 300-325; 1-300; 25-300;
50-300; 75-300; 100-300; 125-300; 150-300; 175-300; 200-300; 225-300; 250-300;
275-300; 1-275; 25-275; 50-275; 75-275; 100-275; 125-275; 150-275; 175-275;
200-275; 225-275; 250-275; I-250; 25-250; 50-250; 75-250; 100-250; 125-250;
150-2~0; 175-250; 200-250; 225-250; 1-225; 25-225; 50-225; 75-225; 100-225;
125-225; 150-225; 175-22.5; 200-225; 1-200; 25-200; 50-200; 75-200; 100-200;
125-200; 150-200; 175-200; :l-175; 25-175; 50-175; 75-175; 100-175; 125-175;
150-175; 1-150; 25-150; 50-150; 75-150; 100-I50; 125-150; I-125; 25-125; 50-
125;
75-125; 100-125; 1-100; 25-100; SO-100; 75-i00; 1-75; 25-75; 50-75; I-50; 25-
50;
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and 1-25 of SEQ ID NO: I. Preferably, these fragments encode a polypeptide
which
has biological activity, and/or a Brainiac-5 functional activity (e.g.,
activation of the
Notch signaling pathway, mediation of protein-protein interactions (e.g.,
between
Brainiac-5 and Su(H) or a human homolog of Su(H)), and/or binding of an
antibody
specific to Brainiac-5).
More generally, by a fragment of an isolated nucleic acid molecule having the
nucleotide sequence of the deposited cDNA or the nucleotide sequences shown in
Figures IA and IB (SEQ II) NO:I), is intended fragments at least about IS nt,
and
more preferably at least about 20 nt, still more preferably at least about 30
nt, and even
more preferably, at least about 40, 50, 60, 70, 80, 90, 100, I50, 200, 250,
300, 400, or
500 nt in length which are useful, for example, as diagnostic probes and
primers as
discussed herein. Of course, larger fragments 50-300 nt in length are also
useful
according to the present invention as are fragments corresponding to most, if
not all, of
the nucleotide sequence of the deposited cDNA or as shown in Figures IA and 1B
(SEQ ID NO:1 ). By a fragment at least 20 nt in length, for example, is
intended
fragments which include 20 or more contiguous bases from the nucleotide
sequence of
the deposited cDNA or the nucleotide sequence as shown in Figures lA and IB
(SEQ
ID NO:1). By "about" in the phrase "at least about" is meant the recited value
and
values that are larger or smaller by several, a few, a small number, 5, 4, 3,
2 or I.
Preferred nucleic acid fragments of the present invention include nucleic acid
molecules encoding epitope-bearing portions of the Brainiac-5 polypeptides as
identified in Figure 3, and described in more detail below.
By "Brainiac-5 functional activity" is meant, for example, activation of the
Notch signaling pathway, mediation of protein-protein interactions (e.g.,
between
Brainiac-5 and Su(H) or a human homolog of Su(H)), and/or binding of an
antibody
specific to Brainiac-5.
a
The functional activity of Brainiac-5 polypeptides, and fragments, variants
derivatives, and analogs thereof, can be assayed by various methods.
For example, in one embodiment where one is assaying for the ability to bind
or compete with full-length Brainiac-5 polypeptide for binding to anti-
Brainiac-5
antibody, various immunoassays known in the art can be used, including but not
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limited to, competitive and non-competitive assay systems using techniques
such as
radioimmunoassays, ELISA (enzyme linked irnmunosorbent assay), "sandwich"
immunoassays, immunoradiometric assays, gel diffusion precipitation reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or
radioisotope labels, for example), western blots, precipitation reactions,
agglutination
assays (e.g., gel agglutination assays, hemagglutination assays), complement
fixation
assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis
assays, etc. In one embodiment, antibody binding is detected by detecting a
label on
the primary antibody. In another embodiment, the primary antibody is detected
by
detecting binding of a secondary antibody or reagent to the primary antibody.
In a
further embodiment, the secondary antibody is labeled. Many means are known in
the
art for detecting binding in an immunoassay and are within the scope of the
present
invention.
In another embodiment, where a Brainiac-5 ligand is identified or the ability
of
a polypeptide fragment, variant or derivative of the invention to multimerize
is being
evaluated, binding can be assayed, e.g., by means well-known in the art, such
as, for
example, reducing and non-reducing gel chromatography, protein affinity
chromatography, and affinity blotting. See generally, Phizicky, E., et al.,
1995,
Microbiol. Rev. 59:94-123. In another embodiment, physiological correlates of
Brainiac-5 binding to its substrates (signal transduction) can be assayed.
Other methods will be known to the skilled artisan and are within the scope of
the invention.
In additional embodiments, the polynucleotides of the invention encode a
polypeptide comprising, or alternatively consisting of, amino acids 7 to 20, 7
to 33, 61
to 83, 105 to 119, 139 to 14$, 160 to 171, 187 to 196 of SEQ ID N0:2.
Polypeptides
encoded by these polynucleotides are also encompassed by the invention.
f
In additional embodiments, the polynucleotides of the invention encode one,
two, three, four or more functional attributes of Brainiac-5. Preferred
embodiments of
the invention in this regard include fragments that comprise alpha-helix and
alpha-helix forming regions ("'alpha-regions"), beta-sheet and beta-sheet
forming
regions ("beta-regions"), turn and turn-forming regions ("turn-regions"), coil
and
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coil-forming regions ("coil-regions"), hydrophilic regions, hydrophobic
regions, alpha
amphipathic regions, beta amphipathic regions, flexible regions, surface-
forming
regions and high antigenic index regions of Brainiac-5. Polypeptides encoded
by these
polynucleotides are also encompassed by the invention.
The data representing the structural or functional attributes of Brainiac-5
set
forth in Figure 3 and/or Table I, as described above, was generated using the
various
modules 'and algorithms of the DNA*STAR set on default parameters. In a
preferred
embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table I
can be
used to determine regions of Brainiac-5 which exhibit a high degree of
potential for
antigenicity. Regions of high antigenicity are determined from the data
presented in
columns VIII, IX, XIII, and/or IV by choosing values which represent regions
of the
polypeptide which are likely to be exposed on the surface of the polypeptide
in an
environment in which antigen recognition may occur in the process of
initiation of an
immune response.
Certain preferred regions in these regards are set out in Figure 3, but may,
as
shown irj Table I, be represented or identified by using tabular
representations of the
data presented in Figure 3. The DNA*STAR computer algorithm used to generate
Figure 3 (set on the original default parameters) was used to present the data
in Figure
3 in a tabular format (See Table I). The tabular format of the data in Figure
3 may be
used to easily deterrriine specific boundaries of a preferred region.
The above-mentioned preferred regions set out in Figure 3 and in Table I
include, but are not limited to, regions of the aforementioned types
identified by
analysis of the amino acid sequence set out in Figures lA and 1B. As set out
in Figure
3 and in Table I, such preferred regions include Gamier-Robson alpha-regions,
beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-
regions,
and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic regions,
Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible
regions,
Emini surface-forming regions and Jameson-Wolf regions of high antigenic
index.
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Table
I
Res Position z zz zzz rv v yr vzr vrzr rx x
xr x rz z
xzz xrv
Val 1 A A
. . . . . -0.2 0 -0.57 0.60 0.81
Ala 2 * . .
A A . . . . . 0.19 -0.21 0.30 0.55
Glu 3 * . .
A A . . . . . 0.69 -0.64 0.60 0.75
Asp 4 A * .
A . . . . . 1.19 -1.07 0.75 1.97
Phe 5 *
A A . . . . . 1.58 -1.71 . F 0.90 3.81
Glu 6 *
A A . . . . . 1.84 -1.81 . F 0.90 3.81
Arg 7 A *
A . . . . . 1.58 -1.31 * F 0.90 2.31
Arg 8 A *
A . . . . . 1.69 -0.67 . F 0.90 1.98
Gln 9 .
A A . . . . . 1.69 -1.46 * F 0.90 2.24
Ala 10 .
A A . . . . . 2.08 -1.06 * 0.75 1.98
Val 11 .
A A . . . . 1.79 -0.57 . F 0.90 2.46
Arg 12 .
. A B . . . . 1.33 0.34 . F -0.15
Gln 13 * 0.88
. A B . . . 0.63 0.37 * F -0.15
Thr 14 * 0.87
A . . T . 0.63 0.37 * F 0.40 1.18
Trp 15 *
. A . . . C 0.88 -0.27 * F 1.07 1.04
Gly 16 *
. . . . . T C 1.84 0.16 * F 0.99 0.60
Ala 17 *
. . . . . T C 0.88 -0.24 * F 1.86 0.81
Glu 18 *
. . . . . T C 0.88 -0.09 * F 2.13 0.57
Gly 19 .
T C 0.84 -0.60 * F 2.70 1.00
Arg 20 .
. . . B T . . 0.54 -0.60 * F 2.23 0.9$
Val 21 .
A . B . . . 0.08 --0.60 * F 1.56 0.57
Gln 22 .
. . B B . . . -0.19 0.09 * F 0.39 0.48
Gly 23 *
. . B B . . . -0.08 0.30 * -0.030.18
Ala 24 * .
. . B B . . , 0.38 0.30 * -0.300.48
Leu 25 *
. . B B . . . -0.59 -0.34 * 0.30 0.54
Val 26 * .
B E3 . . -0.43 -0.10 0.30 0.40
Arg 27 *
. . B B . . . -1.24 0.26 -0.300.35
Arg 28 * . .
. . B Es . . -1.71 0.44 -0.600.35
Val 29 . * .
. . B B , . -1.47 0.44 -0.600.38
Phe 30 *
. . B B . . . -1.51 0.23 -0.300.19
Leu 31 * . .
. . B B . . . -0.87 0.87 _0.600.07
Leu 32 * .
B B . . . -0.87 1.30 -0.600.15
Gly 33 *
. . B B . -1.32 0.66 * -0.600.35
Val 34 *
. . B .. . T . -1.06 0.30 0.10 0.42
Pro 35 *
. . B . . T . -0.70 0.11 . F 0.25 0.51
Arg 36 *
. . . . T T . -0.19 -0.14 . F 1.25 0.51
Gly 37 *
. . . . T T . 0.28 -0.19 . F 1.25 0.92
Ala 38 *
. . . . . C 0.28 -0.40 . F 1.12 0.59
Gly 39 *
. . . . . T C 0.59 -0.40 * F 1.59 0.30
Ser 40 *
. . . . . T C 0.76 0.10 * F 1.26 0.30
Gly 41 *
. . . . . T ._ 0.64 -0.33 * F 2.13 0.50
Gly 42 *
. . . . T C 0.13 -0.83 . F 2.70 0.88
Ala 43 .
. . B . . . . 0.38 -0.61 . F 2.03 0.49
Asp 44 *
. . B . . . _ 0.72 -0.57 . F 1.76 0.49
Glu 45 *
. B . . . . 0.68 -1.00 . F 1.49 0.85
Val 46 *
A . . . . . . 0.43 -1.00 . F 1.22 0.83
Gly 47 A *
. . . . . . 0.89 -I.00 . F 0.95 0.50
Gl *
u 48 A . . . . . . 1.17 -7..00 . F 0.95 0.57
Gly 49 A *
. . . . . . 2.13 -0.51 * F 1.10 1.11
Ala 50 *
A . . . . . . 0.84 -0.66 * F 1.10 1
* 53
Arg 51 A . . . . . _ 1.81 -0.17 * F 0 .
* 65 0
93
Thr 52 ; A . . . . 1.57 -0.17 * F . .
* 0 1
80 83
His 53 A A . . . . . 0.76 -0.10 * . .
* . 0 1
45 83
Trp 54 A A . . . . . 0.29 0.09 * . .
* . -0 0
30 77
Arg 55 A A . . . . . 0.99 0.77 * . .
* . -0 0
60 44
Ala 56 A A . . . . . 0.29 0.29 * . .
* . -0 0
30 64
Leu 57 A A . . . . . 0.60 0.29 * . .
* . -0 0
30 61
Leu 58 A A . . . . . 0.33 -0.63 * . .
* . 0 0
60 54
Arg 59 A A . . . . . -0.19 -0.24 * . .
* 0 0
30 72
Ala 60 A A . . . . . -0.89 -0.06 * . . .
* 0 0
30 72
Glu 61 A A . . . . . -0.54 -0.24 * . .
. 0 0
30 88
Ser 62 A A . . . . . -0.32 -0.17 * . . .
. 0.30 0.70
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Table I (continued)
Res PositionI II III IV V VI VII VIII IX X
XI XII XIII
XIV
Leu 63 A A . . . . . 0 *
49 0
33
. -0.30 0.70
Ala 64 A A .
. . . . . -0.51 -0.17 * 0.30 0.68
Tyr 65 A .
A . . . . . -0.73 0.51 -0.60 0.35
Ala 66 ~ . .
A A . . . . . -1.54 0.81 -0
60 0
35
Asp 67 A A . . . . . -1.53 0.81 .
. .
-0
60 0
29
.
Iie 68 A A . . . . . -1.31 1.23 .
. .
-0
60 0
19
.
Leu 69 A A . . . . -1.42 0.97 .
* . .
-0
60 0
19
Leu 70 . A B . . . . -1.18 1.26 .
* .
-0
60 0
10
.
Trp 71 A A . . . . . -0.59 1.26 .
* .
-0
60 0
24
Ala 72 A A . . . . . -0.90 0.57 .
* . .
-0
60 0
48
'
Phe 73 A . . . . T . -0.71 0.37 .
. .
0
10 0
85
.
Asp 74 A . . . T . -0.60 0.47 .
* .
-0
20 0
70
Asp 75 A . . . . T . 0.21 0.34 * . .
.
F 0
25 0
60
Thr 76 A . . . T . -0.31 0.24 .
. . .
0
25 1
11
'
Phe 77 A A . . . , -0.03 0.14 .
.
-0
30 0
55
.
Phe 78 A A . . . . . -0.14 0.63 * .
* .
-0
60 0
47
Asn 79 A A . . . . . -0.10 1.31 * .
.
-0
60 0
27
.
Leu 80 A A . . . . . -0.10 0.83 * .
* .
-0
60 0
63
Thr 81 A A . . . . . -0.68 0.04 .
* . .
-0
15 1
25
Leu 82 A A . . . . . -0.01 -0.06 .
* .
0
30 0
55
Lys 83 A A . . . . . -0.01 0.04 * .
* .
-0
30 0
90
Glu 84 A A . . . . . -0.82 0.14 .
* .
-0
30 0
54
.
Ile 85 A A . . . . . -0.60 0.34 .
. .
-0
30 0
54
His 86 A A . . . . . -0.58 0.16 .
. .
-0
30 0
27
'
Phe 87 A A . . . . . -0.36 1.07 .
. . .
-0
60 0
17
.
Leu SS A A . . . . . -0.70 1.57 .
. .
-0
60 0
24
'
Ala 89 A A . . . . . -1.29 1.27 * .
.
-0
60 0
24
.
Trp 90 A A . , . . . -1.10 1.27 .
. . .
-0
60 0
27
Ala 91 A A . . . . . -1.73 1.27 .
.
-0
60 0
29
Ser 92 A A . . . . -1.24 1.16 . .
. .
. -0
60 0
15
Ala 93 . A . . T . . -0.43 1.09 .
.
-0
20 0
23
.
Phe 94 . A . T . . -0.70 0.17 * .
* .
0.10 0
37
Cys 95 . . B . . T . -0.30 0.31 * .
* 0.10 0
21
.
Pro 96 . . . . T T . -0.41 -0.07 * .
* 1.10 0
40
.
Asp 97 . . . . T T . -0.97 0.21 * .
* 0
50 0
40
.
Val 98 A . T . -1.08 0.07 * .
* .
0
10 0
55
Arg 99 . . B B . . . -0.33 0.29 * .
* .
-0
30 0
31
Phe 100 . . B B . . . -0.01 -0.14 * .
* .
. 0
64 0
37
Val 101 . . B B . . 0.20 0.29 * * .
.
. 0
38 0
49
Phe 102 . . B , . T . -0.39 -0.36 * .
* .
1.72 0
42
Lys 103 A . T . 0.47 0.14 * * .
F 1
61 0
49
Gly 104 . . . . T T . -0.50 -0.64 * .
* .
F 3.40 1
11
Asp 105 . . . . T T -0.50 -0.64 . * .
F 2
91 0
95
Ala 106 A B . . . -0.50 -0.64 * .
. * .
F 1
77 0
41
Asp 107 A . B . . . 0.20 0.00 * * .
.
. 0
38 0
31
Val 108 . . B B . . . -0.70 -0.03 * .
* .
0
64 0
30
.
Phe 109 . . B B . . . -0.70 0.61 * .
* .
-0
60 0
22
.
Val 110 . . B B . -0.70 0.54 * .
* .
-0
60 0
13
.
Asn 111 B . . T . -0.92 0.94 * .
* .
-0
20 0
28
.
Val 112 . . B . . T . -1.73 0.99 .
* ' .
-0
20 0
27
~
Gly 113 . . . . . T C -0.88 0.89 .
* . .
0
00 0
30
Asn 114 A . . . T . -0.88 0.24 .
* .
0
10 0
32
Leu 115 A A . . . . . -0.83 0.63 .
* ' .
-0
60 0
37
Leu 116 A A . . . . -1.42 0.67 .
* .
-0
60 0
31
Glu 117 . A B . . . . -0.78 0.74 . .
* .
. -0
60 0
19
Phe 118 . A B . . . . -0.32 0.77 .
. . .
-0
60 0
37
Leu 119 . A B . . . . -0.32 0.09 .
.
-0
30 0
87
Ala 120 . A B . . . . 0.28 -0.60 . .
. .
. 0.60 0
84
Pro 121 A A . . . . 0.50 -0.17 . .
. F 0
60 1
49
Arg 122 A . . T . . 0.50 -0.46 . .
. .
F 1.00 1
B3
Asp 123 A . . . . T . 1.20 -0.74 . .
* F 1.30 3.14
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Table I (continued)
Res PositionI II III TV V VI VII VIII IX X XI
X II XIV
XIII
Pro 124 A . . . . T . 1.20 -1.24 . F 1 3
* 30 39
Ala 125 A . . . . T . 0.98 -0.99 . F . .
* 1 1
30 43
Gln 226 A . . T . 0.60 -0.30 . F . .
* 0 0
85 70
Asp 127 . A B . . . . 0.14 0.20 . F . .
* -0 0
15 46
Leu 128 A A . . . . . 0.14 0.20 . .
-0 0
30 45
Leu 129 A A . . . . . -0.50 -0.30 . . . .
. 0 0
30 43
Ala 130 A A . . . . -0.80 -0,06 . . . .
. 0 0
30 19
Gly 131 A . . B . . . -1.66 0.63 . .
. -0 0
60 16
Asp 132 A B . . . -1.69 0.59 . .
. . -0 0
60 15
Val 133 . . B B . . . -1.47 0.40 * . .
. -0 0
60 20
Ile 134 . . B B . . . -0.54 0.40 * . .
* . -0 0
60 20
Val 135 . . B B . . . -0.17 --0.03 * . .
* . 0 0
30 24
His 136 . . B B . . . -0.71 0.40 * . .
* . -0 0
60 50
Ala 137 . . B B . . . -0.60 0.44 * . .
* -0 0
60 50
Arg 138 . . B B . . . -0.06 --0.24 * . .
. 0.45 1
31
Pro 139 . . B B . . 0.94 -0.40 * F 0 .
. 60 1
39
Ile 140 B T . . 1.21 --0.90 * F . .
. 1.30 2
69
Arg 141 . . B B . . , 0.94 -0.90 * F 1 .
* 18 1
39
Thr 142 . . B B . . . 1.58 -0.51 * F . .
* 1.46 1
20
Arg 143 . . B B . . 1.22 -0.94 * F 1 .
* 74 3
44
Ala 144 . . B I3 T . 1.19 -0.87 * F . .
. 2.42 2
75
Ser 145 . T T . 1.19 -0.11 * F 2.80 .
. 2
98
Lys 146 . . . T T . 0.87 0.09 * 1 .
77 1
07
Tyr 147 , . B . . T . 1.18 0.51 . . . .
* 0.79 1
63
Tyr 148 . . B . . T . 0.48 0.01 0 .
* . 81 2
11
Ile 149 . . B . . . . 0.22 0.13 . .
. . 0.33 2
07
Pro 150 . B . . . . 0.27 0.77 -0 .
. . 40 0
51
Glu 151 . . B . . . . -0.12 0.77 . .
* . . -0 0
40 51
Ala 152 . B . . . . -0.69 0.44 . .
. * . 1 -0 0
40 71
Val 153 . B . . . . -0.66 0.44 . .
* -0.400
38
Tyr 154 . . B . . . . -0.36 0.44 . . -0 .
* 40 0
34
Gly 155 . B . . . . -0.39 0.94 . .
. 1 . -0.400
34
Leu 156 . . B . . . . -0.60 1.20 -0.40.
. . 0
72
Pro 157 . . B .. . . . -0.60 0.99 -0 .
. . 40 0
71
Ala 158 . B . . . . 0.01 0.73 . .
. . . , -0.400
72
Tyr 159 . B . . T . -0.33 1.06 -0 .
05 1
37
Pro 160 . . B . . T . -0.33 0.87 . . . .
. -0.200
90
Ala 161 B . . T . 0.13 0.87 -0 .
20 0
88
Tyr 162 . , B . . T . -0.00 0.80 . .
. -0 0
20 56
Ala 163 . . B . . . . 0.24 0.47 . .
-0 0
40 36
Gly 164 . . . . T T . -0.21 0.47 . F . .
. 0.35 0
35
Gly 165 . T T . -0.86 0.76 . F 0.35 .
. 0
19
Gly 166 . B . . T . -1.08 0.64 . F -0 .
. 05 0
14
Gly 167 . B , . T . -1.13 0.83 . F . .
. -0 0
05 12
Phe 168 . B . . . . -0.89 0.79 . . . .
. . -0.400
16
Val 169 . B . . . . -1.13 0.79 . . -0 .
. . 40 0
16
Leu 170 . B . . T - -1.10 0.86 . .
. . -0.200
16
Ser 171 . B . . T -1.57 0.91 . -0 .
. 20 0
27
Gly 172 . . B . . T .. -1.26 0.81 . F . .
* -0 0
05 30
Ala 173 A . . . T . -0.44 0.67 . F . .
* -0.050
50
Thr 174 A A . . . . . -0.40 -0.01 0.30 .
. . 0
73
Leu 175 A A . . . . . -0.18 0.29 -0 .
. 30 0
61
His 276 A A . . . - -0.22 0.36 . .
* . . -0 0
30 61
Arg 177 . A B . . . . -0.47 0.29 . .
* . . -0.300
42
Leu 178 A A . . . . . -0.54 0.30 -0.30.
* ' 0
51
Ala 179 A A . . . - . -0.82 0.19 -0 .
* 30 0
20
Gly 180 A A . . . - . -0.01 0.19 . . . .
* -0 0
30 10
Ala 181 A A . . . . . -0.83 0.59 * . .
* -0 0
60 22
Cys 182 A A . . . . . -0.94 0.54 * . .
-0 0
60 16
Ala 183 A A . . . . . -0.94 0.04 * . . .
. -0.300.28
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Table I (continued)
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Gln 184 A A . . . . -1.06 0.30 * -0.300
. . 23
Val 185 . A B . . . . -0.92 0.59 . -0.60.
. . 0
37
Glu 186 . A B . . . . -1.22 0.44 * -0.60.
. 0
56
Leu 187 . A B . . . . -0.56 0.63 -0.60.
. 0
23
Phe 188 . A B . . . . 0.03 0.23 . -0.30.
. 0
51
Pro 189 . A B . . . -0.82 -0.41 0.30 .
. . 0
49
Ile 190 A A . B , . . -0.67 0.23 . -0.30.
. 0
45
Asp 191 A A B . . . -1.48 0.33 . F -0.15.
. 0
45
Asp 192 A A . B . . . -1.01 0.23 . F' -0 .
. 15 0
24
Val 193 A A B . . . -0.91 0.23 . .
, . -0 0
30 34
Phe 194 A A B . . . -1.37 0.16 . . .
. . -0.300
20
Leu 195 A A B . . . -1.29 0.73 . . -0.60.
. . 0
06
Gly 196 A A B . . . -1.29 1.41 . -0.60.
. . . 0
07
Met 197 A A B . . . -1.18 1.17 -0.60.
. . 0
14
Cys 198 A A . B . . . -1.13 0.39 * -0.30.
* 0
34
Leu 199 A A B . . . -0.32 0.39 * -0 .
* 30 0
28
Gln 200 . A B B . . . -0.32 -0.04 * . . .
. 0.30 0
56
Arg 201 . A B B . . . -0.29 0.03 * -0.30.
. 0
85
Leu 202 . A B B . . . 0.10 -0.06 * 0.69 .
. 1
49
Arg 203 . A B B . . . 0.77 -0.31 * . 0.93 .
* 1
33
Leu 204 . . . B . . C 1.37 -0.71 * 1.67 .
. 1
18
Thr 205 . . . . . T C 1.33 -0.29 * F 2.16 .
. 2
21
Pro 206 . . . . . T C 1-01 --0.47 * F 2.40 .
. 1
54
Glu 207 . . . . . T C 1.23 --0.04 * F 2.16 .
* 2
88
Pro 208 . . . . . T C 0.42 --0.23 * F 1.92 .
* 2
02
His 209 . . . . . . C 1.34 0.07 * F 0.88 .
* 1
13
Pro 210 . . . C 1.34 --0.36 * 1.09 .
* 1
28
Ala 211 . . B B T . . 0.86 0.13 * 0 .
* . 25 1
19
Phe 212 . . B B . . . 0.51 0.49 . . .
* -0.600
76
Arg 213 . . .
B B . -0.17 0.41 . -0.600
* 49
Thr 214 . . B B . . . -0.34 U,67 . -0 .
* 60 0
34
Phe 215 . . B B . . . -0.13 0.60 . . . .
. -0.600
60
Gly 216 . . . B T . . 0.24 0.21 . 0.10 .
. 0
53
Ile 217 . . . B , . C 0.64 0.64 . F -0.25.
. 57
0
Pro 218 . . . E3 . . C -0.06 0.54 . F -0.25.
. 0
88
Gln 219 . . . . . T C -0.33 0.26 . F 0.45 .
. 0
90
Pro 220 . . . . . T C 0.16 0.33 . F 0.60 .
. 1
30
Ser 221 . . . . T C 0.47 0.07 . F 0.60 .
. 1
30
Ala 222 . B . . T . 0.54 0.14 . . 0.25 .
. 1
02
Ala 223 . . B . . . . 0.46 0.43 . . -0.40.
. 0
54
Pro 224 B . . . . 0.14 0.39 . . -0 .
. 10 0
54
His 225 . . B . . . . -0.34 0.49 . . . .
. --0.400.78
Leu 226 B . , . . -0.04 0.77 . . -0.400
. 67
Ser 227 . . B . . . . 0.33 0.27 . F 0.05 .
. 0
72
Thr 228 . T . . 0.26 0.27 . F 0.45 .
. 0
82
Phe 229 . B . . . . -0.23 0.34 . F 0.05 .
. 0
53
Asp 230 . . B . . T . -0.44 0.44 . F -0.05.
* 0
34
Pro 231 . . . . T T . 0.48 0.81 0.20 .
* 0
37
Cys 232 . . T T . 0.78 0.33 . . 0.50 .
* 0
84
Phe 233 . . B . . T . 0.28 -0.46 . . 0.70 .
* 0
87
Tyr 234 A . . . . . . 0.12 0.23 -0.10.
. 0
47
Arg 239 A . . B . . . -0.73 0.44 -0.60.
* 0
65
Glu 236 A . . B . . . -1.38 0.51 . . -0.60.
* 0
55
Leu 237 A . B . . . -0-74 0.37 -0.30.
* 0
26
Val 238 . . B B . . . -0.39 0.11 . . -0.30.
* 0
18
Val 239 . B B . . . -0.96 0.54 . . -0.60.
. 0
10
Val 240 . B B . . . -1.37 1,23 . -0.60.
. 0
10
His 241 A B . . . -1,96 0.93 . . -0.60.
. . 0
19
Gly 242 A . B . . . -1.73 0.79 . . -0.60.
. 0
26
Leu 243 A A . . . . . -0.88 0.64 . . -0.60.
. 0.35
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Table I (continued)
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Ser 244 A A . . . . . -0.91 0.00 -0 0
. . 30 43
'
Ala 245 A A . . . -0.34 0.19 . .
. . -0 0
30 30
Ala 246 A A B . . . -1.12 0.67 . .
' . -0 0
60 39
Asp 247 A A B . . . -1.38 0.67 . .
-0 0
60 24
Ile 248 A A B . . . -0.86 0.90 * . .
. . -0.60 0
23
Trp 249 A A B . . . -0.44 1.31 * -0 .
. . 60 0
24
Leu 250 A A B . . . -0.67 0.81 * . .
* -0 0
60 28
Met 251 A A . B . . . -0.89 1.50 * . .
* . -0 0
60 33
Trp 252 A A . B . . . -0.92 1.50 * . .
* . -0 0
60 26
Arg 253 . A B B . . -0.38 1.09 * . .
* . -0 0
60 43
Leu 254 . A B T . . -0.30 0.83 * . .
. -0 0
20 43
Leu 255 . A . B T . . 0.48 0.64 * . .
* -0 0
20 64
His 256 . A . B T . . 0 0 * . .
73 23
. . 0.10 0.44
Gly 257
. . . . T C 0.81 0.66 * F 0.15 0.53
Pro 258 *
. . . . T T . 0.11 0.40 * F 0.35 0.99
His 259 .
. . . . T T . 0.26 0.21 . F 0.65 0.74
Gly 260 .
. . . . T C 0.48 0.29 . F 0 0
. 45 40
Pro 261 . . . . T . . 0.4B 0.36 . F . .
0 0
45 26
Ala 262 . T . . 0.61 0.43 . .
. . 0 0
. 00 26
Cys 263 . . B . . . . 0.82 U.36 . .
. -0 0
. 10 41
Ala 264 . . B . . . 0 0 . .
64 33
. . -0.10 0.96
His 265 B . .
. . . . T . 0.13 0.33 0.10 0.70
Pro 266 .
. . B . . T . -0.24 0.47 F -0.05 0.97
Gln 267
. . B T . -0.24 0.40 F -0.05 0.97
Pro 268 .
. . B . . T . 0.08 U.40 F -0.05 0.72
Val 269 .
'
. . B . . . . 0.46 0.33 -0.10 0.46
Ala 270 . .
. . B . . . . -0.21 0.33 -0.10 0.41
Ala 271 . . .
B . . . 0.00 0.71 -0.40 0.23
Gly 272 . .
. . B .. . T . -0.29 0.69 * -0.20 0.54
Pro 273 .
, . B . . T . -0.08 0.96 * -0.20 0.56
Ph
e 274 . . . . T T . 0.48 0.46 * 0 0
20 92
Gln 275 . T T . 0.68 0.34 * . .
. ' 0 1
65 25
Trp 276 . . B , . , . 0.88 0.34 * . .
. 0 1
05 03
Asp 277 . , . . . . C 0.83 0.34 * . .
0 1
25 53
Ser 278 . . . . . . C 0.66 -0.01* . . .
. 0.85 1.13
a
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Among highly preferred fragments in this regard are those that comprise, or
alternatively consist of, regions of Brainiac-5 that combine several
structural features,
such as several of the features set out above.
In another embodiment, the invention provides an isolated nucleic acid
molecule comprising a polynucleotide which hybridizes under stringent
hybridization
conditions to a portion of the polynucleotide in a nucleic acid molecule of
the
invention described above, for instance, the sequence complementary to the
coding
sequence and/or noncoding sequence depicted in SEQ ID NO:1, the Brainiac-5
eDNA
clone contained in ATCC Deposit No. 203572, or fragments (such as, for
example, the
open reading frame or a fragment thereof) of these sequences, as described
herein. By
"stringent hybridization conditions" is intended overnight incubation at
42°C in a
solution comprising: 50% formamide, Sx SSC (750 mM NaCI, 75 mM trisodium
citrate), 50 mM sodium phosphate (pH 7.6), Sx Denhardt's solution, 10% dextran
sulfate, and 20 lrg/ml denatured, sheared salmon sperm DNA, followed by
washing the
filters in O.lx SSC at about 65°C.
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is
intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15
nucleotides (nt), and more preferably at least about 20 nt, still more
preferably at least
about 30 nt, and even more preferably about 30-70 (e.g., 50) nt of the
reference
polynucleotide. These are useful as diagnostic probes and primers as discussed
above
and in more detail below.
By a portion of a polynucleotide of "at least 20 nt in length," for example,
is
intended 20 or more contiguous nucleotides from the nucleotide sequence of the
reference polynucleotide (e.g., the deposited cDNA or the nucleotide sequence
as
shown in Figures lA and 1B (SEQ ID NO:1). Of course, a polynucleotide which
hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of
the
Brainiac-5 cDNA shown in Figures 1 A and 1 B (SEQ ID NO: l ), or to a
complementary stretch of T (or U) residues, would not be included in a
polynucleotide
of the invention used to hybridize to a portion of a nucleic acid of the
invention, since
such a polynucleotide would hybridize to any nucleic acid molecule containing
a poly
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(A) stretch or the complement thereof (e.g., practically any double-stranded
cDNA
clone).
As indicated, nucleic acid molecules of the present invention which encode a
Brainiac-5 polypeptide may include, but are not limited to those encoding the
amino
acid sequence of the mature polypeptide, by itself; and the coding sequence
for the
mature polypeptide and additional sequences, such as those encoding a leader
or
secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the
coding
sequence of the mature polypeptide, with or without the aforementioned
additional
coding sequences.
Also encoded by nucleic acids of the invention are the above polypeptide
sequences together with additional, non-coding sequences, including for
example, but
not limited to introns and non-coding 5' and 3' sequences, such as the
transcribed,
non-translated sequences that play a role in transcription, mRNA processing,
including
splicing and polyadenylation signals, for example - ribosome binding and
stability of
mRNA; an additional coding sequence which codes for additional amino acids,
such as
those which provide additional functionalities.
Thus, the sequence encoding the polypeptide may be fused to a marker
sequence, such as a sequence encoding a peptide which facilitates purification
of the
fused polypeptide or which may function in secretion of the fused polypeptide
from a
cell. In certain preferred embodiments of this aspect of the invention, the
marker
amino acid sequence is a hexa-histidine peptide, such as the tag provided in a
pQE
vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311 ), among others,
many of which are commercially available. As described by Gentz and colleagues
(Proc. Natl. Acad. Sci. USA 86:821-824 ( 1989)), for instance, hexa-histidine
provides
for convenient purification of the fusion protein. The "HA" tag is another
peptide
useful for purification which corresponds to an epitope derived from the
influenza
r
hemagglutinin protein, which has been described by Wilson and coworkers (Cell
37:767 ( 1984)). As discussed below, other such fusion proteins include the
Brainiac-5
polypeptides fused to Fc at the N- or C-terminus.
The present invention further relates to variants of the nucleic acid
molecules
of the present invention, which encode portions, analogs or derivatives of the
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Brainiac-S polypeptides. Variants may occur naturally, such as a natural
allelic
variant. By an "allelic variant" is intended one of several alternate forms of
a gene
occupying a given locus on a chromosome of an organism (Genes II, Lewin, B.,
ed.,
John Wiley & Sons, New York (1985)). Non-naturally occurring variants may be
S produced using art-known mutagenesis techniques, which include, but are not
limited
to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis,
site
directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res. 13:4331 (
1986); and
Zoller et al., Nucl. Acids Res. 10:6487 (1982)), cassette mutagenesis (see
e.g., Wells et
al., Gene 34:315 (1985)), restriction selection mutagenesis (see e.g., Wells
er al.,
Philos. Trans. R. Soc. London SerA 317:41 S ( 1986)).
Such variants include those produced by nucleotide substitutions, deletions or
additions. The substitutions, deletions or additions may involve one or more
nucleotides. The variants may be altered in coding regions, non-coding
regions, or
both. Alterations in the coding regions may produce conservative or non-
conservative
1 S amino acid substitutions, deletions or additions. Especially preferred
among these are
silent substitutions, additions and deletions, which do not alter the
properties and
activities of the Brainiac-S polypeptides or portions thereof. Also especially
preferred
in this regard are conservative substitutions.
Additional embodiments of the invention are directed to isolated nucleic acid
molecules comprising, or alternatively consisting of, a polynucleotide which
encodes
the amino acid sequence of a Brainiac-S polypeptide (e.g., a Brainiac-S
fragment
described herein) having an amino acid sequence which contains at least one
conservative amino acid substitution, but not more than SO conservative amino
acid
substitutions, even more preferably, not more than 40 conservative amino acid
2S substitutions, still more preferably, not more than 30 conservative amino
acid
substitutions, and still even more preferably, not more than 20 conservative
amino acid
substitutions, 10-20 conservative amino acid substitutions, S-10 conservative
amino
acid substitutions, 1-S conservative amino acid substitutions, 3-S
conservative amino
acid substitutions, or I-3 conservative amino acid substitutions. Of course,
in order of
ever-increasing preference, it is highly preferable for a polynucleotide which
encodes
the amino acid sequence of a Brainiac-5 polypeptide to have an amino acid
sequence
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which contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative
amino acid
substitutions.
Most highly preferred are nucleic acid molecules encoding a mature
polypeptide having the amino acid sequence shown in SEQ ID N0:2 or the mature
Brainiac-5 amino acid sequence encoded by the deposited cDNA clone.
Thus, one embodiment of the invention provides an isolated nucleic acid
molecule comprising, or alternatively consisting of, a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) a nucleotide
sequence
encoding the Brainiac-5 polypeptide having the complete amino acid sequence in
SEQ
ID N0:2 (i.e., positions 1 to 278 of SEQ ID N0:2); (b) a nucleotide sequence
encoding the Brainiac-5 polypeptide having the complete amino acid sequence
encoded by the cDNA clone contained in ATCC Deposit No. 203572; (c) a
nucleotide
sequence encoding the mature Brainiac-S polypeptide having the amino acid
sequence
encoded by the cDNA clone contained in ATCC Deposit No. 203572; and (d) a
nucleotide sequence complementary to any of the nucleotide sequences in (a),
(b) or
(c), above. Polypeptides encoded by these nucleic acid molecules are also
encompassed by the invention.
Further embodiments of the invention include isolated nucleic acid molecules
that comprise, or alternatively consist of, a polynucleotide having a
nucleotide
sequence at least 90% identical, and more preferably at least 92%, 95%, 96%,
97%,
98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c) or
(d), above,
or a polynucleotide which hybridizes under stringent hybridization conditions
to a
polynucleotide in (a), (b), (c) or (d), above. This polynucleotide which
hybridizes
does not hybridize under stringent hybridization conditions to a
polynucleotide having
a nucleotide sequence consisting of only A residues or of only T residues.
An additional nucleic acid embodiment of the invention relates to an isolated
nucleic acid molecule comprising a polynucleotide which encodes the amino acid
sequence of an epitope-bearing portion of a Brainiac-5 polypeptide having an
amino
acid sequence in (a), (b) or (c), above. A further nucleic acid embodiment of
the
invention relates to an isolated nucleic acid molecule comprising a
polynucleotide
which encodes the amino acid sequence of a Brainiac-5 polypeptide having an
amino
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acid sequence which contains at least one conservative amino acid
substitution, but not
more than SO conservative amino acid substitutions, even more preferably, not
more
than 40 conservative amino acid substitutions, still more preferably, not more
than 30
conservative amino acid substitutions, and still even more preferably, not
more than 20
conservative amino acid substitutions. Of course, in order of ever-increasing
preference, it is highly preferable for a polynucleotide which encodes the
amino acid
sequence of a Brainiac-5 polypeptide to have an amino acid sequence which
contains
not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid
substitutions.
Polypeptides encoded by these nucleic acid molecules are also encompassed by
the
invention.
The present invention also relates to recombinant vectors, which include the
isolated nucleic acid molecules of the present invention, and to host cells
containing
the recombinant vectors, as well as to methods of making such vectors and host
cells
and for using them for production of Brainiac-5 polypeptides or peptides by
recombinant techniques.
In one embodiment of the invention, by a polynucleotide having a nucleotide
sequence at least, for example, 95% "identical" to a reference nucleotide
sequence
encoding a Brainiac-5 polypeptide is intended that the nucleotide sequence of
the
polynucleotide is identical to the reference sequence except that the
polynucleotide
sequence may include up to five point mutations per each 100 nucleotides of
the
reference nucleotide sequences encoding the Brainiac-5 polypeptides. In other
words,
to obtain a polynucleotide having a nucleotide sequence at least 95% identical
to a
reference nucleotide sequence, up to 5% of the nucleotides in the reference
sequence
may be deleted or substituted with another nucleotide, or a number of
nucleotides up
to S% of the total nucleotides in the reference sequence may be inserted into
the
reference sequence. These mutations of the reference sequence may occur at the
5' or
a
3' terminal positions of the reference nucleotide sequence or anywhere between
those
terminal positions, interspersed either individually among nucleotides in the
reference
sequence or m one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular nucleic acid molecule, or a
portion or fragment thereof, is at least 90%, 92%, 95%, 9b%, 97%, 98% or 99%
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identical to, for instance, the nucleotide sequences shown in Figures lA and
1B or to
the nucleotides sequence of the deposited cDNA clone can be determined
conventionally using known computer programs such as the Bestfit program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, 575 Science Drive, Madison, WI 53711).
Bestfit
uses the local homology algorithm of Smith and Waterman to find the best
segment of
homology between two sequences (Advances in Applied Mathematics 2:482-489
( 1981 )). When using Besttit or any other sequence alignment program to
determine
whether a particular sequence is, for instance, 95% identical to a reference
sequence
according to the present invention, the parameters are set, of course, such
that the
percentage of identity is calculated over the full length of the reference
nucleotide
sequence and that gaps in homology of up to 5% of the total number of
nucleotides in
the reference sequence are allowed. A preferred method for determining the
best
overall match between a query sequence (a sequence of the present invention)
and a
subject sequence, also referred to as a global sequence alignment, can be
determined
using the FASTDB computer program based on the algorithm of Brutlag and
colleagues (Comp. App. Biosci. 6:237-245 ( 1990)). In a sequence alignment the
query
and subject sequences are both DNA sequences. An RNA sequence can be compared
by converting U's to T's. The result of said global sequence alignment is in
percent
identity. Preferred parameters used in a FASTDB alignment of DNA sequences to
calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1,
Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap
Penalty=S,
Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide
sequence, whichever is shorter.
If the subject sequence is shorter than the query sequence because of 5' or 3'
deletions, not because of internal deletions, a manual correction must be made
to the
results. This is because the FASTDB program does not account for 5' and 3'
truncations of the subject sequence when calculating percent identity. For
subject
sequences truncated at the S' or 3' ends, relative to the query sequence, the
percent
identity is corrected by calculating the number of bases of the query sequence
that are
5' and 3' of the subject sequence, which are not matched/aligned, as a percent
of the
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total bases of the query sequence. Whether a nucleotide is matched/aligned is
determined by results of the FASTDB sequence alignment. This percentage is
then
subtracted from the percent identity, calculated by the above FASTDB program
using
the specified parameters, to arrive at a final percent identity score. This
corrected
score is what is used for the purposes of the present invention. Only bases
outside the
5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment,
which are not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the S' end of
the
subject sequence and therefore, the FASTDB alignment does not show a
matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases
represent
10% of the sequence (number of bases at the 5' and 3' ends not matched/total
number
of bases in the query sequence) so 10% is subtracted from the percent identity
score
calculated by the FASTDB program. If the remaining 90 bases were perfectly
matched the final percent identity would be 90%. In another example, a 90 base
subject sequence is compared with a 100 base query sequence. This time the
deletions
are internal deletions so that there are no bases on the 5' or 3' of the
subject sequence
which are not matched/aligned with the query. In this case the percent
identity
calculated by FASTDB is not manually corrected. Once again, only bases 5' and
3' of
the subject sequence which are not matched/aligned with the query sequence are
manually corrected for. No other manual corrections are to made for the
purposes of
the present invention.
The present application is directed to nucleic acid molecules, comprising or
alternatively consisting of, a polynucleotide sequence at least 90%, 92%, 95%,
96%,
97%, 98% or 99% identical to the nucleic acid sequence shown in Figures lA and
1B
t
(SEQ ID NO: l ) or to the nucleic acid sequence of the deposited cDNA,
irrespective of
whether they encode a polypeptide having Brainiac-5 activity. This is because
even
where a particular nucleic acid molecule does not encode a polypeptide having
Brainiac-5 activity, one of skill in the art would still know how to use the
nucleic acid
molecule, for instance, as a hybridization probe or a polymerase chain
reaction (PCR)
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primer. Uses of the nucleic acid molecules of the present invention that do
not encode
a polypeptide having Brainiac-S activity include, inter alia, ( 1 ) isolating
the Brainiac-5
gene or allelic variants thereof in a cDNA library; (2) in situ hybridization
(e.g.,
"FISH") to metaphase chromosomal spreads to provide precise chromosomal
location
of the Brainiac-5 gene, as described by Verma and colleagues (Xuman
Chromosomes:
A Manual of Basic Techniques, Pergamon Press, New York ( 1988)); and Northern
Blot analysis for detecting Brainiac-5 mRNA expression in specific tissues.
Preferred, however, are nucleic acid molecules having (i.e., comprising, or
alternatively consisting of, sequences at least 90%, 92%, 95%, 96%, 97%, 98%
or
99% identical to the nucleic acid sequence shown in Figures lA and 1B (SEQ ID
NO:1 ) or to the nucleic acid sequence of the deposited cDNA which do, in
fact,
encode a polypeptide having Brainiac-5 polypeptide functional activity. By "a
polypeptide having Brainiac-5 polypeptide functional activity" is intended
polypeptides exhibiting activity similar, but not necessarily identical, to an
activity of
the mature Brainiac-5 polypeptide of the invention, as measured in a
particular
biological assay.
For example, the Brainiac-5 polypeptides of the present invention modulate
cellular growth and differentiation. Thus, biological activity of Brainiac-S
polypeptides can be examined in organ culture assays or in colony assay
systems in
agarose culture. Stimulation or inhibition of cellular proliferation may be
measured by
a variety of assays. For observing cell growth inhibition, one can use a solid
or liquid
medium. In a solid medium, cells undergoing growth inhibition can easily be
selected
from the subject cell group by comparing the sizes of colonies formed. In a
liquid
medium, growth inhibition can be screened by measuring culture broth turbidity
or
incorporation of labeled thymidine in DNA. Typically, the incorporation of a
nucleoside analog into newly synthesized DNA is employed to measure
proliferation
(i.e., active cell growth) in a population of cells. For example,
bromodeoxyuridine
(BrdU) can be employed as a DNA labeling reagent and anti-BrdU mouse
monoclonal
antibody (clone BMC 9318 IgG,) can be employed as a detection reagent. This
antibody binds only to cells containing DNA which has incorporated
bromodeoxyuridine. A number of detection methods may be used in conjunction
with
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this assay including immunofluorescence, immunohistochemical, ELISA, and
colorimetric methods. Kits that include bromodeoxyuridine (BrdU) and anti-BrdU
mouse monoclonal antibody are commercially available from Boehringer Mannheim
(Indianapolis, IN).
The effect upon cellular differentiation can be measured by contacting
embryonic cells with various amounts of a Brainiac-5 polypeptide and observing
the
effect upon differentiation of the embryonic cells. Tissue-specific antibodies
and
microscopy may be used to identify the resulting cells.
Brainiac-5 polypeptides modulate immune and/or nervous system cell
proliferation and differentiation in a dose-dependent manner in the above-
described
assays. Thus, "a polypeptide having Brainiac-5 polypeptide activity" includes
polypeptides that also exhibit any of the same growth and differentiation
regulating
activities in the above-described assays in a dose-dependent manner. Although
the
degree of dose-dependent activity need not be identical to that of the
Brainiac-5
polypeptide, preferably, "a polypeptide having Brainiac-5 polypeptide
activity" will
exhibit substantially similar dose-dependence in a given activity as compared
to the
Brainiac-5 polypeptide (i.e., the candidate polypeptide will exhibit greater
activity or
not more than about 25-fold less and, preferably, not more than about tenfold
less
activity relative to the reference Brainiac-5 polypeptide).
Of course, due to the degeneracy of the genetic code, one of ordinary skill in
the art will immediately recognize that a large number of the nucleic acid
molecules
having a sequence at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to
the
nucleic acid sequence of the deposited cDNA or the nucleic acid sequence shown
in
Figures 1 A and 1 B (SEQ ID NO:1 ) will encode a polypeptide "having Brainiac-
5
polypeptide functional activity." In fact, since degenerate variants of these
nucleotide
sequences all encode the same polypeptide, this will be clear to the skilled
artisan even
without performing the above described comparison assay. It will be further
recognized in the art that, for such nucleic acid molecules that are not
degenerate
variants, a reasonable number will also encode a polypeptide having Brainiac-5
polypeptide activity. This is because the skilled artisan is fully aware of
amino acid
substitutions that are either less likely or not likely to significantly
effect polypeptide
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function (e.g., replacing one aliphatic amino acid with a second aliphatic
amino acid),
as further described below.
Vectors and Host Cells
The present invention also relates to vectors which include the isolated DNA
molecules of the present invention, host cells which are genetically
engineered with
the recombinant vectors, and the production of Brainiac-5 polypeptides or
fragments
thereof by recombinant techniques. The vector may be, for example, a phage,
plasmid, viral or retroviral vector. Retroviral vectors may be replication
competent or
replication defective. In the latter case, viral propagation generally will
occur only in
complementing host cells.
The polynucleotides may be joined to a vector containing a selectable marker
for propagation in a host. Generally, a plasmid vector is introduced in a
precipitate,
such as a calcium phosphate precipitate, or in a complex with a charged lipid.
If the
vector is a virus, it may be packaged in vitro using an appropriate packaging
cell line
and then transduced into host cells.
The DNA insert should be operatively linked to an appropriate promoter, such
as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters,
the
SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
Other suitable promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the
transcribed region, a ribosome binding site for translation. The coding
portion of the
transcripts expressed by the constructs will preferably include a translation
initiating
codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately
positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one
selectable marker. Such markers include dihydrofolate reductase, 6418 or
neomycin
resistance for eukaryotic cell culture and tetracycline, kanamycin or
ampicillin
resistance genes for culturing in E. coli and other bacteria. Representative
examples of
appropriate hosts include, but are not limited to, bacterial cells, such as E.
coli,
Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast
cells (e.g.,
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Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178));
insect
cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as
CHO, COS,
293 and Bowes melanoma cells; and plant cells. Appropriate culture mediums and
conditions for the above-described host cells are known in the art.
Vectors preferred for use in bacteria include pHE4-5 (ATCC Accession No.
209311; and variations thereofj, pQE70, pQE60 and pQE-9 (QIAGEN, Inc., supra);
pBS vectors, Phagescript vectors, Bluescript vectors, pNHBA, pNHl6a, pNHl8A,
pNH46A (Stratagene); and ptrc99a, pKK223-3, pKK233-3, pDRS40, pRTTS
(Pharmacia). Preferred expression vectors for use in yeast systems include,
but are not
limited to, pYES2, pYDI, pTEFI/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha,
pPIC9, pPIC3.5, pHIL-D2, pHIL-S l, pPIC3.5K, pPIC9K, and PA0815 (all available
from Invitrogen, Carlsbad, CA). Among preferred eukaryotic vectors are pWLNEO,
pSV2CAT, pOG44, pXTI, and pSG (Stratagene); and pSVK3, pBPV, pMSG and
pSVL (Pharmacia). Other suitable vectors will be readily apparent to the
skilled
artisan.
Following transformation of a suitable host strain and growth of the host
strain
to an appropriate cell density, the selected promoter is induced by
appropriate means
(e.g., temperature shift or chemical induction) and cells are cultured for an
additional
period. Cells are typically harvested by centrifugation, disrupted by physical
or
chemical means, and the resulting crude extract retained for further
purification.
Microbial cells employed in expression of proteins can be disrupted by any
convenient method, including freeze-thaw cycling, sonication, mechanical
disruption,
or use of cell lysing agents, such methods are well know to those skilled in
the art.
In one embodiment, the yeast Pichia pastoris is used to express Brainiac-5
Protein in a eukaryotic system. Pichia pastoris is a methylotrophic yeast
which can
metabolize methanol as its sole carbon source. .A main step in the methanol
metabolization pathway is the oxidation of methanol to formaldehyde using Oz.
This
reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize
methanol
as its sole carbon source, Pichia pastoris must generate high levels of
alcohol oxidase
due, in part, to the relatively low affinity of alcohol oxidase for O2.
Consequently, in a
growth medium depending on methanol as a main carbon source, the promoter
region
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of one of the two alcohol oxidase genes (AOXI ) is highly active. In the
presence of
methanol, alcohol oxidase produced from the AOXI gene comprises up to
approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis,
S.B., et
al., Mol. Cell. Biol. 5:1111-21 (1985}; Koutz, P.J, et al., Yeast 5:167-77
(1989);
Tschopp, J.F., et al., Nucl. Acids Res. 15:3859-76 ( 1987). Thus, a
heterologous coding
sequence, such as, for example, a Brainiac-5 polynucleotide of the present
invention,
under the transcriptional regulation of all or part of the AOXI regulatory
sequence is
expressed at exceptionally high levels in Pichia yeast grown in the presence
of
methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding
a Brainiac-5 polypeptide of the invention, as set forth herein, in a Pichea
yeast system
essentially as described in "Pichia Protocols: Methods in Molecular Biology,"
D.R.
Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression
vector allows expression and secretion of a Brainiac-5 protein of the
invention by
virtue of the strong AOXI promoter linked to the Pichia pastoris alkaline
phosphatase
(PHO) secretory signal peptide (i.e., leader) located upstream of a multiple
cloning
site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2,
pYDI, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5,
PHIL-D2, pHIL-S l, pPIC3.5K, and PA0815, as one skilled in the art would
readily
appreciate, as long as the proposed expression construct provides
appropriately located
signals for transcription, translation, secretion (if desired), and the like,
including an
in-frame AUG as required.
In another embodiment, high-level expression of a heterologous coding
sequence, such as, for example, a Brainiac-5 polynucleotide of the present
invention,
d
may be achieved by cloning the heterologous polynucleotide of the invention
into an
expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the
yeast culture in the absence of methanol.
Transcription of the DNA encoding the polypeptides of the present invention
by higher eukaryotes is increased by inserting an enhancer sequence into the
vector.
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Enhancers are cis-acting elements of DNA, usually about from 10 to 300 by that
act on
a promoter to increase its transcription. Examples including the SV40 enhancer
on the
late side of the replication origin by 100 to 270, a cytomegalovirus early
promoter
enhancer, the polyoma enhancer on the late side of the replication origin, and
adenovirus enhancers.
Various mammalian cell culture systems can also be employed to express
recombinant protein. Examples of mammalian expression systems include the COS-
7
lines of monkey kidney fibroblasts, described by Gluzman {Cell 23:175 ( 1981
)), and
other cell lines capable of expressing a compatible vector, for example, the
C127, 3T3,
CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an
origin of replication, a suitable promoter and enhancer, and also any
necessary
ribosome binding sites, polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking nontranscribed
sequences. DNA
sequences derived from the S V40 splice, and polyadenylation sites may be used
to
provide the required nontranscribed genetic elements.
In addition to encompassing host cells containing the vector constructs
discussed herein, the invention also encompasses primary, secondary, and
immortalized host cells of vertebrate origin, particularly mammalian origin,
that have
been engineered to delete or replace endogenous genetic material (e.g.,
Brainiac-5
coding sequence), and/or to include genetic material (e.g., heterologous
polynucleotide
sequences) that is operably associated with Brainiac-5 polynucleotides of the
invention, and which activates, alters, and/or amplifies endogenous Brainiac-5
polynucleotides. For example, techniques known in the art may be used to
operably
associate heterologous control regions (e.g., promoter and/or enhancer) and
endogenous Brainiac-5 polynucleotide sequences via homologous recombination
(see,
e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; International
Publication No.
E
WO 96/2941 l, published September 26, 1996; International Publication No. WO
94/12650, published August 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA
86:8932-8935 (/989); and Zijlstra et al., Nature 342:435-438 (1989), the
disclosures of
each of which are incorporated by reference in their entireties).
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The host cells described infra can be used in a conventional manner to produce
the gene product encoded by the recombinant sequence. Alternatively, cell-free
translation systems can also be employed to produce the polypeptides of the
invention
using RNAs derived from the DNA constructs of the present invention.
Introduction of the construct into the host cell can be effected by calcium
phosphate transfection, DEAF-dextran mediated transfection, cationic lipid-
mediated
transfection, electroporation, transduction, infection or other methods. Such
methods
are described in many standard laboratory manuals (for example, Davis, et al.,
Basic
Methods In Molecular Biology { 1986)).
The polypeptide may be expressed in a modified form, such as a fusion protein,
and may include not only secretion signals, but also additional heterologous
functional
regions. For instance, a region of additional amino acids, particularly
charged amino
acids, may be added to the N-terminus of the polypeptide to improve stability
and
persistence in the host cell, during purification, or during subsequent
handling and
1 S storage. Also, peptide moieties may be added to the polypeptide to
facilitate
purification. Such regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to engender
secretion or
excretion, to improve stability and to facilitate purification, among others,
are familiar
and routine techniques in the art. A preferred fusion protein comprises a
heterologous
region from immunoglobulin that is useful to stabilize and purify
polypeptides. For
example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion
proteins
comprising various portions of constant region of immunoglobulin molecules
together
with another human protein or part thereof. In many cases, the Fc part in a
fusion
protein is thoroughly advantageous for use in therapy and diagnosis and thus
results,
for example, in improved pharmacokinetic properties (EP-A 0232 262). On the
other
hand, for some uses it would be desirable to be able to delete the Fc part
after the
fusion protein has been expressed, detected and purified in the advantageous
manner
described. This is the case when Fc portion proves to be a hindrance to use in
therapy
and diagnosis, for example when the fusion protein is to be used as antigen
for
immunizations. In drug discovery, for example, human proteins, such as hIL-5,
have
been fused with Fc portions for the purpose of high-throughput screening
assays to
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identify antagonists of hIL-5 (Bennett, D., et al., J. Molecular Recognition
8:52-58
( 1995); Johanson, K., et al., J. Biol. Chem. 270:9459-9471 ( 1995)).
In one embodiment, polynucleotides encoding Brainiac-5 polypeptides of the
invention may be fused to the pelB pectate lyase signal sequence to increase
the
efficiency to expression and purification of such polypeptides in Gram-
negative
bacteria. See, U.S. Patent Nos. 5,576,195 and 5,846,818, the contents of which
are
herein incorporated by reference in their entireties.
A preferred fusion protein of the invention comprises a heterologous region
from immunoglobulin that is useful to stabilize and purify proteins. For
example,
EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins
comprising
various portions of constant region of immunoglobulin molecules together with
another human protein or part thereof. In many cases, the Fc part in a fusion
protein is
thoroughly advantageous for use in therapy and diagnosis and thus results, for
example, in improved pharmacokinetic properties (EP-A 0232 262). On the other
hand, for some uses it would be desirable to be able to delete the Fc part
after the
fusion protein has been expressed, detected and purified in the advantageous
manner
described. This is the case when Fc portion proves to be a hindrance to use in
therapy
and diagnosis, for example when the fusion protein is to be used as antigen
for
immunizations. In drug discovery, for example, human proteins, such as hIL-5
has
been fused with Fc portions for the purpose of high-throughput screening
assays to
identify antagonists of hIL-5. See, D. Bennett et al., J. Molecular
Recognition 8:52-58
( 1995) and K. Johanson et al., J. Biol. Chem. 270:9459-9471 ( 1995).
Polypeptides of the present invention include naturally purified products,
products of chemical synthetic procedures, and products produced by
recombinant
techniques from a prokaryotic or eukaryotic host, including, for example,
bacterial,
yeast, higher plant, insect and mammalian cells. Depending upon the host
employed
in a recombinant production procedure, the polypeptides of the present
invention may
be glycosylated or may be non-glycosylated. In addition, polypeptides of the
invention
may also include an initial modified methionine residue, in some cases as a
result of
host-mediated processes.
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Polypeptides of the invention can be chemically synthesized using techniques
known in the art (e.g., see Creighton, 1983, Proteins: Structures and
Molecular
Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al., 1984,
Nature
310:105-111). For example, a peptide corresponding to a fragment of the
complete
Brainiac-5 polypeptides of the invention can be synthesized by use of a
peptide
synthesizer. Furthermore, if desired, nonclassical amino acids or chemical
amino acid
analogs can be introduced as a substitution or addition into the Brainiac-5
polynucleotide sequence. Non-classical amino acids include, but are not
limited to, to
the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino
isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx,
6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid,
ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline,
homocitrulline,
cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,
cycIohexylalanine, b-
alanine, fluoro-amino acids, designer amino acids such as b-methyl amino
acids, Ca-
methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
Furthermore, the amino acid can be D (dextrorotary) or L (levorotary)
The invention encompasses Brainiac-S polypeptides which are differentially
modified during or after translation, e.g., by glycosylation, acetylation,
phosphorylation, amidation, derivatization by known protecting/blocking
groups,
proteolytic cleavage, linkage to an antibody molecule or other cellular
ligand, etc.
Any of numerous chemical modifications may be carried out by known techniques,
including but not limited, to specific chemical cleavage by cyanogen bromide,
trypsin,
chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation,
reduction, metabolic synthesis in the presence of tunicamycin, etc.
Additional post-translational modifications encompassed by the invention
include, for example, e.g., N-linked or O-linked carbohydrate chains,
processing of
a
N-terminal or C-terminal ends), attachment of chemical moieties to the amino
acid
backbone, chemical modifications of N-linked or O-linked carbohydrate chains,
and
addition or deletion of an N-terminal methionine residue as a result of
procaryotic host
cell expression. The polypeptides may also be modified with a detectable
label, such
as an enzymatic, fluorescent, isotopic or affinity label to allow for
detection and
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isolation of the protein. In addition, polypeptides of the invention may be
modified by
iodination.
In one embodiment, Brainiac-5 polypeptides of the invention may also be
labeled with biotin. In other related embodiments, biotinylated Brainiac-5
polypeptides of the invention may be used, for example, as an imaging agent or
as a
means of identifying one or more Brainiac-5 receptors) or other coreceptor or
coligand molecules.
Also provided by the invention are chemically modified derivatives of
Brainiac-5 which may provide additional advantages such as increased
solubility,
stability and circulating time of the polypeptide, or decreased immunogenicity
(see U.
S. Patent No. 4,179,337). The chemical moieties for derivitization may be
selected
from water soluble polymers such as polyethylene glycol, ethylene
glycoUpropylene
glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the
like.
The polypeptides may be modified at random positions within the molecule, or
at
predetermined positions within the molecule and may include one, two, three or
more
attached chemical moieties.
The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about
I kDa and about 100 kDa (the term "about" indicating that in preparations of
polyethylene glycol, some molecules will weigh more, some less, than the
stated
molecular weight) for ease in handling and manufacturing. Other sizes may be
used,
depending on the desired therapeutic profile (e.g., the duration of sustained
release
desired, the effects, if any on biological activity, the ease in handling, the
degree or
lack of antigenicity and other known effects of the polyethylene glycol to a
therapeutic
protein or analog).
The polyethylene glycol molecules (or other chemical moieties) should be
attached to the protein with consideration of effects on functional or
antigenic domains
of the protein. There are a number of attachment methods available to those
skilled in
the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to
G-CSF),
see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation
of
GM-CSF using tresyl chloride). For example, polyethylene glycol may be
covalently
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bound through amino acid residues via a reactive group, such as, a free amino
or
carboxyl group. Reactive groups are those to which an activated polyethylene
glycol
molecule may be bound. The amino acid residues having a free amino group may
include lysine residues and the N-terminal amino acid residues; those having a
free
carboxyl group may include aspartic acid residues, glutamic acid residues, and
the
C-terminal amino acid residue. Sulfhydryl groups may also be used as a
reactive
group for attaching the polyethylene glycol molecules. Preferred for
therapeutic
purposes is attachment at an amino group, such as attachment at the N-terminus
or
lysine group.
One may specifically desire proteins chemically modified at the N-terminus.
Using polyethylene glycol as an illustration, one may select from a variety of
polyethylene glycol molecules (by molecular weight, branching, etc.), the
proportion
of polyethylene glycol molecules to protein (or peptide) molecules in the
reaction mix,
the type of pegylation reaction to be performed, and the method of obtaining
the
selected N-terminally pegylated protein. The method of obtaining the N-
terminally
pegylated preparation (i.e., separating this moiety from other monopegylated
moieties
if necessary) may be by purification of the N-terminally pegylated material
from a
population of pegylated protein molecules. Selective proteins chemically
modified at
the N-terminus modification may be accomplished by reductive alkylation which
exploits differential reactivity of different types of primary amino groups
(lysine
versus the N-terminal) available for derivatization in a particular protein.
Under the
appropriate reaction conditions, substantially selective derivatization of the
protein at
the N-terminus with a carbonyl group containing polymer is achieved.
The Brainiac-5 polypeptides of the invention can be recovered and purified by
well-known methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography, phosphocellulose
E
chromatography, hydrophobic interaction chromatography, affinity
chromatography,
hydroxylapatite chromatography and lectin chromatography. Most preferably,
high
performance liquid chromatography ("HPLC") is employed for purification.
Polypeptides of the present invention include: products purified from natural
sources,
including bodily fluids, tissues and cells, whether directly isolated or
cultured;
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products of chemical synthetic procedures; and products produced by
recombinant
techniques from a prokaryotic or eukaryotic host, including, for example,
bacterial,
yeast, higher plant, insect and mammalian cells. Depending upon the host
employed
in a recombinant production procedure, the polypeptides of the present
invention may
be glycosylated or may be non-glycosylated. In addition, polypeptides of the
invention
may also include an initial modified methionine residue, in some cases as a
result of
host-mediated processes. Thus, it is well known in the art that the N-terminal
methionine encoded by the translation initiation codon generally is removed
with high
efficiency from any protein after translation in all eukaryotic cells. While
the
N-terminal methionine on most proteins also is efficiently removed in most
prokaryotes, for some proteins this prokaryotic removal process is
inefficient,
depending on the nature of the amino acid to which the N-terminal methionine
is
covalently linked.
Polypeptides
The invention further provides an isolated Brainiac-5 polypeptide having
(i.e.,
comprising, or alternatively consisting of) the amino acid sequence encoded by
the
deposited cDNA, or the amino acid sequence in SEQ ID N0:2, or a peptide or
polypeptide comprising a portion of the above polypeptides.
20. The Brainiac-5 polypeptides of the invention may be in monomers or
multimers (i.e., dimers, trimers, tetramers and higher multimers).
Accordingly, the
present invention relates to monomers and multimers of the Brainiac-5
polypeptides of
the invention, their preparation, and compositions (preferably, pharmaceutical
compositions) containing them. In specific embodiments, the polypeptides of
the
invention are monomers, dinners, trimers or tetramers. In additional
embodiments, the
multimers of the invention are at least dimers, at least trimers, or at least
tetrarners.
Multimers encompassed by the invention may be homomers or heteromers. As
used herein, the term homomer, refers to a multimer containing only Brainiac-5
polypeptides of the invention (including Brainiac-5 fragments, variants, and
fusion
proteins, as described herein). These homomers may contain Brainiac-5
polypeptides
having identical or different amino acid sequences. In a specific embodiment,
a
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homomer of the invention is a multimer containing only Brainiac-5 polypeptides
having an identical amino acid sequence. In another specific embodiment, a
homomer
of the invention is a multimer containing Brainiac-5 polypeptides having
different
amino acid sequences. In specific embodiments, the multimer of the invention
is a
homodimer (e.g., containing Brainiac-5 polypeptides having identical or
different
amino acid sequences) or a homotrimer (e.g., containing Brainiac-5
polypeptides
having identical or different amino acid sequences). In a preferred
embodiment, the
multimer of the invention is a homotrimer. In additional embodiments, the
homomeric
multimer of the invention is at least a homodirner, at least a homotrimer, or
at least a
homotetramer.
As used herein, the term heteromer refers to a multimer containing
heterologous polypeptides (i.e., polypeptides of a different protein) in
addition to the
Brainiac-5 polypeptides of the invention. In a specific embodiment, the
multimer of
the invention is a heterodimer, a heterotrimer, or a heterotetramer. In
additional
embodiments, the heteromeric multimer of the invention is at least a
heterodimer, at
least a heterotrimer, or at least a heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic,
ionic
and/or covalent associations and/or may be indirectly linked, by for example,
liposome
formation. Thus, in one embodiment, multimers of the invention, such as, for
example, homodimers or homotrimers, are formed when polypeptides of the
invention
contact one another in solution. In another embodiment, heteromultimers of the
invention, such as, for example, heterotrimers or heterotetramers, are formed
when
polypeptides of the invention contact antibodies to the polypeptides of the
invention
(including antibodies to the heterologous polypeptide sequence in a fusion
protein of
the invention) in solution. In other embodiments, multimers of the invention
are
formed by covalent associations with and/or between the Brainiac-5
polypeptides of
the invention. Such covalent associations may involve one or more amino acid
residues contained in the polypeptide sequence (e.g., that recited in SEQ ID
N0:2 or
contained in the polypeptide encoded by the clone deposited in connection with
this
application). In one instance, the covalent associations are cross-linking
between
cysteine residues located within the polypeptide sequences which interact in
the native
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(i.e., naturally occurring) polypeptide. In another instance, the covalent
associations
are the consequence of chemical or recombinant manipulation. Alternatively,
such
covalent associations may involve one or more amino acid residues contained in
the
heterologous polypeptide sequence in a Brainiac-S fusion protein. In one
example,
covalent associations are between the heterologous sequence contained in a
fusion
protein of the invention (see, e.g., US Patent Number 5,478,925). In a
specific
example, the covalent associations are between the heterologous sequence
contained in
a Brainiac-5-Fc fusion protein of the invention (as described herein). In
another
specific example, covalent associations of fusion proteins of the invention
are between
heterologous polypeptide sequence from another protein that is capable of
forming
covalently associated multimers, such as for example, oseteoprotegerin (see,
e.g.,
International Publication No. WO 98/49305, the contents of which are herein
incorporated by reference in its entirety). In another embodiment, two or more
Brainiac-S polypeptides of the invention are joined through synthetic linkers
(e.g.,
peptide, carbohydrate or soluble polymer linkers). Examples include those
peptide
linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by
reference).
Proteins comprising multiple Brainiac-5 polypeptides separated by peptide
linkers may
be produced using conventional recombinant DNA technology.
Another method for preparing multimer Brainiac-S polypeptides of the
invention involves use of Brainiac-5 polypeptides fused to a leucine zipper
polypeptide sequence. Leucine zipper domains are polypeptides that promote
multimerization of the proteins in which they are found. Leucine zippers were
originally identified in several DNA-binding proteins (Landschulz et al.,
Science
240:1759, ( 1988)), and have since been found in a variety of different
proteins.
Among the known leucine zippers are naturally occurring peptides and
derivatives
thereof that dimerize or trimerize. Examples of leucine zipper domains
suitable for
producing soluble multimeric Brainiac-5 proteins are those described in PCT
application WO 94/10308, hereby incorporated by reference. Recombinant fusion
proteins comprising a soluble Brainiac-5 polypeptide fused to a peptide that
dimerizes
or trimerizes in solution are expressed in suitable host cells, and the
resulting soluble
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multimeric Brainiac-5 is recovered from the culture supernatant using
techniques
known in the art.
Trimeric Brainiac-S may offer the advantage of enhanced biological activity.
Preferred leucine zipper moieties are those that preferentially form trimers.
One
example is a leucine zipper derived from lung surfactant protein D (SPD), as
described
in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application
Ser. No.
08/446,922, hereby incorporated by reference. Other peptides derived from
naturally
occurring trimeric proteins may be employed in preparing trimeric Brainiac-5.
In another example, proteins of the invention are associated by interactions
between the Flag~ polypeptide sequence contained in Flag~-Brainiac-5 fusion
proteins of the invention. In a further embodiment, proteins of the invention
are
associated by interactions between the heterologous polypeptide sequence
contained in
Flag-Brainiac-5 fusion proteins of the invention and anti-Flag~ antibody.
The multimers of the invention may be generated using chemical techniques
known in the art. For example, polypeptides desired to be contained in the
multimers
of the invention may be chemically cross-linked using linker molecules and
linker
molecule length optimization techniques known in the art (see, e.g., US Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
Additionally, multimers of the invention may be generated using techniques
known in
the art to form one or more inter-molecule cross-links between the cysteine
residues
located within the sequence of the polypeptides desired to be contained in the
multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
reference in its entirety). Further, polypeptides of the invention may be
routinely
modified by the addition of cysteine or biotin to the C terminus or N-terminus
of the
polypeptide and techniques known in the art may be applied to generate
multimers
containing one or more of these modified polypeptides (see, e.g., US Patent
Number
a
5,478,925, which is herein incorporated by reference in its entirety).
Additionally,
techniques known in the art may be applied to generate liposomes containing
the
polypeptide components desired to be contained in the multimer of the
invention (see,
e.g., US Patent Number 5,478,925, which is herein incorporated by reference in
its
entirety).
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Alternatively, multimers of the invention may be generated using genetic
engineering techniques known in the art. In one embodiment, polypeptides
contained
in multimers of the invention are produced recombinantly using fusion protein
technology described herein or otherwise known in the art (see, e.g., US
Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
In a
specific embodiment, polynucleotides coding for a homodimer of the invention
are
generated by ligating a polynucleotide sequence encoding a polypeptide of the
invention to a sequence encoding a linker polypeptide and then further to a
synthetic
polynucleotide encoding the translated product of the polypeptide in the
reverse
orientation from the original C-terminus to the N-terminus (lacking the leader
sequence) (see, e.g., US Patent Number 5,478,925, which is herein incorporated
by
reference in its entirety). In another embodiment, recombinant techniques
described
herein or otherwise known in the art are applied to generate recombinant
polypeptides
of the invention which contain a transmembrane domain and which can be
incorporated by membrane reconstitution techniques into liposomes (see, e.g.,
US
Patent Number 5,478,925, which is herein incorporated by reference in its
entirety).
In one embodiment, the invention provides an isolated Brainiac-5 polypeptide
having (i.e., comprising, or alternatively consisting of) the amino acid
sequence
encoded by the cDNA clone contained in ATCC No. 203572, or the amino acid
sequence in Figures 1 A and 1 B (SEQ ID N0:2), or a peptide or polypeptide
comprising a portion (i.e., a fragment) of the above polypeptides.
Polypeptide fragments of the present invention include polypeptides
comprising or alternatively, consisting of, an amino acid sequence contained
in SEQ
ID N0:2, encoded by the cDNA contained in the plasmid having ATCC accession
number 203572, or encoded by nucleic acids which hybridize (e.g., under
stringent
hybridization conditions) to the nucleotide sequence contained in the
deposited clone,
A
or the complementary strand of the nucleotide sequence shown in Figures 1 A-B
(SEQ
ID NO:1 ).
Polypeptide fragments may be "free-standing," or comprised within a larger
polypeptide of which the fragment forms a part or region, most preferably as a
single
continuous region. Representative examples of polypeptide fragments of the
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invention, include, for example, fragments that comprise or alternatively,
consist of,
from about amino acid residues: 1 to 15, 16-30, 31-46, 47-55, 56-72, 73-104,
105-163,
163-188, 186-210 and 2101-278 of the amino acid sequence disclosed in SEQ ID
N0:2.
Moreover, polypeptide fragments can be at least 10, 20, 30, 40, 50, 60, 70,
80, 90, 100,
110, 120, 130, 140, 150, 175 or 200 amino acids in length. In this context,
"about"
means several, a few, S, 4, 3, 2 or 1. Polynucleotides encoding these
polypeptide
fragments are also encompassed by the invention.
Additional polypeptide fragments of the invention comprise, or alternatively
consist of, amino acids 7 to 20, 7 to 33, 61 to 83, 105 to 119, 139 to 148,
160 to 17I,
187 to 196 of SEQ ID N0:2. Polypeptides encoded by these polynucleotides are
also
encompassed by the invention.
Additional embodiments encompass Brainiac-5 polypeptide fragments
comprising, or alternatively consisting of, one, two, three, four, five or
more functional
regions of polypeptides of the invention, such as the Gamier-Robson alpha-
regions,
beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-
regions,
and coil-regions, Kyte-Doolittle hydrophilic regions and hydrophobic regions,
Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible
regions,
Emini surface-forming regions and Jameson-Wolf regions of high antigenic index
set
out in Figures 3 and in Table I and as described herein. In a preferred
embodiment,
the polypeptide fragments of the invention are antigenic. The data presented
in
columns VIII, IX, XIII, and XIV of Table I can be used to routinely determine
regions
of Brainiac-5 which exhibit a high degree of potential for antigenicity.
Regions of
high antigenicity are determined from the data presented in columns VIII, IX,
XIII,
and/or IV by choosing values which represent regions of the polypeptide which
are
likely to be exposed on the surface of the polypeptide in an environment in
which
antigen recognition may occur in the process of initiation of an immune
response (e.g.,
a polypeptide comprising amino acid residues from about Val-1 to about Val-11
in
SEQ ID N0:2; a polypeptide comprising amino acid residues from about Thr-14 to
about Gln-22 in SEQ ID N0:2; a polypeptide comprising amino acid residues from
about Val-34 to about His-53 in SEQ ID N0:2; a polypeptide comprising amino
acid
residues from about Phe-94 to about Val-108 in SEQ ID N0:2; a polypeptide
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comprising amino acid residues from about Ala-120 to about Gln-126 in SEQ ID
N0:2; a polypeptide comprising amino acid residues from about Arg-138 to about
Ile-149 in SEQ ID N0:2; a polypeptide comprising amino acid residues from
about
Leu-202 to about Ala-211 in SEQ ID N0:2; and a polypeptide comprising amino
acid
residues from about Phe-274 to about Ser-278 in SEQ ID N0:2). Among highly
preferred fragments of the invention are those that comprise regions of
Brainiac-5 that
combine several structural features, such as several (e.g., 1, 2, 3 or 4) of
the features
set out above. Polynucleotides encoding these polypeptides are also
encompassed by
the invention.
In another embodiment, the invention provides a peptide or polypeptide
comprising an epitope-bearing portion of a polypeptide of the invention. The
epitope
of this polypeptide portion is an immunogenic or antigenic epitope of a
polypeptide of
the invention. An "immunogenic epitope" is defined as a part of a polypeptide
that
elicits an antibody response when the complete or whole polypeptide is the
immunogen. On the other hand, a region of a protein molecule to which an
antibody
can bind is defined as an "antigenic epitope." The number of immunogenic
epitopes
of a protein generally is less than the number of antigenic epitopes {see, for
instance,
Geysen, et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 ( 1983)).
As to the selection of peptides or polypeptides bearing an antigenic epitope
(i.e., that contain a region of a polypeptide molecule to which an antibody
can bind), it
is well known in that art that relatively short synthetic peptides that mimic
part of a
polypeptide sequence are routinely capable of eliciting an antiserum that
reacts with
the partially mimicked polypeptide (see, for instance, Sutcliffe, J. G., et
al., Science
219:660-666 ( 1983)). Peptides capable of eliciting protein-reactive sera are
frequently
represented in the primary sequence of a polypeptide, can be characterized by
a set of
simple chemical rules, and are confined neither to immunodominant regions of
intact
polypeptides (i.e., immunogenic epitopes) nor to the amino or carboxyl
terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore
useful to raise antibodies, including monoclonal antibodies, that bind
specifically to a
polypeptide of the invention (see, for instance, Wilson, et al., Cell 37:767-
778 ( 1984)).
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Antigenic epitope-bearing peptides and polypeptides of the invention
preferably contain a sequence of at least seven, more preferably at least nine
and most
preferably between about 15 to about 30 amino acids contained within the amino
acid
sequence of a polypeptide of the invention. Non-limiting examples of antigenic
polypeptides or peptides that can be used to generate Brainiac-5-specific
antibodies
include: a polypeptide comprising amino acid residues from about Val-1 to
about
Val-11 in SEQ ID N0:2; from about Thr-14 to about Gln-22 in SEQ ID N0:2; from
about VaI-34 to about His-53 in SEQ ID N0:2; from about Phe-94 to about Val-
108 in
SEQ ID N0:2; from about .Ala-120 to about Gln-126 in SEQ ID N0:2; from about
Arg-138 to about Ile-149 in SEQ ID N0:2; ; from about Leu-202 to about Ala-211
in
SEQ ID N0:2; and from about Phe-274 to about Ser-278 in SEQ ID N0:2. These
polypeptide fragments have been determined to bear antigenic epitopes of the
Brainiac-5 polypeptide by the analysis of the Jameson-Wolf antigenic index, as
shown
in Figure 3 and/or Table I, above.
The epitope-bearing peptides and polypeptides of the invention may be
produced by any conventional means (see, for example, Houghten, R. A., et al.,
Proc.
Natl. Acad. Sci. USA 82:5131-5135 (1985); and U.S. Patent No. 4,631,211 to
Houghten, et al. ( I 986)).
Epitope-bearing peptides and polypeptides of the invention are used to induce
antibodies according to methods well known in the art (see, for instance,
Sutcliffe, et
al., supra; Wilson, et al., supra; Chow, M., et al., Proc. Natl. Acad. Sci.
USA
82:910-914; and Bittle, F. J., et al., J. Gen. Virol. 66:2347-2354 ( 1985)).
Immunogenic epitope-bearing peptides of the invention, i.e., those parts of a
protein
that elicit an antibody response when the whole protein is the immunogen, are
identified according to methods known in the art (see, for instance, Geysen,
et al.,
supra). Further still, U.S. Patent No. 5,194,392, issued to Geysen, describes
a general
method of detecting or determining the sequence of monomers (amino acids or
other
compounds) which is a topological equivalent of the epitope (i.e., a
"mimotope")
which is complementary to a particular paratope (antigen binding site) of an
antibody
of interest. More generally, tl.S. Patent No. 4,433,092, issued to Geysen,
describes a
method of detecting or determining a sequence of monomers which is a
topographical
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equivalent of a ligand which is complementary to the ligand binding site of a
particular
receptor of interest. Similarly, U.S. Patent No. 5,480,971, issued to Houghten
and
colleagues, on Peralkylated Oligopeptide Mixtures discloses linear C1-C7-alkyl
peralkylated oligopeptides and sets and libraries of such peptides, as well as
methods
for using such oligopeptide sets and libraries for determining the sequence of
a
peralkylated oligopeptide that preferentially binds to an acceptor molecule of
interest.
Thus, non-peptide analogs of the epitope-bearing peptides of the invention
also can be
made routinely by these methods.
Brainiac-5 polypeptide-specific antibodies for use in the present invention
can
be raised against the intact Brainiac-5 polypeptide or an antigenic
polypeptide
fragment thereof, which may be presented together with a carrier protein, such
as an
albumin, to an animal system (such as rabbit or mouse) or, if it is long
enough (at least
about 25 amino acids), without a carrier.
As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is
I S meant to include intact molecules as well as antibody fragments (such as,
for example,
Fab and F(ab')2 fragments) which are capable of specifically binding to
Brainiac-5
polypeptides. Fab and F(ab')2 fragments lack the Fc fragment of intact
antibody, clear
more rapidly from the circulation, and may have less non-specific tissue
binding of an
intact antibody (Wahl, et al., J. Nucl. Med. 24:316-325 ( 1983)). Thus, these
fragments
are preferred.
The antibodies of the present invention may be prepared by any of a variety of
methods. For example, cells expressing the Brainiac-5 polypeptides or an
antigenic
fragment thereof can be administered to an animal in order to induce the
production of
sera containing polyclonal antibodies. In a preferred method, a preparation of
Brainiac-5 polypeptide is prepared and purified to render it substantially
free of natural
contaminants. Such a preparation is then introduced into an animal in order to
produce
polyclonal antisera of greater specific activity.
In the most preferred method, the antibodies of the present invention are
monoclonal antibodies (or Brainiac-5 polypeptide-binding fragments thereof).
Such
monoclonal antibodies can be prepared using hybridoma technology (Kohler, et
al.,
Nature 256:495 ( 1975); Kohler, et al., Eur. J. Immunol. 6:511 ( 1976);
Kohler, et al.,
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Eur. J. Immunol. 6:292 ( 1976); Hammerling, et al., in: Monoclonal Antibodies
and
T Cell Hybridomas, Elsevier, N.Y., ( 1981 ) pp. 563-681 )). In general, such
procedures
involve immunizing an animal (preferably a mouse) with a Brainiac-5
polypeptide
antigen or, more preferably, with a Brainiac-5 polypeptide-expressing cell.
Suitable
cells can be recognized by their capacity to bind anti-Brainiac-5 polypeptide
antibody.
Such cells may be cultured in any suitable tissue culture medium; however, it
is
preferable to culture cells in Earle's modified Eagle's medium supplemented
with 10%
fetal bovine serum (inactivated at about 56° C), and supplemented with
about 10 Ng/I
of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100
pg/ml of
streptomycin. The splenocytes of such mice are extracted and fused with a
suitable
myeloma cell line. Any suitable myeloma cell line may be employed in
accordance
with the present invention; however, it is preferable to employ the parent
myeloma cell
line (SP20), available from the American Type Culture Collection, Manassas,
Virginia. After fusion, the resulting hybridoma cells are selectively
maintained in
HAT medium, and then cloned by limiting dilution as described by Wands and
colleagues (Gastroenterolo,gy 80:225-232 ( 1981 )). The hybridoma cells
obtained
through such a selection are then assayed to identify clones which secrete
antibodies
capable of binding the Brainiac-5 polypeptide antigen.
Alternatively, additional antibodies capable of binding to the Brainiac-5
polypeptide antigen may be produced in a two-step procedure through the use of
anti-idiotypic antibodies. Such a method makes use of the fact that antibodies
are
themselves antigens, and that, therefore, it is possible to obtain an antibody
which
binds to a second antibody. In accordance with this method, Brainiac-S
polypeptide-specific antibodies are used to immunize an animal, preferably a
mouse.
The splenocytes of such an animal are then used to produce hybridoma cells,
and the
hybridoma cells are screened to identify clones which produce an antibody
whose
ability to bind to the Brainiac-5 polypeptide-specific antibody can be blocked
by the
Brainiac-5 polypeptide antigen. Such antibodies comprise anti-idiotypic
antibodies to
the Brainiac-5 polypeptide-specific antibody and can be used to immunize an
animal
to induce formation of further Brainiac-S polypeptide-specific antibodies.
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It will be appreciated that Fab and F(ab')2 and other fragments of the
antibodies of the present invention may be used according to the methods
disclosed
herein. Such fragments are typically produced by proteolytic cleavage, using
enzymes
such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments).
S Alternatively, Brainiac-S polypeptide-binding fragments can be produced
through the
application of recombinant DNA technology or through synthetic chemistry.
For in vivo use of anti-Brainiac-5 in humans, it may be preferable to use
"humanized" chimeric monoclonal antibodies. Such antibodies can be produced
using
genetic constructs derived from hybridoma cells producing the monoclonal
antibodies
described above. Methods for producing chimeric antibodies are known in the
art
(Morrison, Science 229:1202 (1985); Oi, et al., BioTechniques 4:214 (1986);
Cabilly,
et al., U.S. Patent No. 4,816,567; Taniguchi, et al., EP 171496; Morrison, et
al., EP
173494; Neuberger, et al., WO 8601533; Robinson, et al., WO 8702671;
Boulianne, et
al., Nature 312:643 (1984); Neuberger, et al., Nature 314:268 (1985).
The present invention encompasses polypeptides comprising, or alternatively
consisting of, an epitope of the polypeptide having an amino acid sequence of
SEQ ID
N0:2, or an epitope of the polypeptide sequence encoded by a polynucleotide
sequence contained in deposited clone 203572 or encoded by a polynucleotide
that
hybridizes to the complement of the sequence of SEQ ID NO:1 or contained in
deposited clone HOGCC45 under stringent hybridization conditions or lower
stringency hybridization conditions as defined supra. The present invention
further
encompasses polynucleotide sequences encoding an epitope of a polypeptide
sequence
of the invention (such as, for example, the sequence disclosed in SEQ ID NO:1
),
polynucleotide sequences of the complementary strand of a polynucleotide
sequence
encoding an epitope of the invention, and polynucleotide sequences which
hybridize to
the complementary strand under stringent hybridization conditions or lower
stringency hybridization conditions defined supra.
The term "epitopes," as used herein, refers to portions of a polypeptide
having
antigenic or immunogenic activity in an animal, preferably a mammal, and most
preferably in a human. In a preferred embodiment, the present invention
encompasses
a polypeptide comprising an epitope, as well as the polynucleotide encoding
this
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polypeptide. An "immunogenic epitope," as used herein, is defined as a portion
of a
protein that elicits an antibody response in an animal, as determined by any
method
known in the art, for example, by the methods for generating antibodies
described
infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-
4002
S ( 1983)). The term "antigenic epitope," as used herein, is defined as a
portion of a
protein to which an antibody can immunospecifically bind its antigen as
determined by
any method well known in the art, for example, by the immunoassays described
herein. Immunospecific binding excludes non-specific binding but does not
necessarily exclude cross-reactivity with other antigens. Antigenic epitopes
need not
necessarily be immunogenic.
Fragments that function as epitopes may be produced by any conventional
means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985},
further described in U.S. Patent No. 4,631,211 }.
In the present invention, antigenic epitopes preferably contain a sequence of
at
least 4, at least 5, at least 6, at least 7, more preferably at least 8, at
least 9, at least 10,
at least 15, at least 20, at least 25, and, most preferably, between about 15
to about 30
amino acids. Preferred polypeptides comprising immunogenic or antigenic
epitopes
are at least 10, 15, 20, 25, 30, 35, 40, 45, S0, 55, 60, 65, 70, 75, 80, 85,
90, 95, or 100
amino acid residues in length. Antigenic epitopes are useful, for example, to
raise
antibodies, including monoclonal antibodies, that specifically bind the
epitope.
Antigenic epitopes can be used as the target molecules in immunoassays. (See,
for
instance, Wilson et al., Cell 37:767-778 ( I 984); Sutcliffe et al., Science
219:660-666
( 1983)).
Similarly, immunogenie epitopes can be used, for example, to induce
antibodies according to methods well known in the art. (See, for instance,
Sutcliffe et
al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA
82:910-914;
and Bittle et al., J. Gen. Viral. 66:2347-2354 ( 1985). A preferred
immunogenic
epitope includes the secreted protein. The polypeptides comprising one or more
immunogenic epitopes may be presented for eliciting an antibody response
together
with a carrier protein, such as an albumin, to an animal system (such as, for
example,
rabbit or mouse), or, if the polypeptide is of sufficient length (at least
about 25 amino
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acids), the polypeptide may be presented without a carrier. However,
immunogenic
epitopes comprising as few as 8 to 10 amino acids have been shown to be
sufficient to
raise antibodies capable of binding to, at the very least, linear epitopes in
a denatured
polypeptide (e.g., in Western blotting).
Epitope-bearing polypeptides of the present invention may be used to induce
antibodies according to methods well known in the art including, but not
limited to, in
vivo immunization, in vitro immunization, and phage display methods. See,
e.g.,
Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-
2354 (1985). If in vivo immunization is used, animals may be immunized with
free
peptide; however, anti-peptide antibody titer may be boosted by coupling the
peptide
to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or
tetanus
toxoid. For instance, peptides containing cysteine residues may be coupled to
a carrier
using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),
while
other peptides may be coupled to carriers using a more general linking agent
such as
glutaraldehyde. Animals such as, for example, rabbits, rats, and mice are
immunized
with either free or carrier-coupled peptides, for instance, by intraperitoneal
and/or
intradermal injection of emulsions containing about 100 micrograms of peptide
or
carrier protein and Freund's adjuvant or any other adjuvant known for
stimulating an
immune response. Several booster injections may be needed, for instance, at
intervals
of about two weeks, to provide a useful titer of anti-peptide antibody that
can be
detected, for example, by EL,ISA assay using free peptide adsorbed to a solid
surface.
The titer of anti-peptide antibodies in serum from an immunized animal may be
increased by selection of anti-peptide antibodies, for instance, by adsorption
to the
peptide on a solid support and elution of the selected antibodies according to
methods
well known in the art.
As one of skill in the art will appreciate, and as discussed above, the
polypeptides of the present invention comprising an immunogenic or antigenic
epitope
can be fused to other polypeptide sequences. For example, the polypeptides of
the
present invention may be fused with the constant domain of immunoglobulins
(IgA,
IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof
and
portions thereof] resulting in chimeric polypeptides. Such fusion proteins may
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facilitate purification and may increase half life in vivo. This has been
shown for
chimeric proteins consisting of the first two domains of the human CD4-
polypeptide
and various domains of the constant regions of the heavy or light chains of
mammalian
immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86
(1988).
IgG Fusion proteins that have a disulfide-linked dimeric structure due to the
IgG
portion desulfide bonds have also been found to be more efficient in binding
and
neutralizing other molecules than monomeric polypeptides or fragments thereof
alone.
See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 ( 1995). Nucleic
acids
encoding the above epitopes can also be recombined with a gene of interest as
an
epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid in
detection and
purification of the expressed polypeptide. For example, a system described by
Janknecht et al. allows for the ready purification of non-denatured fusion
proteins
expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA
88:8972- 897). In this system, the gene of interest is subcloned into a
vaccinia
recombination plasmid such that the open reading frame of the gene is
translationally
fused to an amino-terminal tag consisting of six histidine residues. The tag
serves as a
matrix-binding domain for the fusion protein. Extracts from cells infected
with the
recombinant vaccinia virus are loaded onto Ni'-+ nitriloacetic acid-agarose
column and
histidine-tagged proteins can be selectively eluted with imidazole-containing
buffers.
Additional fusion proteins of the invention may be generated through the
techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-
shuffling
(collectively referred to as "DNA shuffling";y. DNA shuffling may be employed
to
modulate the activities of polypeptides of the invention, such methods can be
used to
generate polypeptides with altered activity, as well as agonists and
antagonists of the
polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238;
5,830,721;
5,834,252; and 5,$37,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-
33
( 1997); Harayama, Trends Biotechnol. 16(2):76-82 ( 1998); Hansson, et al., J.
Mol.
Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308- 13
(1998) (each of these patents and publications are hereby incorporated by
reference in
its entirety). In one embodiment, alteration of polynucleotides corresponding
to SEQ
ID NO:l and the polypeptides encoded by these polynucleotides may be achieved
by
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DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments
by homologous or site-specific recombination to generate variation in the
polynucIeotide sequence. In another embodiment, polynucleotides of the
invention, or
the encoded polypeptides, may be altered by being subjected to random
mutagenesis
by error-prone PCR, random nucleotide insertion or other methods prior to
recombination. In another embodiment, one or more components, motifs,
sections,
parts, domains, fragments, etc., of a polynucleotide coding a polypeptide of
the
invention may be recombined with one or more components, motifs, sections,
parts,
domains, fragments, etc. of one or more heterologous molecules.
In another embodiment, the invention provides a peptide or polypeptide
comprising, or alternatively consisting of, an epitope-bearing portion of a
polypeptide
of the invention. Polynucleotides encoding these peptides or polypeptides are
also
encompassed by the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention. An
"immunogenic epitope" is defined as a part of a protein that elicits an
antibody
response when the whole protein is the immunogen. On the other hand, a region
of a
protein molecule to which an antibody can bind is defined as an "antigenic
epitope."
The number of immunogenic epitopes of a protein generally is less than the
number of
antigenic epitopes. See, for instance, Geysen et al., Proc. Natl. Acad. Sci.
USA
81:3998- 4002 (1983).
As to the selection of peptides or polypeptides bearing an antigenic epitope
(i.e., that contain a region of a protein molecule to which an antibody can
bind), it is
well known in that art that relatively short synthetic peptides that mimic
part of a
protein sequence are routinely capable of eliciting an antiserum that reacts
with the
partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T.
M., Green,
N. and Learner, R. A. ( 1983) "Antibodies that react with predetermined sites
on
proteins", Science, 219:660-666. Peptides capable of eliciting protein-
reactive sera are
frequently represented in the primary sequence of a protein, can be
characterized by a
set of simple chemical rules, and are confined neither to immunodominant
regions of
intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl
terminals.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore
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useful to raise antibodies, including monoclonal antibodies, that bind
specifically to a
polypeptide of the invention. See, for instance, Wilson et al., Cell 37:767-
778 (1984)
at 777.
Antigenic epitope-bearing peptides and polypeptides of the invention
S preferably contain a sequence of at least six, at least seven, at least
eight, more
preferably at least nine, at least 10, at least 1 S, at least 20, at least 30,
at least 40, at
least S0, at least 7S, and most preferably between about 1 S to about 30 amino
acids
contained within the amino acid sequence of a polypeptide of the invention.
Non-limiting examples of antigenic polypeptides or peptides that can be used
to
generate Brainiac-S-specific antibodies include: a polypeptide comprising, or
alternatively consisting of, amino acid residues from about Val-1 to about Val-
11 in
SEQ ID N0:2; a polypeptide comprising, or alternatively consisting of, amino
acid
residues from about Thr-14 to about Gln-22 in SEQ ID NO:2; a polypeptide
comprising, or alternatively consisting of, amino acid residues from about Val-
34 to
1 S about His-S3 in SEQ ID NO:2; a polypeptide comprising, or alternatively
consisting
of, amino acid residues from about Phe-94 to about Val-108 in SEQ ID N0:2; a
polypeptide comprising, or alternatively consisting of, amino acid residues
from about
Ala-120 to about Gln-126 in SEQ ID N0:2; a polypeptide comprising, or
alternatively
consisting of, amino acid residues from about Arg-138 to about Ile-149 in SEQ
ID
N0:2; a polypeptide comprising, or alternatively consisting of, amino acid
residues
from about Leu-202 to about Ala-211 in SEQ ID N0:2; and a polypeptide
comprising,
or alternatively consisting of, amino acid residues from about Phe-274 to
about
Ser-278 in SEQ ID N0:2). These polypeptide fragments have been determined to
bear
antigenic epitopes of the Brainiac-S polypeptide by the analysis of the
Jameson-Wolf
2S antigenic index, as shown in Figure 3 and Table I, above. Polynucleotides
encoding
these polypeptides are also encompassed by the invention. By "about" is meant,
the
particularly recited ranges and ranges larger or smaller at the N- and/or C-
terminus by
several, a few, S, 4, 3, 2 or 1 residue.
The epitope-bearing peptides and polypeptides of the invention may be
produced by any conventional means. See, e.g., Houghten, R. A. (1985) General
method for the rapid solid-phase synthesis of large numbers of peptides:
specificity of
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antigen-antibody interaction at the level of individual amino acids. Proc.
Natl. Acad.
Sci. USA 82:5131-5135; this "Simultaneous Multiple,Peptide Synthesis (SMPS)"
process is further described in U. S. Patent No. 4,631,211 to Houghton et al.
(1986).
Epitope-bearing peptides and polypeptides of the invention are used to induce
antibodies according to methods well known in the art. See, for instance,
Sutcliffe et
al., supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA
82:910-914; and Bittle, F. J. et al., J. Gen. Viral. 66:2347-2354 (1985).
Immunogenic
epitope-bearing peptides of the invention, i.e., those parts of a protein that
elicit an
antibody response when the whole protein is the immunogen, are identified
according
to methods known in the art. See, for instance, Geysen et al., supra. Further
still, U.S.
Patent No. 5,194,392 to Geysen ( 1990) describes a general method of detecting
or
determining the sequence of monomers (amino acids or other compounds) which is
a
topological equivalent of the epitope (i.e., a "mimotope") which is
complementary to a
particular paratope (antigen binding site) of an antibody of interest. More
generally,
U.S. Patent No. 4,433,092 to Geysen ( 1989) describes a method of detecting or
determining a sequence of monomers which is a topographical equivalent of a
ligand
which is complementary to the ligand binding site of a particular receptor of
interest.
Similarly, U.S. Patent No. 5,480,971 to Houghton, R. A. et al. ( 1996) on
Peralkylated
Oligopeptide Mixtures discloses linear C1-C7-alkyl peralkylated oligopeptides
and
sets and libraries of such peptides, as well as methods for using such
oligopeptide sets
and libraries for determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus, non-peptide
analogs of
the epitope-bearing peptides of the invention also can be made routinely by
these
methods.
As one of skill in the art will appreciate, Brainiac-S polypeptides of the
present
invention and the epitope-bearing fragments thereof described above can be
combined
with heterologous polypeptide sequences. For example, the polypeptides of the
present invention may be fused with the constant domain of immunoglobulins
(IgA,
IgE, IgG, IgM) or portions thereof (CH 1, CH2, CH3, and any combination
thereof,
including both entire domains and portions thereof), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and show an
increased
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half-life in vivo. This has been shown, e.g., for chimeric proteins consisting
of the first
two domains of the human CD4-polypeptide and various domains of the constant
regions of the heavy or light chains of mammalian immunoglobulins (EP A
394,827;
Traunecker et al., Nature 331:84-86 ( 1988;)). Fusion proteins that have a
S disulfide-linked dimeric structure due to the IgG part can also be more
efficient in
binding and neutralizing other molecules than the monomeric Brainiac-S
polypeptides
or polypeptide fragments alone (Fountoulakis et al., J. Biochem. 270:3958-3964
( 1995)).
As one of skill in the art will appreciate, Brainiac-S polypeptides of the
present
invention and the epitope-bearing fragments thereof described above can be
combined
with parts of the constant domain of immunoglobulins (IgG), resulting in
chimeric
polypeptides. These fusion proteins facilitate purification and show an
increased
half-life in vivo. This has been shown, e.g., for chimeric proteins consisting
of the first
two domains of the human CD4-polypeptide and various domains of the constant
1S regions of the heavy or light chains of mammalian immunoglobulins (EP A
394,827;
Traunecker, et al., Nature 331:84-86 (1988)). Fusion proteins that have a
disulfide-linked dimeric structure due to the IgG part can also be more
efficient in
binding and neutralizing other molecules than the monomeric Brainiac-S
polypeptide
or polypeptide fragment alone (Fountoulakis, et al., J. Biochem. 270:3958-3964
( 1995)).
In another embodiment, the Brainiac-5 polypeptides of the present invention
and the epitope-bearing fragments thereof are fused with a heterologous
antigen (e.g.,
polypeptide, carbohydrate, phospholipid, or nucleic acid). In specific
embodiments,
the heterologous antigen is an immunogen.
2S The techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or
codon-shuffling (collectively referred to as "DNA shuffling") may be employed
to
modulate the activities of Brainiac-S thereby effectively generating agonists
and
antagonists of Brainiac-S. See generally, U.S. Patent Nos. 5,605,793,
5,811,238,
5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion
Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16{2):76-82
(1998);
Hansson, L. O., et al., J. Mol. Biol. 287:265-76 { 1999); and Lorenzo, M. M.
and
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Blasco, R. Biotechniques 24(2}:308-13 ( 1998) (each of these patents and
publications
are hereby incorporated by reference). In one embodiment, alteration of
Brainiac-5
polynucleotides and corresponding polypeptides may be achieved by DNA
shuffling.
DNA shuffling involves the assembly of two or more DNA segments into a desired
Brainiac-5 molecule by homologous, or site-specific, recombination. In another
embodiment, Brainiac-5 palynucleotides and corresponding polypeptides may be
altered by being subjected to random mutagenesis by error-prone PCR, random
nucleotide insertion or other methods prior to recombination. In another
embodiment,
one or more components, motifs, sections, parts, domains, fragments, etc., of
Brainiac-
5 may be recombined with one or more components, motifs, sections, parts,
domains,
fragments, etc. of one or mare heterologous molecules. In preferred
embodiments, the
heterologous molecules are., for example, TNF-alpha, lymphotoxin-alpha (LT-
alpha,
also known as TNF-beta), LT-beta (found in camplex heterotrimer LT-alpha2-
beta),
OPGL, Fast, CD27L, CD30L, CD40L, 4-1 BB L, DcR3, OX40L, TNF-gamma
(International Publication No. WO 96/14328), AIM-I (International Publication
No.
WO 97/33899}, AIM-II (International Publication No. WO 97/34911 ), APRIL (J.
Exp.
Med. 188(6):1185-1190), endokine-alpha (International Publication No. WO
98/07880), OPG, OX40, and nerve growth factor (NGF), and soluble forms of Fas,
CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095),
DR3 (International Publication No. WO 97/33904}, DR4 (International
Publication
No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6
(International Publication No. W0 98/30694),TR7 (International Publication No.
WO
98/41629), TRANK, TR9 (International Publication No. WO 98/56892},TR10
(International Publication No. WO 98/54202),312C2 (International Publication
No.
WO 98/06842), TR I2, CAD, and v-FLIP. In further preferred embodiments, the
heterologous molecules are any member of the TNF family.
To improve or alter the characteristics of Brainiac-5 polypeptides, protein
engineering may be employed. Recombinant DNA technology known to those skilled
in the art can be used to create novel mutant polypeptides or muteins
including single
or multiple amino acid substitutions, deletions, additions or fusion
polypeptides. Such
modified polypeptides can show, e.g., enhanced activity or increased
stability. In
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addition, they may be purified in higher yields and show better solubility
than the
corresponding natural polypeptide, at least under certain purification and
storage
conditions. For instance, f'or many proteins, including the extracellular
domain of a
membrane associated protein or the mature forms) of a secreted protein, it is
known in
the art that one or more amino acids may be deleted from the N-terminus or C-
terminus without substantial loss of biological function. For instance, Ron
and
colleagues (J. Biol. Chem., 268:2984-2988 (1993)) reported modified KGF
proteins
that had heparin binding activity even if 3, 8, or 27 N-terminal amino acid
residues
were missing.
In the present case, since the Brainiac-S polypeptide of the invention is a
member of the Brainiac polypeptide family, deletions of N-terminal amino acids
up to
the arginine at position 8 of SEQ ID N0:2 may retain some biological activity
such as
the ability to modulate cell growth and differentiation. Polypeptides having
further N-
terminal deletions including the arginine-8 residue in SEQ ID N0:2 may not
retain
such biological activities, or may exhibit an alterred biological activity,
because it is
known that this residue in a Brainiac-related polypeptide is in the beginning
of the
conserved domain believed to be required for biological activities.
However, even if deletion of one or more amino acids from the N-terminus of a
protein results in modification or loss of one or more biological functions of
the
protein, other biological activities may still be retained. Thus, the ability
of the
shortened polypeptide to induce and/or bind to antibodies which recognize the
complete or mature form of the polypeptide generally will be retained when
less than
the majority of the residues of the complete or mature form of the polypeptide
are
removed from the N-terminus. Whether a particular polypeptide lacking N-
terminal
residues of a complete polypeptide retains such immunologic activities can
readily be
determined by routine methods described herein and otherwise known in the art.
Accordingly, the present invention further provides polypeptides having one or
more residues deleted from the amino terminus of the amino acid sequence of
the
Brainiac-S polypeptide shown in SEQ ID N0:2, up to the arginine residue at
position
number 8, and polynucleotides encoding such polypeptides. In particular, the
present
invention provides polypeptides comprising, or alternatively consisting of,
the amino
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acid sequence of residues n'-278 of SEQ ID N0:2, where n' is an integer in the
range
of 1 to 8, and 9 is the position of the first residue from the N-terminus of
the complete
Brainiac-5 polypeptide (shown in SEQ ID N0:2) believed to be required for
modulation of cell growth and differentiation activity of the Brainiac-5
polypeptide.
More in particular, the invention provides nucleic acid molecules encoding
polypeptides having (i.e., comprising, or alternatively consisting of) the
amino acid
sequence of residues of 1-278; 2-278; 3-278; 4-278; S-278; 6-278; 7-278; or 8-
278 of
SEQ ID N0:2. The present application is also directed to nucleic acid
molecules
comprising, or alternatively, consisting of, a polynucleotide sequence at
least 90%,
95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding
the
Brainiac-5 polypeptides described above. The present invention also
encompasses the
above nucleic acid molecule sequences fused to a heterologous polynucleotide
sequence. Polypeptides encoded by these nucleic acid molecules are also
provided.
Similarly, many examples of biologically functional C-terminal deletion
muteins are known. For instance, Interferon gamma shows up to ten times higher
activities by deleting 8-10 amino acid residues from the carboxy terminus of
the
protein (Dobeli, et al., J. Biotechnology 7:199-216 ( 1988)).
In the present case, since the Brainiac-5 polypeptide of the invention is a
member of the Brainiac polypeptide family, deletions of C-terminal amino acids
up to
the cysteine at position 263 of 5EQ ID N0:2 may retain some biological
activity such
as the ability to modulate cell growth and differentiation. Polypeptides
having further
C-terminal deletions including the cysteine residue at positian 263 of SEQ ID
N0:2
may not retain such biological activities because this residue is in the
beginning of the
conserved domain required far biological activities.
However, even if deletion of one or more amino acids from the C-terminus of a
protein results in modification of loss of one or more biological functions of
the
protein, other biological activities may still be retained. Thus, the ability
of the
shortened protein to induce and/or bind to antibodies which recognize the
complete or
mature form of the polypeptide generally will be retained when less than the
majority
of the residues of the complete or mature form of the polypeptide are removed
from
the C-terminus. Whether a particular polypeptide lacking C-terminal residues
of a
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complete protein retains such immunologic activities can readily be determined
by
routine methods described herein and otherwise known in the art.
Accordingly, the present invention further provides polypeptides having one or
more residues from the carboxy terminus of the amino acid sequence of the
Brainiac-5
polypeptide shown in SEQ ID N0:2, up to the cysteine residue at position 263
of SEQ
ID N0:2, and polynucleotides encoding such polypeptides. In particular, the
present
invention provides polypeptides having (i.e., comprising, or alternatively
consisting
of) the amino acid sequence of residues 1-m' of the amino acid sequence in SEQ
ID
N0:2, where m' is any integer in the range of 263 to 278, and residue 262 is
the
position of the first residue from the C- terminus of the complete Brainiac-5
polypeptide (shown in SEQ ID N0:2) believed to be required for the cell growth
and
differentiation modulatory activities of the Brainiac-S polypeptide.
More in particular, the invention provides nucleic acid molecules encoding
polypeptides having (i.e., comprising, or alternatively consisting of) the
amino acid
sequence of residues 1-278; 1-277; 1-276; 1-275; 1-274; 1-273; 1-272;1-271; 1-
270;
1-269; 1-268; 1-267; 1-266; 1-265; 1-264; or 1-263 of SEQ ID N0:2. The present
application is also directed to nucleic acid molecules comprising, or
alternatively,
consisting of, a polynucleotide sequence at least 90%, 95%, 96%, 97%, 98% or
99%
identical to the polynucleotide sequence encoding the Brainiac-5 polypeptides
described above. The present invention also encompasses the above
polynucleotide
sequences fused to a heterologous polynucleotide sequence. Polypeptides
encoded by
these nucleic acid molecules are also are provided.
The invention also provides nucleic acid molecules encoding polypeptides
having (i.e., comprising, or alternatively consisting of) one or more amino
acids
deleted from both the amino and the carboxyl termini, which may be described
generally as having residues n'-m' of SEQ ID N0:2, where n' and m' are
integers as
descried above e.g., a polypeptide comprising, or alternatively consisting of,
amino
acids 8 to 263 of SEQ ID NC>:2). The present application is also directed to
nucleic
acid molecules comprising, or alternatively, consisting of, a polynucleotide
sequence
at least 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide
sequence
encoding the Brainiac-5 polypeptides described above. The present invention
also
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encompasses the above poiynucleotide sequences fused to a heterologous
polynucleotide sequence. Polypeptides encoded by these nucleic acid molecules
are
also encompassed by the invention.
Also included are a nucleotide sequence encoding a polypeptide consisting of a
portion of the complete Brainiac-5 amino acid sequence encoded by the cDNA
clone
contained in ATCC Deposit No. 203572, where this portion excludes any integer
from
1 to about 7 amino acids from the amino terminus of the complete amino acid
sequence encoded by the cDNA clone contained in ATCC Deposit No. 203572, or
any
integer from 1 to about 15 amino acids from the carboxy terminus, or any
combination
of the above amino terminal and carboxy terminal deletions, of the complete
amino
acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 203572.
In this context, "about" means the recited value and values that are larger or
smaller by
several, a few, a small number, 5, 4, 3, 2 or 1.
As mentioned above, even if deletion of one or more amino acids from the
N-terminus of a protein results in modification of loss of one or more
functional
activities of the protein (e.g., activation of the Notch signaling pathway,
mediation of
protein-protein interactions (e.g., between Brainiac-5 and Su(H) or a human
homolog
of Su(H)), and/or binding of an antibody specific to Brainiac-5), other
functional
and/or biological activities may still be retained. Thus, the ability of
shortened
Brainiac-5 muteins to induce and/or bind to antibodies which recognize the
complete
or mature forms of the polypeptides generally will be retained when less than
the
majority of the residues of the complete or mature polypeptide are removed
from the
N-terminus. Whether a particular polypeptide lacking N-terminal residues of a
complete polypeptide retains such immunologic activities can readily be
determined
by routine methods described herein and otherwise known in the art. It is not
unlikely
that a Brainiac-5 mutein with a large number of deleted N-terminal amino acid
residues may retain some biological or immunogenic activities. In fact,
peptides
composed of as few as six Brainiac-5 amino acid residues may often evoke an
immune
response.
Accordingly, the present invention further provides polypeptides having one or
more residues deleted from the amino terminus of the Brainiac-5 amino acid
sequence
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shown in Figures lA and 1B (i.e., SEQ ID NO:2), up to the proline residue at
position
number 273 and polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid sequence of
residues n2-278 of Figures lA and 1B (SEQ ID N0:2), where n' is an integer in
the
range of 2 to 273, and 273 is the position of the first residue from the N-
terminus of
the complete Brainiac-5 polypeptide believed to be required for at least
immunogenic
activity of the Brainiac-S polypeptide.
More in particular, the invention provides nucleic acid molecules encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of
residues of A-2 to S-278; E-3 to S-278; D-4 to S-278; F-S to S-278; E-6 to S-
278; R-7
to S-278; R-8 to S-278; Q-~) to S-278; A-10 to S-278; V-11 to S-278; R-12 to S-
278;
Q-13 to S-278; T-14 to S-2'78; W-15 to S-278; G-16 to S-278; A-17 to S-278; E-
18 to
S-278; G-19 to S-278; R-20 to S-278; V-21 to S-278; Q-22 to S-278; G-23 to S-
278;
A-24 to S-278; L-25 to S-278; V-26 to S-278; R-27 to S-278; R-28 to S-278; V-
29 to
S-278; F-30 to S-278; L-31 to S-278; L-32 to S-278; G-33 to S-278; V-34 to S-
278;
P-35 to S-278; R-36 to S-278; G-37 to S-278; A-38 to S-278; G-39 to S-278; S-
40 to
S-278; G-41 to S-278; G-42 to S-278; A-43 to S-278; D-44 to S-278; E-45 to S-
278;
V-46 to S-278; G-47 to S-278; E-48 to S-278; G-49 to S-278; A-50 to S-278; R-
51 to
S-278; T-52 to S-278; H-53 to S-278; W-54 to S-278; R-55 to S-278; A-56 to S-
278;
L-57 to S-278; L-58 to S-278; R-59 to S-278; A-60 to S-278; E-61 to S-278; S-
62 to
S-278; L-63 to S-278; A-64 to S-278; Y-65 to S-278; A-66 to S-278; D-67 to S-
278;
I-68 to S-278; L-69 to S-278; L-70 to S-278; W-71 to S-278; A-72 to S-278; F-
73 to
S-278; D-74 to S-278; D-75 to S-278; T-76 to S-278; F-77 to S-278; F-78 to S-
278;
N-79 to S-278; L-80 to S-278; T-81 to S-278; L-82 to S-278; K-83 to S-278; E-
84 to
S-278; I-85 to S-278; H-86 to S-278; F-87 to S-278; L-88 to S-278; A-89 to S-
278;
W-90 to S-278; A-91 to S-278; S-92 to S-278; A-93 to S-278; F-94 to S-278; C-
95 to
a
S-278; P-96 to S-278; D-97 to S-278; V-98 to S-278; R-99 to S-278; F-100 to S-
278;
V-1 O 1 to S-278; F-102 to S-278; K-103 to S-278; G-104 to S-278; D-1 OS to S-
278;
A-106 to S-278; D-107 to S-278; V-108 to S-278; F-109 to S-278; V-110 to S-
278;
N-111 to S-278; V-112 to S-278; G-113 to S-278; N-114 to S-278; L-115 to S-
278;
L-116 to S-278; E-117 to S-278; F-118 to S-278; L-119 to S-278; A-120 to S-
278;
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P-121 to S-278; R-122 to S-278; D-123 to S-278; P-124 to S-278; A-125 to S-
278;
Q-126 to S-278; D-127 to S-278; L-128 to S-278; L-129 to S-278; A-130 to S-
278;
G-131 to S-278; D-132 to S-278; V-133 to S-278; I-134 to S-278; V-135 to S-
278;
H-136 to S-278; A-137 to S-278; R-138 to S-278; P-139 to S-278; I-140 to S-
278;
R-141 to S-278; T-142 to S-278; R-143 to S-278; A-144 to S-278; S-145 to S-
278;
K-146 to S-278; Y-147 to S-278; Y-148 to S-278; I-149 to S-278; P-I50 to S-
278;
E-151 to S-278; A-152 to S-278; V-153 to S-278; Y-154 to S-278; G-155 to S-
278;
L-156 to S-278; P-157 to S-278; A-158 to S-278; Y-159 to S-278; P-160 to S-
278;
A- I 61 to S-278; Y-162 to S-278; A-163 to S-278; G-164 to S-278; G- I 65 to S-
278;
G-166 to S-278; G-167 to S-278; F-168 to S-278; V-169 to S-278; L-170 to S-
278;
S-171 to S-278; G-172 to S-278; A-173 to S-278; T-174 to S-278; L-175 to S-
278;
H-176 to S-278; R-177 to S-278; L-178 to S-278; A-179 to S-278; G-180 to S-
278;
A-181 to S-278; C-182 to S-278; A-183 to S-278; Q-184 to S-278; V-185 to S-
278;
E-186 to S-278; L-187 to S~-278; F-188 to S-278; P-189 to S-278; I-190 to S-
278;
D-191 to S-278; D-192 to S-278; V-193 to S-278; F-194 to S-278; L-195 to S-
278;
G-196 to S-278; M-197 to S-278; C-198 to S-278; L-199 to S-278; Q-200 to S-
278;
R-201 to S-278; L-202 to S-278; R-203 to S-278; L-204 to S-278; T-205 to S-
278;
P-206 to S-278; E-207 to S-278; P-208 to S-278; H-209 to S-278; P-210 to S-
278;
A-211 to S-278; F-212 to S-278; R-213 to S-278; T-214 to S-278; F-215 to S-
278;
G-216 to S-278; I-217 to S-278; P-218 to S-278; Q-219 to S-278; P-220 to S-
278;
S-221 to S-278; A-222 to S-278; A-223 to S-278; P-224 to S-278; H-225 to S-
278;
L-226 to S-278; S-227 to S-278; T-228 to S-2?8; F-229 to S-278; D-230 to S-
278;
P-231 to S-278; C-232 to S-278; F-233 to S-278; Y-234 to S-278; R-235 to S-
278;
E-236 to S-278; L-237 to S-278; V-238 to S-278; V-239 to S-278; V-240 to S-
278;
H-241 to S-278; G-242 to S-278; L-243 to S-2?8; S-244 to S-278; A-245 to S-
278;
A-246 to S-278; D-247 to S-278; I-248 to S-278; W-249 to S-278; L-250 to S-
278;
M-251 to S-278; W-252 to S-278; R-253 to S-278; L-254 to S-278; L-255 to S-
278;
H-256 to S-278; G-257 to S-278; P-258 to S-278; H-259 to S-278; G-260 to S-
278;
P-261 to S-278; A-262 to S-278; C-263 to S-278; A-264 to S-278; H-265 to S-
278;
P-266 to S-278; Q-267 to S-278; P-268 to S-278; V-269 to S-278; A-270 to S-
278;
A-271 to S-278; G-272 to S-278; or P-273 to S-278 of the Brainiac-5 sequence
shown
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in Figures IA and 1B (SEQ ID N0:2). The present application is also directed
to
nucleic acid molecules comprising, or alternatively, consisting of, a
polynucleotide
sequence at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the
polynucleotide sequence encoding the Brainiac-5 polypeptides described above.
The
present invention also encompasses the above polynucleotide sequences fused to
a
heterologous polynucleotide sequence. Polypeptides encoded by these nucleic
acid
molecules are also encompassed by the invention.
Also as mentioned above, even if deletion of one or more amino acids from the
C-terminus of a protein results in modification of loss of one or more
biological
functions of the protein, other biological activities may still be retained.
Thus, the
ability of the shortened Brainiac-5 mutein to induce and/or bind to antibodies
which
recognize the complete or mature forms of the polypeptide generally will be
retained
when less than the majority of the residues of the complete or mature
polypeptide are
removed from the C-terminus. Whether a particular polypeptide lacking C-
terminal
residues of a complete polypeptide retains such immunologic activities can
readily be
determined by routine methods described herein and otherwise known in the art.
It is
not unlikely that a Brainiac--5 mutein with a large number of deleted C-
terminal amino
acid residues may retain some biological or immunogenic activities. In fact,
peptides
composed of as few as six Brainiac-5 amino acid residues may often evoke an
immune
response.
Accordingly, the present invention further provides polypeptides having one or
more residues deleted from the carboxy terminus of the amino acid sequence of
the
Brainiac-5 polypeptide shown in Figures lA and 1B (SEQ ID N0:2), up to the
glutamic acid residue at position number 6, and polynucleotides encoding such
polypeptides. In particular, the present invention provides polypeptides
comprising
the amino acid sequence of residues 1-mz of Figures lA and 1B (SEQ ID N0:2),
where m'- is an integer in the range of 6 to 278, and 6 is the position of the
first residue
from the C-terminus of the complete Brainiac-5 polypeptide believed to be
required
for at least immunogenic activity of the Brainiac-5 polypeptide.
More in particular, the invention provides nucleic acid molecules encoding
polypeptides comprising, or alternatively consisting of, the amino acid
sequence of
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residues V-1 to D-277; V-1 to W-276; V-I to Q-275; V-1 to F-274; V-1 to P-273;
V-1
to G-272; V-1 to A-271; V-1 to A-270; V-1 to V-269; V-1 to P-268; V-1 to Q-
267;
V-I to P-266; V-I to H-265; V-1 to A-264; V-1 to C-263; V-1 to A-262; V-1 to P-
261;
V-1 to G-260; V-1 to H-259; V-1 to P-258; V-1 to G-257; V-1 to H-256; V-I to
L-255; V-l to L-254; V-t to R-253; V-1 to W-252; V-1 to M-251; V-1 to L-250; V-
1
to W-249; V-1 to I-248; V-1 to D-247; V-1 to A-246; V-1 to A-245; V-1 to S-
244;
V-1 to L-243; V-1 to G-242; V-1 to H-241; V-1 to V-240; V-1 to V-239; V-I to
V-238; V-1 to L-237; V-:l to E-236; V-1 to R-235; V-1 to Y-234; V-1 to F-233;
V-1 to
C-232; V-1 to P-231; V-1 to D-230; V-1 to F-229; V-1 to T-228; V-1 to S-227; V-
1 to
L-226; V-1 to H-225; V-1 to P-224; V-1 to A-223; V-1 to A-222; V-1 to S-221; V-
1 to
P-220; V-1 to Q-219; V-1 to P-218; V-1 to I-217; V-I to G-216; V-I to F-215; V-
1 to
T-214; V-1 to R-213; V-1 to F-212; V-1 to A-211; V-1 to P-210; V-1 to H-209; V-
1 to
P-208; V-1 to E-207; V-1 to P-206; V-1 to T-205 ; V-1 to L-204; V-1 to R-203;
V-1 to
L-202; V-1 to R-201; V-1 to Q-200; V-1 to L-199; V-1 to C- I 98; V-1 to M-197;
V-1
to G-196; V-1 to L-195; V-I to F-194; V-1 to V-193; V-1 to D-192; V-I to D-
191;
V-1 to I-190; V-1 to P-189; V-1 to F-188; V-1 to L-187; V-1 to E-186; V-1 to V-
185;
V-1 to Q-184; V-1 to A-183; V-1 to C-182; V-1 to A-181; V-1 to G-180; V-1 to
A-179; V-1 to L-178; V-1 to R-177; V-1 to H-176; V-1 to L-175; V-1 to T-174; V-
I to
A-173; V-1 to G-172; V-1 to S-171; V-1 to L-170; V-1 to V-169; V-1 to F-168; V-
1 to
G-167; V-1 to G-166; V-1 to G-165; V-1 to G-164; V-1 to A-163; V-1 to Y-162; V-
1
to A-161; V-1 to P-160; V-1 to Y-159; V-1 to A-158; V-1 to P-157; V-1 to L-
156; V-I
to G-155; V-1 to Y-154; V-1 to V-153; V-1 to A-152; V-1 to E-151; V-1 to P-
150;
V-I to I-149; V-I to Y-148; V-1 to Y-147; V-1 to K-146; V-1 to S-145; V-1 to A-
144;
V-1 to R-143; V-1 to T-142; V-1 to R-141; V-I to I-140; V-1 to P-139; V-1 to R-
138;
V-1 to A-137; V-1 to H-136; V-1 to V-135; V-1 to I-134; V-1 to V-133; V-1 to D-
132;
V-1 to G-131; V-I to A-130; V-1 to L-129; V-1 to L-128; V-1 to D-127; V-1 to
Q-126; V-1 to A-125; V-1 to P-124; V-1 to D-123; V-1 to R-122; V-1 to P-121; V-
1 to
A-120; V-1 to L-1 I9; V-1 to :F-118; V-1 to E-117; V-1 to L-116; V-I to L-115;
V-1 to
N-114; V-1 to G-113; V-1 to V-112; V-1 to N-111; V-1 to V-110; V-1 to F-109; V-
1
to V-108; V-1 to D-107; V-1 to A-106; V-1 to D-105; V-1 to G-104; V-1 to K-
103;
V-1 to F-102; V-1 to V-101; V-1 to F-100; V-1 to R-99; V-1 to V-98; V-1 to D-
97;
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V-1 to P-96; V-1 to C-95; 'V-1 to F-94; V-I to A-93; V-1 to S-92; V-I to A-91;
V-1 to
W-90; V-1 to A-89; V-1 to L-88; V-1 to F-87; V-1 to H-86; V-1 to I-85; V-1 to
E-84;
V-1 to K-83; V-1 to L-82; 'V-1 to T-81; V-1 to L-80; V-1 to N-79; V-1 to F-78;
V-1 to
F-77; V-1 to T-76; V-1 to D-75; V-1 to D-74; V-1 to F-73; V-1 to A-72; V-1 to
W-71;
V-1 to L-70; V-1 to L-69; V-1 to I-68; V-1 to D-67; V-1 to A-66; V-1 to Y-65;
V-1 to
A-64; V-1 to L-63; V-1 to S-62; V-I to E-61; V-1 to A-60; V-1 to R-59; V-1 to
L-58;
V-1 to L-57; V-1 to A-56; V-1 to R-55; V-1 to W-54; V-1 to H-53; V-I to T-52;
V-1
to R-51; V-1 to A-50; V-I to G-49; V-1 to E-48; V-1 to G-47; V-1 to V-46; V-1
to
E-45; V-1 to D-44; V-1 to A-43; V-1 to G-42; V-1 to G-41; V-I to S-40; V-I to
G-39;
V-1 to A-38; V-1 to G-37; V-1 to R-36; V-I to P-35; V-1 to V-34; V-I to G-33;
V-1 to
L-32; V-1 to L-31; V-1 to F-30; V-1 to V-29; V-1 to R-28; V-1 to R-27; V-1 to
V-26;
V-1 to L-25; V-1 to A-24; V-1 to G-23; V-1 to Q-22; V-1 to V-21; V-1 to R-20;
V-1
to G-19; V-I to E-18; V-1 to A-17; V-1 to G-16; V-1 to W-15; V-1 to T-14; V-1
to
Q-13; V-1 to R-12; V-1 to V-11; V-1 to A-10; V-1 to Q-9; V-I to R-8; V-1 to R-
7; or
V-I to E-6 of the sequence of the Brainiac-5 sequence shown in Figures IA and
1B
(SEQ ID N0:2). The present application is also directed to nucleic acid
molecules
comprising, or alternatively, consisting of, a polynucleotide sequence at
least 90%,
92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence
encoding
the Brainiac-5 polypeptides described above. The present invention also
encompasses
the above polynucleotide sequences fused to a heterologous polynucleotide
sequence.
Polypeptides encoded by these nucleic acid molcules are also provided.
The invention also provides nucleic acid molcules encoding polypeptides
having (i.e., comprising, or alternatively consisting of) one or more amino
acids
deleted from both the amino and the carboxyl termini of a Brainiac-5
polypeptide,
which may be described generally as having residues n'-rn2 of Figures lA and
1B
(SEQ ID N0:2), where n2 and m'- are integers as described above. The present
application is also directed to nucleic acid molecules comprising, or
alternatively,
consisting of, a polynucleotide sequence at least 90%, 92%, 95%, 96%, 97%, 98%
or
99% identical to the polynucleotide sequence encoding the Brainiac-5
polypeptides
described above. The present invention also encompasses the above
polynucleotide
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sequences fused to a heterologous polynucleotide sequence. Polypeptides
encoded by
these nucleic acid molecules are also provided by the invention.
In addition to terminal deletion forms of the protein discussed above, it also
will be recognized by one of ordinary skill in the art that some amino acid
sequences
of the Brainiac-5 polypeptides can be varied without significant effect of the
structure
or function of the protein. :ff such differences in sequence are contemplated,
it should
be remembered that there will be critical areas on the protein which determine
activity.
Thus, the invention further includes variations of the Brainiac-5 polypeptides
which show substantial Brainiac-5 polypeptide activity or which include
regions of
Brainiac-5 polypeptides such as the polypeptide portions discussed below. Such
mutants include deletions, insertions, inversions, repeats, and type
substitutions
selected according to general rules known in the art so as have little effect
on activity.
For example, guidance concerning how to make phenotypically silent amino acid
substitutions is provided wherein the authors indicate that there are two main
approaches for studying the tolerance of an amino acid sequence to change
(Bowie, J.
U., et al., Science 247:1306-1310 (1990)),. The first method relies on the
process of
evolution, in which mutations are either accepted or rejected by natural
selection. The
second approach uses genetic engineering to introduce amino acid changes at
specific
positions of a cloned gene and selections or screens to identify sequences
that maintain
functionality.
As the authors state, these studies have revealed that proteins are
surprisingly
tolerant of amino acid substitutions. The authors further indicate which amino
acid
changes are likely to be permissive at a certain position of the protein. For
example,
most buried amino acid residues require nonpolar side chains, whereas few
features of
surface side chains are generally conserved. Other such phenotypically silent
substitutions are described by Bowie and coworkers (supra) and the references
cited
therein. Typically seen as conservative substitutions are the replacements,
one for
another, among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of
the
hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu,
substitution between the amide residues Asn and Gln, exchange of the basic
residues
Lys and Arg and replacements among the aromatic residues Phe, Tyr.
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Thus, the fragment, derivative or analog of the polypeptide of SEQ ID N0:2,
or those encoded by the deposited cDNA, may be (i) one in which one or more of
the
amino acid residues are substituted with a conserved or non-conserved amino
acid
residue (preferably a conserved amino acid residue) and such substituted amino
acid
S residue may or may not be one encoded by the genetic code, or (ii) one in
which one
or more of the amino acid residues includes a substituent group, or (iii) one
in which
either the Brainiac-5 mature polypeptide is fused with another compound, such
as a
compound to increase the half life of the polypeptide (for example,
polyethylene
glycol), or (iv) one in which the additional amino acids are fused to the
above form of
the polypeptide, such as an IgG Fc fusion region peptide or leader or
secretory
sequence or a sequence which is employed for purification of the above form of
the
polypeptide or a proprotein sequence. Such fragments, derivatives and analogs
are
deemed to be within the scope of those skilled in the art from the teachings
herein.
Thus, the Brainiac-S polypeptides of the present invention may include one or
more amino acid substitutions, deletions or additions, either from natural
mutations or
human manipulation. As indicated, changes are preferably of a minor nature,
such as
conservative amino acid substitutions that do not significantly affect the
folding or
activity of the protein (see Table I:I).
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TABLE II. Conservative Amino Acid Substitutions.
Aromatic Phenylalanine
Tryptophan
Tyrosine
Hydrophobic Leucine
Isoleucine
Va:line
Polar ~ Glutamine
Asparagine
Basic Arginine
Lysine
Histidine
Acidic ~ Aspartic Acid
Glutamic Acid
Small Alanine
Serine
Threonine
Methionine
GI tine
Amino acids in the Brainiac-5 polypeptides of the present invention that are
essential for function can be identified by methods known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and
Wells,
Science 244:1081-1085 ( 1989)). The latter procedure introduces single alanine
mutations at every residue in the molecule. The resulting mutant molecules are
then
tested for biological activity such as receptor binding or in vitro
proliferative activity.
Of special interest are substitutions of charged amino acids with other
charged
or neutral amino acids which may produce proteins with highly desirable
improved
characteristics, such as less aggregation. Aggregation may not only reduce
activity but
also be problematic when preparing pharmaceutical formulations, because
aggregates
can be immunogenic (Pinckard, et al., Clin. Exp. Immunol. 2:331-340 (1967);
Robbins, et al., Diabetes 36:838-845 ( 1987); Cleland, et al., Crit. Rev.
Therapeutic
Drug Carrier Systems 10:307-377 (1993)).
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Replacement of amino acids can also change the selectivity of the binding of a
ligand to cell surface receptors (for example, Ostade, et al., Nature 361:266-
268
( 1993)) describes certain mutations resulting in selective binding of TNF-
alpha to only
one of the two known types of TNF receptors. Sites that are critical for
ligand-
receptor binding can also be determined by structural analysis such as
crystallization,
nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol.
Biol.
224:899-904 ( 1992); de Vos, et al. Science 255:306-312 ( 1992)).
Thus, the invention also encompasses Brainiac-5 derivatives and analogs that
have one or more amino acid residues deleted, added, or substituted to
generate
Brainiac-5 polypeptides that are better suited for expression, scale up, etc.,
in the host
cells chosen. For example, cysteine residues can be deleted or substituted
with another
amino acid residue in order to eliminate disulfide bridges; N-linked
glycosylation
sites can be altered or eliminated to achieve, for example, expression of a
homogeneous product that is more easily recovered and purified from yeast
hosts
which are known to hyperglycosylate N-linked sites. To this end, a variety of
amino
acid substitutions at one or both of the first or third amino acid positions
on any one or
more of the glycosylation recognitions sequences in the Brainiac-5
polypeptides of the
invention, and/or an amino acid deletion at the second position of any one or
more
such recognition sequences will prevent glycosylation of the Brainiac-5 at the
modified tripeptide sequence (see, e.g., Miyajimo et al., EMBO J 5(6):1193-
1197).
Additionally, one or more of the amino acid residues of the polypeptides of
the
invention (e.g., arginine and lysine residues) may be deleted or substituted
with
another residue to elminate undesired processing by proteases such as, for
example,
furins or kexins.
The polypeptides of the present invention are preferably provided in an
isolated
form, and preferably are substantially purified. Recombinantly produced
versions of
the Brainiac-5 polypeptides can be substantially purified by the one-step
method
described by Smith and Johnson (Gene 67:31-40 ( 1988)). Polypeptides of the
invention also can be purified from natural or recombinant sources using anti-
Brainiac-5 antibodies of the invention in methods which are well known in the
art of
protein purification.
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Recombinant DNA technology known to those skilled in the art (see, for
instance, DNA shuffling supra) can be used to create novel mutant proteins or
muteins
including single or multiple amino acid substitutions, deletions, additions or
fusion
proteins. Such modified polypeptides can show, e.g., enhanced activity or
increased
S stability. In addition, they may be purified in higher yields and show
better solubility
than the corresponding natural polypeptide, at least under certain
purification and
storage conditions.
The invention also provides an isolated Brainiac-5 polypeptide comprising, or
alternatively consisting of, an amino acid sequence selected from the group
consisting
of: (a) the amino acid sequence of the Brainiac-5 polypeptide having the
complete
amino acid sequence shown in SEQ ID N0:2 (i.e., positions 1-278 of SEQ ID
N0:2);
(b) the complete amino acid sequence encoded by the eDNA clone contained in
the
ATCC Deposit No. 203572; (c) the complete amino acid sequence of the predicted
mature Brainiac-5 polypeptide encoded by the cDNA clone contained in the ATCC
I S Deposit No. 203572. The polypeptides of the present invention also include
polypeptides having an amino acid sequence at least 80% identical, more
preferably at
least 90% identical, and still more preferably 92%, 95%, 96%, 97%, 98% or 99%
identical to those described in (a), (b) or (c), above, as well as
polypeptides having an
amino acid sequence with at least 90% similarity, and more preferably at least
95%
similarity, to those above.
Further polypeptides of the present invention include polypeptides which have
(i.e., comprise, or alternatively consist of) at least 90% similarity, more
preferably at
least 92% similarity, more preferably at least 95% similarity, and still more
preferably
at least 96%, 97%, 98% or 99% similarity to those described above. The
polypeptides
of the invention also comprise those which are at least 80% identical, more
preferably
at least 90%, 92% or 95% identical, still more preferably at least 96%, 97%,
98% or
99% identical to the polypeptide encoded by the deposited cDNA or to the of a
tide
P YP P
of SEQ ID N0:2, and also include portions of such polypeptides with at least
15
amino acids, more preferably at least 30 amino acids, even more preferably at
least 40
amino acids, still even more preferably at least 50 amino acids, still more
preferably at
least 60 amino acids, and yet even more preferably at least 75 amino acids.
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A further embodiment of the invention relates to a peptide or polypeptide
which comprises the amino acid sequence of a Brainiac-5 polypeptide having an
amino acid sequence which contains at least one conservative amino acid
substitution,
but not more than 50 conservative amino acid substitutions, even more
preferably, not
more than 40 conservative amino acid substitutions, still more preferably, not
more
than 30 conservative amino acid substitutions, and still even more preferably,
not more
than 20 conservative amino acid substitutions. Of course, in order of ever-
increasing
preference, it is highly preferable for a peptide or polypeptide to have an
amino acid
sequence which comprises the amino acid sequence of a Brainiac-S polypeptide,
which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
conservative
amino acid substitutions.
Representative examples of polypeptide fragments of the invention, include,
for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80,
81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-
260,
261-278 or 261 to the end of the coding region of SEQ ID N0:2 or to a
polypeptide
expressed from the deposited cDNA clone which expresses Brainiac-5. Moreover,
polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,
120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or 270 amino
acids in
length. In this context "about" includes the particularly recited ranges or
values, and
ranges or values larger or smaller by several (5, 4, 3, 2, or 1 ) amino acids,
at either
extreme or at both extremes. The invention also provides an isolated
polypeptide
comprising an amino acid sequence at least 90% identical to a sequence of at
least
about 10, 30 or 100 contiguous amina acids in the amino acid sequence of SEQ
ID
N0:2.
By "% similarity" for two polypeptides is intended a similarity score produced
by comparing the amino acid sequences of the two polypeptides using the
Bestfit
a
program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, 575 Science Drive, Madison, WI
53711)
and the default settings for determining similarity. Bestfit uses the local
homology
algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489,
1981 ) to find the best segment of similarity between two sequences.
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In one embodiment of the invention, by a polypeptide having an amino acid
sequence at least, for example, 95% "identical" to a reference amino acid
sequence of
a Brainiac-5 polypeptide is intended that the amino acid sequence of the
polypeptide is
identical to the reference sequence except that the polypeptide sequence may
include
up to five amino acid alterations per each 100 amino acids of the reference
amino acid
of the Brainiac-5 polypeptide. In other words, to obtain a polypeptide having
an
amino acid sequence at least 95% identical to a reference amino acid sequence,
up to
5% of the amino acid residues in the reference sequence may be deleted or
substituted
with another amino acid, or a number of amino acids up to 5% of the total
amino acid
residues in the reference sequence rnay be inserted into the reference
sequence. These
alterations of the reference sequence may occur at the amino or carboxy
terminal
positions of the reference amino acid sequence or anywhere between those
terminal
positions, interspersed either individually among residues in the reference
sequence or
in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 90%,
92%~,
95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence
shown in Figures lA and LB (SEQ ID N0:2), the amino acid sequence encoded by
deposited cDNA clone HOGCC45, or fragments thereof, can be determined
conventionally using known computer programs such the Bestfit program
(Wisconsin
Sequence Analysis Package, Version 8 for LJnix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, WI 53711 ). When using
Bestfit or any other sequence alignment program to determine whether a
particular
sequence is, for instance, 95% identical to a reference sequence according to
the
present invention, the parameters are set, of course, such that the percentage
of identity
is calculated over the full length of the reference amino acid sequence and
that gaps in
homology of up to 5% of the total number of arriino acid residues in the
reference
sequence are allowed.
In a specific embodiment, the identity between a reference (query) sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, is determined using the FASTDB computer program based on
the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred
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parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2,
Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff
Score=l, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05,
Window Size=500 or the length of the subject amino acid sequence, whichever is
shorter. According to this embodiment, if the subject sequence is shorter than
the
query sequence due to N- or C-terminal deletions, not because of internal
deletions, a
manual correction is made to the results to take into consideration the fact
that the
FASTDB program does not account for N- and C-terminal truncations of the
subject
sequence when calculating global percent identity. For subject sequences
truncated at
the N- and C-termini, relative to the query sequence, the percent identity is
corrected
by calculating the number of residues of the query sequence that are N- and C-
terminal
of the subject sequence, which are not matched/aligned with a corresponding
subject
residue, as a percent of the total bases of the query sequence. A
determination of
whether a residue is matched/aligned is determined by results of the FASTDB
sequence alignment. This percentage is then subtracted from the percent
identity,
calculated by the above FASTDB program using the specified parameters, to
arrive at
a final percent identity score. This final percent identity score is what is
used for the
purposes of this embodiment. Only residues to the N- and C-termini of the
subject
sequence, which are not matched/aligned with the query sequence, are
considered for
the purposes of manually adjusting the percent identity score. That is, only
query
residue positions outside the farthest N- and C-terminal residues of the
subject
sequence. For example, a 90 amino acid residue subject sequence is aligned
with a
100 residue query sequence to determine percent identity. The deletion occurs
at the
N-terminus of the subject sequence and therefore, the FASTDB alignment does
not
show a matching/alignment of the first 10 residues at the N-terminus. The 10
unpaired
residues represent 10% of the sequence (number of residues at the N- and C-
termini
not matched/total number of residues in the query sequence) so 10% is
subtracted from
the percent identity score calculated by the FASTDB program. If the remaining
90
residues were perfectly matched the final percent identity would be 90%. In
another
example, a 90 residue subject sequence is compared with a 100 residue query
sequence. This time the deletions are internal deletions so there are no
residues at the
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N- or C-termini of the subject sequence which are not matched/aligned with the
query.
In this case the percent identity calculated by FASTDB is not manually
corrected.
Once again, only residue positions outside the N- and C-terminal ends of the
subject
sequence, as displayed in the FASTDB alignment, which are not matched/aligned
with
the query sequence are manually corrected for. No other manual corrections are
made
for the purposes of this embodiment.
The polypeptide of the present invention could be used, for example, as a
molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration
columns using methods well known to those of skill in the art.
As described in detail below, the polypeptides of the present invention can
also
be used to raise polyclonal and monoclonal antibodies, which are useful in
assays for
detecting Brainiac-5 polypeptide expression as described below or as agonists
and
antagonists capable of enhancing or inhibiting Brainiac-5 polypeptide
function.
Further, such polypeptides can be used in the yeast two-hybrid system to
"capture"
Brainiac-S polypeptide-binding polypeptides which are also candidate agonists
and
antagonists according to the present invention. The yeast two hybrid system is
described by Fields and Sonl; (Nature 340:245-246 (1989)).
Transgenics and "knock-outs"
The polypeptides of the invention can also be expressed in transgenic animals.
Animals of any species, including, but not limited to, mice, rats, rabbits,
hamsters,
guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates,
e.g.,
baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
In a
specific embodiment, techniques described herein or otherwise known in the
art, are
used to express polypeptides of the invention in humans, as part of a gene
therapy
protocol.
Any technique known in the art may be used to introduce the transgene (i.e.,
polynucleotides of the invention) into animals to produce the founder lines of
transgenic animals. Such techniques include, but are not limited to,
pronuclear
microinjection (Paterson, et al., Appl. Microbiol. Biotechnol. 40:691-698
(1994);
Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al.,
Biotechnology
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(NY) 9:830-834 ( 1991 ); and Hoppe et al., U.S. Pat. No. 4,873,191 ( 1989));
retrovirus
mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl.
Acad. Sci.,
USA 82:6148-6152 ( 1985);1, blastocysts or embryos; gene targeting in
embryonic stem
cells (Thompson et al., Cell 56:313-321 ( 1989)); electroporation of cells or
embryos
(Lo, 1983, Mol Cell. Biol. 3:1803-1814 ( 1983)); introduction of the
polynucleotides of
the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (
1993);
introducing nucleic acid constructs into embryonic pleuripotent stem cells and
transferring the stem cells back into the blastocyst; and sperm-mediated gene
transfer
(Lavitrano et al., Cell 57:71?-723 ( 1989); etc. For a review of such
techniques, see
Gordon, "Transgenic Animals," Intl. Rev. Cytol. 115:171-229 (1989), which is
incorporated by reference herein in its entirety. See also, U.S. Patent No.
5,464,764
(Capecchi, et al., Positive-Negative Selection Methods and Vectors); U.S.
Patent No.
5,631,153 (Capecchi, et aL, Cells and Non-Human Organisms Containing
Predetermined Genomic Modifications and Positive-Negative Selection Methods
and
Vectors for Making Same); U.S. Patent No. 4,736,866 (Leder, et al., Transgenic
Non-
Human Animals); and U.S. Patent No. 4,873,191 (Wagner, et al., Genetic
Transformation of Zygotes); each of which is hereby incorporated by reference
in its
entirety.
Any technique known in the art may be used to produce transgenic clones
containing polynucleotides of the invention, for example, nuclear transfer
into
enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells
induced to
quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature
385:810-
8 I 3 ( 1997)).
The present invention provides for transgenic animals that carry the transgene
in all their cells, as well as animals which carry the transgene in some, but
not all their
cells, i.e., mosaic or chimeric animals. The transgene may be integrated as a
single
6
transgene or as multiple copies such as in concatamers, e.g., head-to-head
tandems or
head-to-tail tandems. The transgene may also be selectively introduced into
and
activated in a particular cell type by following, for example, the teaching of
Lasko et
al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 ( 1992)). The
regulatory
sequences required for such a cell-type specific activation will depend upon
the
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particular cell type of interest, and will be apparent to those of skill in
the art. When it
is desired that the polynucle.otide transgene be integrated into the
chromosomal site of
the endogenous gene, gene targeting is preferred. Briefly, when such a
technique is to
be utilized, vectors containing some nucleotide sequences homologous to the
endogenous gene are designed for the purpose of integrating, via homologous
recombination with chromosomal sequences, into and disrupting the function of
the
nucleotide sequence of the endogenous gene. The transgene may also be
selectively
introduced into a particular cell type, thus inactivating the endogenous gene
in only
that cell type, by following, :for example, the teaching of Gu et al. (Gu et
al., Science
265:103-106 (1994)). The regulatory sequences required for such a cell-type
specific
inactivation will depend upon the particular cell type of interest, and will
be apparent
to those of skill in the art. In addition to expressing the polypeptide of the
present
invention in a ubiquitous or tissue specific manner in transgenic animals, it
would also
be routine for one skilled in the art to generate constructs which regulate
expression of
the polypeptide by a variety of other means (for example, developmentally or
chemically regulated expression).
Once transgenic animals have been generated, the expression of the
recombinant gene may be assayed utilizing standard techniques. Initial
screening may
be accomplished by Southern blot analysis or PCR techniques to analyze animal
tissues to verify that integration of the transgene has taken place. The level
of mRNA
expression of the transgene in the tissues of the transgenic animals may also
be
assessed using techniques which include, but are not limited to, Northern blot
analysis
of tissue samples obtained from the animal, in situ hybridization analysis,
and reverse
transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may
also
be evaluated immunocytochemically or immunohistochemically using antibodies
specific for the transgene product.
Once the founder animals are produced, they may be bred, inbred, outbred, or
crossbred to produce colonies of the particular animal. Examples of such
breeding
strategies include, but are not limited to: outbreeding of founder animals
with more
than one integration site in order to establish separate lines; inbreeding of
separate
lines in order to produce compound transgenics that express the transgene at
higher
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levels because of the effects of additive expression of each transgene;
crossing of
heterozygous transgenic animals to produce animals homozygous for a given
integration site in order to both augment expression and eliminate the need
for
screening of animals by DNA analysis; crossing of separate homozygous lines to
S produce compound heterozygous or homozygous lines; and breeding to place the
transgene on a distinct background that is appropriate for an experimental
model of
interest.
Transgenic and "knock-out" animals of the invention have uses which include,
but are not limited to, animal model systems useful in elaborating the
biological
function of Brainiac-S polypeptides, studying conditions and/or disorders
associated
with aberrant Brainiac-S expression, and in screening for compounds effective
in
ameliorating such conditions and/or disorders.
In further embodiments of the invention, cells that are genetically engineered
to express the polypeptides of the invention, or alternatively, that are
genetically
1S engineered not to express the polypeptides of the invention (e.g.,
knockouts) are
administered to a patient in vivo. Such cells may be obtained from the patient
(i.e.,
animal, including human) or an MIiC compatible donor and can include, but are
not
limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes),
adipocytes,
muscle cells, endothelial cells etc. The cells are genetically engineered in
vitro using
recombinant DNA techniques to introduce the coding sequence of polypeptides of
the
invention into the cells, or alternatively, to disrupt the coding sequence
and/or
endogenous regulatory sequence associated with the polypeptides of the
invention,
e.g., by transduction (using viral vectors, and preferably vectors that
integrate the
transgene into the cell genome) or transfection procedures, including, but not
limited
2S to, the use of plasmids, cosmids, YACs, naked DNA, electroporation,
liposomes, etc.
The coding sequence of the polypeptides of the invention can be placed under
the
S
control of a strong constitutive or inducible promoter or promoter/enhancer to
achieve
expression, and preferably secretion, of the polypeptides of the invention.
The
engineered cells which express and preferably secrete the polypeptides of the
invention can be introduced into the patient systemically, e.g., in the
circulation, or
intraperitoneally.
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Alternatively, the cells can be incorporated into a matrix and implanted in
the
body, e.g., genetically engineered fibroblasts can be implanted as part of a
skin graft;
genetically engineered endathelial cells can be implanted as part of a
lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Patent No. 5,399,349;
and
Mulligan & Wilson, U.S. Patent No. 5,460,959 each of which is incorporated by
reference herein in its entire.ty).
When the cells to be administered are non-autologous or non-MHC compatible
cells, they can be administered using well known techniques which prevent the
development of a host immune response against the introduced cells. For
example, the
cells may be introduced in an encapsulated form which, while allowing for an
exchange of components with the immediate extracellular environment, does not
allow
the introduced cells to be recognized by the host immune system.
Antibodies
The present invention further relates to antibodies and T-cell antigen
receptors
(TCR) which immunospecifically bind a polypeptide, preferably an epitope, of
the
present invention (as determined by immunoassays well known in the art for
assaying
specific antibody-antigen binding). Antibodies of the invention include, but
are not
limited to, polyclonal, monoclonal, multispecific, human, humanized or
chimeric
antibodies, single chain antibodies, Fab fragments, F(ab') fragments,
fragments
produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies
(including,
e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding
fragments
of any of the above. The terns "antibody," as used herein, refers to
immunoglobulin
molecules and immunologically active portions of immunoglobulin molecules,
i.e.,
molecules that contain an antigen binding site that immunospecifically binds
an
antigen. The immunoglobulin molecules of the invention can be of any type
(e.g.,
IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI, IgG2, IgG3, IgG4, IgAI
and
IgA2) or subclass of immunoglobulin molecule.
Most preferably the antibodies are human antigen-binding antibody fragments
of the present invention and include, but are not limited to, Fab, Fab' and
F(ab')2, Fd,
single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv)
and
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fragments comprising either a VL or VH domain. Antigen-binding antibody
fragments, including single-chain antibodies, may comprise the variable
region{s)
alone or in combination with the entirety or a portion of the following: hinge
region,
CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding
S fragments also comprising any combination of variable regions) with a hinge
region,
CHI, CH2, and CH3 domains. The antibodies of the invention may be from any
animal origin including birds and mammals. Preferably, the antibodies are
human,
murine, donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As
used
herein, "human" antibodies include antibodies having the amino acid sequence
of a
human immunoglobulin and include antibodies isolated from human immunoglobulin
libraries or from animals transgenic for one or more human immunoglobulin and
that
do not express endogenous immunoglobulins, as described infra and, for example
in,
U.S. Patent No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific,
I S trispecific or of greater multispecificity. Multispecific antibodies may
be specific for
different epitopes of a golypeptide of the present invention or may be
specific fir both
a polypeptide of the present invention as well as for a heterologous epitope,
such as a
heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/I771S; WO 92/08802; WO 91/00360; WO 92/OS793; Tutt, et al., J. Immunol.
147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; S_573,920;
5,601,819; Kostelny et al., J. Immunol. 148:1547-ISS3 (1992).
Antibodies of the present invention may be described or specified in terms of
the epitope(s) or portions) of a polypeptide of the present invention that
they
recognize or specifically bind. The epitope(s) or polypeptide portions) may be
2S specified as described herein, e.g., by N-terminal and C-terminal
positions, by size in
contiguous amino acid residues, or listed in the Tables and Figures.
Antibodies that
specifically bind any epitope or polypeptide of the present invention may also
be
excluded. Therefore, the present invention includes antibodies that
specifically bind
polypeptides of the present invention, and allows for the exclusion of the
same.
Antibodies of the present invention may also be described or specified in
terms
of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or
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homolog of a polypeptide of the present invention are included. Antibodies
that bind
polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at
least 75%,
at least 70%, at least 65°l0, at least 60%, at least 55%, and at least
50% identity (as
calculated using methods known in the art and described herein) to a
polypeptide of
S the present invention are also included in the present invention. Antibodies
that do not
bind polypeptides with less than 95%, less than 90%, less than 85%, less than
80%,
less than 75%, less than 70%, less than 65%, less than 60%, less than SS%, and
less
than 50% identity (as calculated using methods known in the art and described
herein)
to a polypeptide of the present invention are also included in the present
invention.
Further included in the present invention are antibodies that bind
polypeptides encoded
by polynucleotides which hybridize to a polynucleotide of the present
invention under
stringent hybridization conditions (as described herein). Antibodies of the
present
invention may also be described or specified in terms of their binding
affinity to a
polypeptide of the invention. Preferred binding affinities include those with
a
dissociation constant or Kd less than SX 10~2M, l O~zM, SX 10-3M, 10-~M, SX
10~4M, 10-
~M, SX 10~5M, 10-SM, SX 10~°M, 10-6M, SX 10~'M, 10~'M, SX 10-gM, 10~$M,
SX 10'9M, 10-
9M, SX10-'°M, 10-'°M, SX10-"M, 10-"M, SX10~'ZM, 10-'ZM, SX10~"M,
10-'3M, SX10-
'4M, 10-'''M, SX10~'SM, and 10-'SM.
The invention also provides antibodies that competitively inhibit binding of
an
antibody to an epitope of the invention as determined by any method known in
the art
for deternuning competitive binding, for example, the immunoassays described
herein.
In preferred embodiments, the antibody competitively inhibits binding to the
epitope
by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
Antibodies of the present invention may act as agonists or antagonists of the
polypeptides of the present invention. For example, the present invention
includes
antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the
invention either partially or fully. The invention features both receptor-
specific
antibodies and ligand-specific antibodies. The invention also features
receptor-
specific antibodies which do not prevent ligand binding but prevent receptor
activation. Receptor activation (i.e., signaling) may be determined by
techniques
described herein or otherwise known in the art. For example, receptor
activation can
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be determined by detecting the phosphorylation (e.g., tyrosine or
serine/threonine) of
the receptor or its substrate by immunoprecipitation followed by western blot
analysis
(for example, as described supra). In specific embodiments, antibodies are
provided
that inhibit ligand or receptor activity by at least 90%, at least 80%, at
least 70%, at
least 60%, or at least 50% of the activity in absence of the antibody.
The invention also features receptor-specific antibodies which both prevent
ligand binding and receptor activation as well as antibodies that recognize
the
receptor-ligand complex, and, preferably, do not specifically recognize the
unbound
receptor or the unbound ligand. Likewise, included in the invention are
neutralizing
antibodies which bind the ligand and prevent binding of the ligand to the
receptor, as
well as antibodies which bind the ligand, thereby preventing receptor
activation, but
do not prevent the ligand from binding the receptor. Further included in the
invention
are antibodies which activate the receptor. These antibodies may act as
receptor
agonists, i.e., potentiate or activate either all or a subset of the
biological activities of
the ligand-mediated receptor activation. The antibodies may be specified as
agonists,
antagonists or inverse agonists for biological activities comprising the
specific
biological activities of the peptides of the invention disclosed herein. The
above
antibody agonists can be made using methods known in the art. See, e.g., PCT
publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood
92(6):1981-
1988 ( 1998); Chen, et al., Cancer Res. 58( 16):3668-3678 ( 1998); Harrop et
al., J.
Immunol. 161(4):1786-1794 (I998); Zhu et al., Cancer Res. 58(15):3209-3214
( 1998); Yoon, et al., J. Immunol. 160(7):3170-3179 ( 1998); Prat et al., J.
Cell. Sci.
1 I 1 (Pt2):237-247 ( 1998); Pitard et al., J. Immunol. Methods 205(2):177-190
( 1997);
Liautard et al., Cytokine 9(4):233-241 ( 1997); Carlson et al., J. Biol. Chem.
272( 17):11295-11301 ( 1997); Taryman et al., Neuron 14(4):755-762 ( 1995);
Muller
et al., Structure 6(9):1153-1167 ( 1998); Bartunek et al., Cytokine 8( 1 ):14-
20 ( 1996)
(which are all incorporated by reference herein in their entiretiesj.
Antibodies of the present invention may be used, for example, but riot limited
to, to purify, detect, and target the polypeptides of the present invention,
including
both in vitro and in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and quantitatively
measuring
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levels of the polypeptides of the present invention in biological samples.
See, e.g.,
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
As discussed in more detail below, the antibodies of the present invention may
be used either alone or in combination with other compositions. The antibodies
may
further be recombinantly fused to a heterologous polypeptide at the N- or C-
terminus
or chemically conjugated (including covalently and non-covalently
conjugations) to
polypeptides or other compositions. For example, antibodies of the present
invention
may be recombinantly fused or conjugated to molecules useful as labels in
detection
assays and effector molecules such as heterologous polypeptides, drugs, or
toxins. See,
e.g., PCT publications WO 92108495; WO 91/14438; WO 89/12624; U.S. Patent No.
5,314,995; and EP 396,387.
The antibodies of the invention include derivatives that are modified, i.e, by
the
covalent attachment of any type of molecule to the antibody such that covalent
attachment does not prevent the antibody from generating an anti-idiotypic
response.
For example, but not by way of limitation, the antibody derivatives include
antibodies
that have been modified, e.g., by glycosylation, acetylation, pegylation,
phosphylation,
amidation, derivatization by known protectinglblocking groups, proteolytic
cleavage,
linkage to a cellular ligand or other protein, etc. Any of numerous chemical
modifications may be carried out by known techniques, including, but not
limited to
specific chemical cleavage, acetylation, formylation, metabolic synthesis of
tunicamycin, etc. Additionally, the derivative may contain one or more non-
classical
amino acids.
The antibodies of the present invention may be generated by any suitable
method known in the art. Polyclonal antibodies to an antigen-of interest can
be
produced by various procedures well known in the art. For example, a
polypeptide of
a
the invention can be administered to various host animals including, but not
limited to,
rabbits, mice, rats, etc. to induce the production of sera containing
polyclonal
antibodies specific for the a~~tigen. Various adjuvants may be used to
increase the
immunological response, depending on the host species, and include but are not
limited to, Freund's (complete and incomplete), mineral gels such as aluminum
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hydroxide, surface active substances such as lysolecithin, pluronic polyols,
polyanions,
peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially
useful human adjuvants such as BCG (bacille Calmette-Guerin) and
corynebacterium
parvum. Such adjuvants are also well known in the art.
Monoclonal antibodies can be prepared using a wide variety of techniques
known in the art including the use of hybridoma, recombinant, and phage
display
technologies, or a combination thereof. For example, monoclonal antibodies can
be
produced using hybridorna techniques including those known in the art and
taught, for
example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring
Harbor
Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies
and
T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated
by
reference in their entireties). The term "monoclonal antibody" as used herein
is not
limited to antibodies produced through hybridoma technology. The term
"monoclonal
antibody" refers to an antibody that is derived from a single clone, including
any
1 S eukaryotic, prokaryotic, or phage clone, and not the method by which it is
produced.
Methods for producing and screening for specific antibodies using hybridoma
technology are routine and well-known in the art and axe discussed in detail
in
Example 5. Briefly, mice can be immunized with a polypeptide of the invention
or a
cell expressing such peptide. Once an immune response is detected, e.g.,
antibodies
specific for the antigen are detected in the mouse serum, the mouse spleen is
harvested
and splenocytes isolated. 'The splenocytes are then fused by well-known
techniques to
any suitable myeloma cells, for example cells from cell line SP20 available
from the
ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma
clones are then assayed by methods known in the art for cells that secrete
antibodies
capable of binding a polypeptide of the invention. Ascites fluid, which
generally
contains high levels of antibodies, can be generated by immunizing mice with
positive
hybridoma clones.
Accordingly, the present invention provides methods of generating monoclonal
antibodies as well as antibodies produced by the method comprising culturing a
hybridoma cell secreting an antibody of the invention wherein, preferably, the
hybridoma is generated by fusing splenocytes isolated from a mouse immunized
with
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an antigen of the invention with myeloma cells and then screening the
hybridomas
resulting from the fusion far hybridoma clones that secrete an antibody able
to bind a
polypeptide of the invention.
Antibody fragments that recognize specific epitopes may be generated by
known techniques. For example, Fab and F(ab')2 fragments of the invention may
be
produced by proteolytic cleavage of immunoglobulin molecules, using enzymes
such
as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F{ab')2 fragments contain the variable region, the light chain constant region
and the
CH 1 domain of the heavy chain.
For example, the antibodies of the present invention can also be generated
using various phage display methods known in the art. In phage display
methods,
functional antibody domains are displayed on the surface of phage particles
which
carry the polynucleotide sequences encoding them. In a particular, such phage
can be
utilized to display antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or marine). Phage expressing an
antigen
binding domain that binds the antigen of interest can be selected or
identified with
antigen, e.g., using labeled antigen or antigen bound or captured to a solid
surface or
bead. Phage used in these methods are typically filamentous phage including fd
and
M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized
Fv
antibody domains recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make the
antibodies
of the present invention include those disclosed in Brinkman et al., J.
Immunol.
Methods 182:41-50 ( 1995); Ames et al., J. Immunol. Methods 184:177-186 (
1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene
187 9-18
(1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application
No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO
92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.
Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;
5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743
and
5,969,108; each of which is incorporated herein by reference in its entirety.
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As described in the above references, after phage selection, the antibody
coding regions from the phage can be isolated and used to generate whole
antibodies,
including human antibodies, or any other desired antigen binding fragment, and
expressed in any desired host, including mammalian cells, insect cells, plant
cells,
yeast, and bacteria, e.g., as described in detail below. For example,
techniques to
recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed
using
methods known in the art such as those disclosed in PCT publication WO
92/22324;
Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI
34:26-
34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references
incorporated by reference in their entireties).
Examples of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et
al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999
(1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in
vivo use of antibodies in humans and in vitro detection assays, it may be
preferable to
use chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule in
which different portions of the antibody are derived from different animal
species,
such as antibodies having a variable region derived from a murine monoclonal
antibody and a human immunoglobulin constant region. Methods for producing
chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202
( 1985); Oi et al., BioTechniques 4:214 ( 1986); Gillies et al., ( 1989) J.
Immunol.
Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397,
which
are incorporated herein by reference in their entireties. Humanized antibodies
are
antibody molecules from non-human species antibody that binds the desired
antigen
having one or more complementarity determining regions (CDRs) from the non-
human species and framework regions from a human immunoglobulin molecule.
Often, framework residues in the human framework regions will be substituted
with
the corresponding residue from the CDR donor antibody to alter, preferably
improve,
antigen binding. These framework substitutions are identified by methods well
known
in the art, e.g., by modeling of the interactions of the CDR and framework
residues to
identify framework residues important for antigen binding and sequence
comparison
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to identify unusual framework residues at particular positions. (See, e.g.,
Queen et al.,
U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are
incorporated herein by reference in their entireties.) Antibodies can be
humanized
using a variety of techniques known in the art including, for example, CDR-
grafting
(EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539;
5,530,101;
and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan,
Molecular
Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering
7(6):805-
814 ( 1994); Roguska. et al., PNAS 91:969-973 ( 1994)), and chain shuffling
(U.S.
Patent No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic
treatment of human patients. Human antibodies can be made by a variety of
methods
known in the art including phage display methods described above using
antibody
libraries derived from human immunoglobulin sequences. See also, U.S. Patent
Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of
which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice which are
incapable of expressing functional endogenous immunoglobulins, but which can
express human immunoglobulin genes. For example, the human heavy and light
chain
immunoglobulin gene complexes may be introduced randomly or by homologous
recombination into mouse embryonic stem cells. Alternatively, the human
variable
region, constant region, and diversity region may be introduced into mouse
embryonic
stem cells in addition to the human heavy and light chain genes. The mouse
heavy and
light chain immunoglobulin genes may be rendered non-functional separately or
simultaneously with the introduction of human immunoglobulin loci by
homologous
recombination. In particular, homozygous deletion of the JH region prevents
endogenous antibody production. The modified embryonic stem cells are expanded
and microinjected into blastocysts to produce chimeric mice. The chimeric mice
are
then bred to produce homozygous offspring that express human antibodies. The
transgenic mice are immunized in the normal fashion with a selected antigen,
e.g., all
or a portion of a polypeptide of the invention. Monoclonal antibodies directed
against
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the antigen can be obtained from the immunized, transgenic mice using
conventional
hybridoma technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and subsequently
undergo
class switching and somatic: mutation. Thus, using such a technique, it is
possible to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an
overview of
this technology for producing human antibodies, see Lonberg and Huszar (1995,
Int.
Rev. Immunol. 13:65-93). For a detailed discussion of this technology for
producing
human antibodies and human monoclonal antibodies and protocols for producing
such
antibodies, see, e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735;
U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016;
5,545,806;
5,814,318; and 5,939,598, which are incorporated by reference herein in their
entirety.
In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharm (San
Jose, CA) can be engaged to provide human antibodies directed against a
selected
antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can be
generated using a technique referred to as "guided selection." In this
approach a
selected non-human monoclonal antibody, e.g., a mouse antibody, is used to
guide the
selection of a completely human antibody recognizing the same epitope.
(Jespers et
al., Biotechnology 12:899-9173 ( 1988)).
Further, antibodies to the polypeptides of the invention can, in turn, be
utilized
to generate anti-idiotype antibodies that "mimic" polypeptides of the
invention using
techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona,
FASEB J. 7(5):437-444; ( 1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (
1991 )).
For example, antibodies which bind to and competitively inhibit polypeptide
multimerization and/or binding of a polypeptide of the invention to a ligand
can be
used to generate anti-idiotype;s that "mimic" the polypeptide multimerization
and/or
binding domain and, as a consequence, bind to and neutralize polypeptide
and/or its
ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-
idiotypes can be
used in therapeutic regimens t~o neutralize polypeptide ligand. For example,
such anti-
idiotypic antibodies can be used to bind a polypeptide of the invention and/or
to bind
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its ligands/receptors, and thereby block its biological activity.
PolynucIeotides
Encoding Antibodies.
The invention further provides polynucleotides comprising a nucleotide
sequence encoding an antibody of the invention and fragments thereof. The
invention
also encompasses polynucleotides that hybridize under stringent or lower
stringency
hybridization conditions, e.g., as defined supra, to polynucleotides that
encode an
antibody, preferably, that specifically binds to a polypeptide of the
invention,
preferably, an antibody that binds to a polypeptide having the amino acid
sequence of
SEQ ID N0:2.
The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the
nucleotide sequence of the antibody is known, a polynucleotide encoding the
antibody
may be assembled from chemically synthesized oligonucleotides (e.g., as
described in
Kutmeier et al., BioTechniques 17:242 ( 1994)), which, briefly, involves the
synthesis
of overlapping oligonucleotides containing portions of the sequence encoding
the
antibody, annealing and ligation of those oligonucleotides, and then
amplification of
the ligated oligonucleotides by PCR.
Alternatively, a polynucleotide encoding an antibody may be generated from
nucleic acid from a suitable source. If a clone containing a nucleic acid
encoding a
particular antibody is not available, but the sequence of the antibody
molecule is
known, a nucleic acid encoding the immunoglobulin may be obtained from a
suitable
source (e.g., an antibody cDNA library, or a cDNA library generated from, or
nucleic
acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing
the
antibody, such as hybridoma cells selected to express an antibody of the
invention) by
PCR amplification using synthetic primers hybridizable to the 3' and 5' ends
of the
sequence or by cloning using an oligonucleotide probe specific for the
particular gene
6
sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the
antibody. Amplified nucleic acids generated by PCR may then be cloned into
replicable cloning vectors using any method well known in the art.
Once the nucleotide sequence and corresponding amino acid sequence of the
antibody is determined, the nucleotide sequence of the antibody may be
manipulated
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using methods well known in the art for the manipulation of nucleotide
sequences,
e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see,
for
example, the techniques described in Sambrook et al., 1990, Molecular Cloning,
A
Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY
and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John
Wiley &
Sons, NY, which are both incorporated by reference herein in their entireties
}, to
generate antibodies having a different amino acid sequence, for example to
create
amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light
chain variable domains may be inspected to identify the sequences of the
complementarity determining regions (CDRs) by methods that are well know in
the
art, e.g., by comparison to known amino acid sequences of other heavy and
light chain
variable regions to determine the regions of sequence hypervariability. Using
routine
recombinant DNA techniques, one or more of the CDRs may be inserted within
framework regions, e.g., into human framework regions to humanize a non-human
antibody, as described supra. The framework regions may be naturally occurring
or
consensus framework regions, and preferably human framework regions (see,
e.g.,
Chothia et al., J. Mol. Bioi. 278: 457-479 ( 1998) for a listing of human
framework
regions). Preferably, the polynucleotide generated by the combination of the
framework regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one or more
amino acid
substitutions may be made within the framework regions, and, preferably, the
amino
acid substitutions improve binding of the antibody to its antigen.
Additionally, such
methods may be used to make amino acid substitutions or deletions of one or
more
2S variable region cysteine residues participating in an intrachain disulfide
bond to
generate antibody molecules lacking one or more intrachain disulfide bonds.
Other
a
alterations to the polynucleotide are encompassed by the present invention and
within
the skill of the art.
In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al.,
1984,
Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes
from
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a mouse antibody molecule of appropriate antigen specificity together with
genes
from a human antibody molecule of appropriate biological activity can be used.
As
described supra, a chimeric antibody is a molecule in which different portions
are
derived from different animal species, such as those having a variable region
derived
from a murine mAb and a human immunoglobulin constant region, e.g., humanized
antibodies.
Alternatively, techniques described for the production of single chain
antibodies (U.S. Patent No. 4,694,778; Bird, 1988, Science 242:423- 42; Huston
et al.,
1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature
334:544-54) can be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain fragments of the Fv
region
via an amino acid bridge, resulting in a single chain polypeptide. Techniques
for the
assembly of functional Fv fragments in E. coli may also be used (Skerra et
al., 1988,
Science 242:1038- 1041).
1 S Methods of Producing Antibodies.
The antibodies of the invention can be produced by any method known in the
art for the synthesis of antibodies, in particular, by chemical synthesis or
preferably,
by recombinant expression techniques.
Recombinant expression of an antibody of the invention, or fragment,
derivative or analog thereof, e.g., a heavy or light chain of an antibody of
the
invention, requires construction of an expression vector containing a
polynucleotide
that encodes the antibody. Once a polynucleotide encoding an antibody molecule
or a
heavy or light chain of an antibody, or portion thereof (preferably containing
the heavy
or light chain variable domain), of the invention has been obtained, the
vector for the
production of the antibody molecule may be produced by recombinant DNA
technology using techniques well known in the art. Thus, methods for preparing
a
f
protein by expressing a polynucleotide containing an antibody encoding
nucleotide
sequence are described herein. Methods which are well known to those skilled
in the
art can be used to construct expression vectors containing antibody coding
sequences
and appropriate transcriptional and translational control signals. These
methods
include, for example, in vitro recombinant DNA techniques, synthetic
techniques, and
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in vivo genetic recombination. The invention, thus, provides replicable
vectors
comprising a nucleotide sequence encoding an antibody molecule of the
invention, or
a heavy or light chain thereof, or a heavy or light chain variable domain,
operably
linked to a promoter. Such vectors may include the nucleotide sequence
encoding the
constant region of the antibody molecule (see, e.g., PCT Publication WO
86/05807;
PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable
domain of the antibody may be cloned into such a vector for expression of the
entire
heavy or light chain.
The expression vector is transferred to a host cell by conventional techniques
and the transfected cells are then cultured by conventional techniques to
produce an
antibody of the invention. Thus, the invention includes host cells containing
a
polynucleotide encoding an antibody of the invention, or a heavy or light
chain
thereof, operably linked to a heterologous promoter. In preferred embodiments
for the
expression of double-chained antibodies, vectors encoding both the heavy and
light
chains may be co-expressed in the host cell for expression of the entire
immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the
antibody molecules of the invention. Such host-expression systems represent
vehicles
by which the coding sequences of interest may be produced and subsequently
purified,
but also represent cells which may, when transformed or transfected with the
appropriate nucleotide coding sequences, express an antibody molecule of the
invention in situ. These include but are not limited to microorganisms such as
bacteria (e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA,
plasmid DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant
yeast
expression vectors containing antibody coding sequences; insect cell systems
infected
s
with recombinant virus expression vectors (e.g., baculovirus) containing
antibody
coding sequences; plant cell systems infected with recombinant virus
expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti plasmid)
containing
antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK,
293,
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3T3 cells) harboring recombinant expression constructs containing promoters
derived
from the genome of mammalian cells (e.g., metallothionein promoter) or from
mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.SK
promoter). Preferably, bacterial cells such as Escherichia coli, and more
preferably,
eukaryotic cells, especially for the expression of whole recombinant antibody
molecule, are used for the expression of a recombinant antibody molecule. For
example, mammalian cells such as Chinese hamster ovary cells (CHO), in
conjunction
with a vector such as the major intermediate early gene promoter element from
human
cytomegalovirus is an effective expression system for antibodies (Foecking et
al.,
1986, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).
In bacterial systems, a number of expression vectors may be advantageously
selected depending upon the use intended for the antibody molecule being
expressed.
For example, when a large quantity of such a protein is to be produced, for
the
generation of pharmaceutical compositions of an antibody molecule, vectors
which
direct the expression of high levels of fusion protein products that are
readily purified
may be desirable. Such vectors include, but are not limited, to the E. coli
expression
vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791 ), in which the antibody
coding
sequence may be ligated individually into the vector in frame with the lac Z
coding
region so that a fusion protein is produced; pIN vectors (Inouye & Inouye,
1985,
Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
24:5503-5509); and the like. pGEX vectors may also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase (GST). In
general, such
fusion proteins are soluble and can easily be purified from lysed cells by
adsorption
and binding to a matrix glutathione-agarose beads followed by elution in the
presence
of free glutathione. The pGEX vectors are designed to include thrombin or
factor Xa
protease cleavage sites so that the cloned target gene product can be released
from the
6
GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptera
frugiperda cells. The antibody coding sequence rnay be cloned individually
into non-
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essential regions (for example the polyhedrin gene) of the virus and placed
under
control of an AcNPV promoter (for example the polyhedrin promoter).
In mammalian host cells, a number of viral-based expression systems may be
utilized. In cases where an adenovirus is used as an expression vector, the
antibody
coding sequence of interest may be ligated to an adenovirus
transcription/translation
control complex, e.g., the late promoter and tripartite leader sequence. This
chimeric
gene may then be inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non- essential region of the viral genome (e.g.,
region
El or E3) will result in a recombinant virus that is viable and capable of
expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk, 1984, Proc.
Natl.
Acad. Sci. USA 81:355-359). Specific initiation signals may also be required
for
efficient translation of inserted antibody coding sequences. These signals
include the
ATG initiation codon and adjacent sequences. Furthermore, the initiation codon
must
be in phase with the reading frame of the desired coding sequence to ensure
translation
of the entire insert. These exogenous translational control signals and
initiation
codons can be of a variety of origins, both natural and synthetic. The
efficiency of
expression may be enhanced by the inclusion of appropriate transcription
enhancer
elements, transcription terminators, etc. (see Bittner et al., 1987, Methods
in Enzymol.
153:5 I -544).
In addition, a host cell strain may be chosen which modulates the expression
of
the inserted sequences, or modifies and processes the gene product in the
specific
fashion desired. Such modifications (e.g., glycosylation) and processing
(e.g.,
cleavage) of protein products may be important for the function of the
protein.
Different host cells have characteristic and specific mechanisms for the post-
translational processing and modification of proteins and gene products.
Appropriate
cell lines or host systems can be chosen to ensure the correct modification
and
processing of the foreign protein expressed. To this end, eukaryotic host
cells which
possess the cellular machinery for proper processing of the primary
transcript,
glycosyIation, and phosphorylation of the gene product may be used. Such
mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS,
MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for
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example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell
line such as, for example, CRL7030 and Hs578Bst.
For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which stably express the
antibody
molecule may be engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with DNA
controlled by
appropriate expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a selectable
marker.
Following the introduction of the foreign DNA, engineered cells may be allowed
to
grow for 1-2 days in an enriched media, and then are switched to a selective
media.
The selectable marker in the recombinant plasmid confers resistance to the
selection
and allows cells to stably integrate the plasmid into their chromosomes and
grow to
form foci which in turn can be cloned and expanded into cell lines. This
method may
advantageously be used to engineer cell lines which express the antibody
molecule.
Such engineered cell lines may be particularly useful in screening and
evaluation of
compounds that interact directly or indirectly with the antibody molecule.
A number of selection systems may be used, including but not limited to the
herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223),
hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192,
Proc.
Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et
al.,
1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells,
respectively.
Also, antimetabolite resistance can be used as the basis of selection for the
following
genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980,
Natl. Acad.
Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527);
gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc.
Natl.
Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-
418
Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev,
1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science
260:926-
932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993,
TIB TECH 11 (5):155-215); and hygro, which confers resistance to hygromycin
(Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of
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recombinant DNA technology which can be used are described in Ausubel et al.
{eds.),
1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler,
1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;
and
in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in
Human
S Genetics, John Wiley & Sons, NY.; Colberre-Uarapin et al., 1981, J. Mol.
Biol. 150:1,
which are incorporated by reference herein in their entireties.
The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based
on gene amplification for the expression of cloned genes in mammalian cells in
DNA
cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of inhibitor
present in
culture of host cell will increase the number of copies of the marker gene.
Since the
amplified region is associated with the antibody gene, production of the
antibody will
also increase (Grouse et al., 1983, Mol. Cell. Biol. 3:257).
1S The host cell may be co-transfected with two expression vectors of the
invention, the first vector encoding a heavy chain derived polypeptide and the
second
vector encoding a light chain derived polypeptide. The two vectors may contain
identical selectable markers which enable equal expression of heavy and light
chain
polypeptides. Alternatively, a single vector may be used which encodes both
heavy
and light chain polypeptides. In such situations, the light chain should be
placed
before the heavy chain to avoid an excess of toxic free heavy chain
(Proudfoot, 1986,
Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 7?:2197). The coding
sequences for the heavy and light chains may comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been recombinantly expressed,
2S it may be purified by any method known in the art for purification of an
immunoglobulin molecule, for example, by chromatography (e.g., ion exchange,
affinity, particularly by affinity for the specific antigen after Protein A,
and sizing
column chromatography), centrifugation, differential solubility, or by any
other
standard technique for the purification of proteins.
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Antibody conjugates.
The present invention encompasses antibodies recombinantly fused or
chemically conjugated (including both covalently and non-covalently
conjugations) to
a polypeptide (or portion thereof, preferably at least I0, 20 or 50 amino
acids of the
polypeptide) of the present invention to generate fusion proteins. The fusion
does not
necessarily need to be direct, but may occur through linker sequences. The
antibodies
may be specific for antigens other than polypeptides (or portion thereof,
preferably at
least 10, 20 or 50 amino acids of the poIypeptide) of the present invention.
For
example, antibodies may be used to target the polypeptides of the present
invention to
particular cell types, either in vitro or in vivo, by fusing or conjugating
the
polypeptides of the present invention to antibodies specific for particular
cell surface
receptors. Antibodies fused or conjugated to the polypeptides of the present
invention
may also be used in in vitro immunoassays and purification methods using
methods
known in the art. See e.g., Harbor et al., supra, and PCT publication WO
93/21232;
EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Patent
5,474,981;
Gillies et al., PNAS 89:1428-1432 ( 1992); Fell et al., J. Immunol. 146:2446-
2452( 1991 ), which are incorporated by reference in their entireties.
The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other
than the variable regions. For example, the polypeptides of the present
invention may
be fused or conjugated to an antibody Fc region, or portion thereof. The
antibody
portion fused to a polypeptide of the present invention may comprise the
constant
region, hinge region, CH 1 domain, CH2 domain, and CH3 domain or any
combination of whole domains or portions thereof. The polypeptides may also be
fused or conjugated to the above antibody portions to form multimers. For
example,
Fc portions fused to the polypeptides of the present invention can form dimers
through
disulfide bonding between the Fc portions. Higher multimeric forms can be made
by
fusing the polypeptides to portions of IgA and IgM. Methods for fusing or
conjugating the polypeptides of the present invention to antibody portions are
known
in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046;
5,349,053;
5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO
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91/06570; Ashkenazi et al., Proc. Natl. Acid. Sci. USA 88:10535-10539 (1991);
Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl.
Acid. Sci.
USA 89:11337- 11341(1992) (said references incorporated by reference in their
entireties).
As discussed, supra, the polypeptides of the present invention may be fused or
conjugated to the above antibody portions to increase the in vivo half life of
the
polypeptides or for use in immunoassays using methods known in the art.
Further, the
polypeptides of the present invention may be fused or conjugated to the above
antibody portions to facilitate purification. One reported example describes
chimeric
proteins consisting of the first two domains of the human CD4-polypeptide and
various domains of the constant regions of the heavy or light chains of
mammalian
immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The
polypeptides of the present invention fused or conjugated to an antibody
having
disulfide- linked dirneric structures (due to the IgG) may also be more
efficient in
binding and neutralizing other molecules, than the monomeric secreted protein
or
protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (
1995)). In
many cases, the Fc part in a fusion protein is beneficial in therapy and
diagnosis, and
thus can result in, for example, improved pharmacokinetic properties. (EP A
232,262). Alternatively, deleting the Fc part after the fusion protein has
been
expressed, detected, and purified, would be desired. For example, the Fc
portion may
hinder therapy and diagnosis if the fusion protein is used as an antigen for
immunizations. In drug discovery, for example, human proteins, such as hIL-5,
have
been fused with Fc portions for the purpose of high-throughput screening
assays to
identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular
Recognition 8:52-
58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)0.
Moreover, the antibodies or fragments thereof of the present invention can be
fused to marker sequences, such as a peptide to facilitates their
purification. In
preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide,
such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, CA, 91311), among others, many of which are commercially
available.
As described in Gentz et al., Proc. Natl. Acid. Sci. USA 86:821-824 ( 1989),
for
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instance, hexa-histidine provides for convenient purification of the fusion
protein.
Other peptide tags useful for purification include, but are not limited to,
the "HA" tag,
which corresponds to an epitope derived from the influenza hemagglutinin
protein
(Wilson et al., Cell 37:767 (1984)) and the "flag" tag.
The present invention further encompasses antibodies or fragments thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically to, for example, monitor the development or progression of a
tumor as
part of a clinical testing procedure to, e.g., determine the efficacy of a
given treatment
regimen. Detection can be facilitated by coupling the antibody to a detectable
substance. Examples of detectable substances include various enzymes,
prosthetic
groups, fluorescent materials, luminescent materials, bioluminescent
materials,
radioactive materials, positron emitting metals using various positron
emission
tomographies, and nonradioactive paramagnetic metal ions. See, for example,
U.S.
Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for
use as
diagnostics according to the present invention. Examples of suitable enzymes
include
horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin; examples of suitable fluorescent
materials
include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luminescent material includes luminol; examples of bioluminescent materials
include
luciferase, luciferin, and aequorin; and examples of suitable radioactive
material
include 125I, 131I, 11 lIn or 99Tc.
Further, an antibody or fragment thereof may be conjugated to a therapeutic
moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a
therapeutic agent or
a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent
that is
detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin
D,
ethidium bromide, ernetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine,
colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs
thereof.
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Therapeutic agents include, but are not limited to, antimetabolites (e.g.,
methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine
(BSNU)
and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP)
cisplatin),
anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and
anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
The conjugates of the invention can be used for modifying a given biological
response, the therapeutic agent or drug moiety is not to be construed as
limited to
classical chemical therapeutic agents. For example, the drug moiety may be a
protein
or polypeptide possessing a desired biological activity. Such proteins may
include,
for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin;
a protein such as tumor necrosis factor, a-interferon,13-interferon, nerve
growth factor,
platelet derived growth factor, tissue plasminogen activator, a thrombotic
agent or an
anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological
response modifiers
such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-
2"),
interleukin-6 ("IL-6"), granulocyte macrophase colony stimulating factor ("GM-
CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
Antibodies may also be attached to solid supports, which are particularly
useful
for immunoassays or purification of the target antigen. Such solid supports
include,
but are not limited to, glass, cellulose, polyacryIamide, nylon, polystyrene,
polyvinyl
chloride or polypropylene.
Techniques for conjugating such therapeutic moiety to antibodies are well
known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of
Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy,
Reisfeld
et a1. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,
"Antibodies For
Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.),
pp.
623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic
Agents
In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 ( 1985); "Analysis,
Results,
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And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In
Cancer
Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin
et
al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And
Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62:119-58
(1982).
Alternatively, an antibody can be conjugated to a second antibody to form an
antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980,
which
is incorporated herein by reference in its entirety.
An antibody, with or without a therapeutic moiety conjugated to it,
administered alone or in combination with cytotoxic factors) and/or
cytokine(s) can
be used as a therapeutic. Assays For Antibody Binding
The antibodies of the invention may be assayed for immunospecific binding by
any method known in the art. The immunoassays which can be used include but
are
not limited to competitive and non-competitive assay systems using techniques
such as
western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel
diffusion precipitin reactions, immunodiffusion assays, agglutination assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays,
protein A immunoassays, to name but a few. Such assays are routine and well
known
in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in
Molecular Biology,
Vol. l, John Wiley & Sons, Inc., New York, which is incorporated by reference
herein
in its entirety). Exemplary immunoassays are described briefly below (but are
not
intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells
in a lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium
deoxycholate, 0.1 % SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1 %
Trasylol) supplemented with protein phosphatase and/or protease inhibitors
(e.g.,
EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to
the cell
lysate, incubating for a period of time (e.g., l-4 hours) at 4° C,
adding protein A
and/or protein G sepharose beads to the cell lysate, incubating for about an
hour or
more at 4° C, washing the beads in lysis buffer and resuspending the
beads in
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SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a
particular antigen can be assessed by, e.g., western blot analysis. One of
skill in the
art would be knowledgeable as to the parameters that can be modified to
increase the
binding of the antibody to an antigen and decrease the background (e.g., pre-
clearing
the cell lysate with sepharose beads). For further discussion regarding
immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current
Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-
PAGE depending on the molecular weight of the antigen), transferring the
protein
sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF
or
nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or
non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking
the
membrane with primary antibody (the antibody of interest) diluted in blocking
buffer,
washing the membrane in washing buffer, blocking the membrane with a secondary
antibody (which recognizes the primary antibody, e.g., an anti-human antibody)
conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline
phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking
buffer,
washing the membrane in wash buffer, and detecting the presence of the
antigen. One
of skill in the art would be knowledgeable as to the parameters that can be
modified to
increase the signal detected and to reduce the background noise. For further
discussion regarding western blot protocols see, e.g., Ausubel et al, eds,
1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at
10.8.1.
ELISAs comprise preparing antigen, coating the well of a 96 well microtiter
plate with the antigen, adding the antibody of interest conjugated to a
detectable
compound such as an enzymatic substrate (e.g., horseradish peroxidase or
alkaline
phosphatase) to the well and incubating for a period of time, and detecting
the
presence of the antigen. In ELISAs the antibody of interest does not have to
be
conjugated to a detectable compound; instead, a second antibody (which
recognizes
the antibody of interest) conjugated to a detectable compound may be added to
the
well. Further, instead of coating the well with the antigen, the antibody may
be
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coated to the well. In this case, a second antibody conjugated to a detectable
compound may be added following the addition of the antigen of interest to the
coated
well. One of skill in the art would be knowledgeable as to the parameters that
can be
modified to increase the signal detected as well as other variations of ELISAs
known
in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al,
eds, 1994,
Current Protocols in Molecular Biology, Vol. l, John Wiley & Sons, Inc., New
York
at I 1.2. I .
The binding affinity of an antibody to an antigen and the off rate of an
antibody-antigen interaction can be determined by competitive binding assays.
One
example of a competitive binding assay is a radioimmunoassay comprising the
incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest
in the
presence of increasing amounts of unlabeled antigen, and the detection of the
antibody
bound to the labeled antigen. The affinity of the antibody of interest for a
particular
antigen and the binding off-rates can be determined from the data by scatchard
plot
analysis. Competition with a second antibody can also be determined using
radioimmunoassays. In this case, the antigen is incubated with antibody of
interest is
conjugated to a labeled compound (e.g., 3H or 125I) in the presence of
increasing
amounts of an unlabeled second antibody.
Therapeutic Uses.
The present invention is further directed to antibody-based therapies which
involve administering antibodies of the invention to an animal, preferably a
mammal,
and most preferably a human, patient for treating one or more of the described
disorders. Therapeutic compounds of the invention include, but are not limited
to,
antibodies of the invention (including fragments, analogs and derivatives
thereof as
described herein) and nucleic acids encoding antibodies of the invention
(including
fragments, analogs and derivatives thereof as described herein). The
antibodies of the
invention can be used to treat, inhibit or prevent diseases and disorders
associated
with aberrant expression and/or activity of a polypeptide of the invention,
including,
but not limited to, [insert diseases and disorders]. The treatment and/or
prevention of
diseases and disorders associated with aberrant expression and/or activity of
a
polypeptide of the invention includes, but is not limited to, alleviating
symptoms
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associated with those diseases and disorders. Antibodies of the invention may
be
provided in pharmaceutically acceptable compositions as known in the art or as
described herein.
A summary of the ways in which the antibodies of the present invention may
be used therapeutically includes binding polynucleotides or polypeptides of
the
present invention locally or systemically in the body or by direct
cytotoxicity of the
antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some
of these approaches are described in more detail below. Armed with the
teachings
provided herein, one of ordinary skill in the art will know how to use the
antibodies of
the present invention for diagnostic, monitoring or therapeutic purposes
without undue
experimentation.
The antibodies of this invention may be advantageously utilized in
combination with other monoclonal or chimeric antibodies, or with lymphokines
or
hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for
example, which
serve to increase the number or activity of effector cells which interact with
the
antibodies.
The antibodies of the invention may be administered alone or in combination
with other types of treatments (e.g., radiation therapy, chemotherapy,
hormonal
therapy, immunotherapy and anti-tumor agents). Generally, administration of
products of a species origin or species reactivity (in the case of antibodies)
that is the
same species as that of the patient is preferred. Thus, in a preferred
embodiment,
human antibodies, fragments derivatives, analogs, or nucleic acids, are
administered
to a human patient for therapy or prophylaxis.
It is preferred to use high affinity and/or potent in vivo inhibiting and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention, fragments or regions thereof, for both immunoassays directed to and
s
therapy of disorders related to polynucleotides or polypeptides, including
fragments
thereof, of the present invention. Such antibodies, fragments, or regions,
will
preferably have an affinity for polynucleotides or polypeptides, including
fragments
thereof. Preferred binding affinities include those with a dissociation
constant or Kd
less than SX 10-ZM, 10-'-M, 5X 10~;M, 10-~M, SX 1 O~M, 10-'~M, SX 1 O~SM, 10-
SM, SX 10'
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6M, 10-6M, SX 10-'M, 10~'M, SX IO~gM, 10~~M, SX 10~9M, 10-9M, SX 10-
'°M, 10-'°M,
SX10-"M, 10-"M, SX10-''M, 10-'ZM, SX10-"M, 10-"M, SX10-'4M, 10-'~M, SX10-'SM,
and 10-' SM.
Gene Therapy.
In a specific embodiment, nucleic acids comprising sequences encoding
antibodies or functional derivatives thereof, are administered to treat,
inhibit or
prevent a disease or disorder associated with aberrant expression and/or
activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy refers to
therapy
performed by the administration to a subject of an expressed or expressible
nucleic
acid. In this embodiment of the invention, the nucleic acids produce their
encoded
protein that mediates a therapeutic effect.
Any of the methods for gene therapy available in the art can be used according
to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
1993,
Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev,
1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science
260:926-
932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993,
TIBTECH 11(5):155-215). Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al. (eds.), 1993,
Current
Protocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990,
Gene
Transfer and Expression, A Laboratory Manual, Stockton Press, NY.
In a preferred aspect, the compound comprises nucleic acid sequences
encoding an antibody, said nucleic acid sequences being part of expression
vectors
that express the antibody or fragments or chimeric proteins or heavy or light
chains
thereof in a suitable host. In particular, such nucleic acid sequences have
promoters
operably linked to the antibody coding region, said promoter being inducible
or
constitutive, and, optionally, tissue- specific. In another particular
embodiment,
nucleic acid molecules are used in which the antibody coding sequences and any
other
desired sequences are flanked by regions that promote homologous recombination
at a
desired site in the genome, thus providing fox intrachromosomal expression of
the
antibody nucleic acids (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA
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86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438). In specific
embodiments,
the expressed antibody malecule is a single chain antibody; alternatively, the
nucleic
acid sequences include sequences encoding both the heavy and light chains, or
fragments thereof, of the antibody.
Delivery of the nucleic acids into a patient may be either direct, in which
case
the patient is directly exposed to the nucleic acid or nucleic acid- carrying
vectors, or
indirect, in which case, cells are first transformed with the nucleic acids in
vitro, then
transplanted into the patient. These two approaches are known, respectively,
as in
vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly administered
in vivo, where it is expressed to produce the encoded product. This can be
accomplished by any of numerous methods known in the art, e.g., by
constructing
them as part of an appropriate nucleic acid expression vector and
administering it so
that they become intracellular, e.g., by infection using defective or
attenuated
retrovirals or other viral vectors (see U.S. Patent No. 4,980,286), or by
direct injection
of naked DNA, or by use of microparticle bombardment (e.g., a gene gun;
Biolistic,
Dupont), or coating with lipids or cell-surface .receptors or transfecting
agents,
encapsulation in liposomes, microparticles, or microcapsules, or by
administering
them in linkage to a peptide which is known to enter the nucleus, by
administering it
?0 in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g.,
Wu and Wu,
1987, J. Biol. Chem. 262:4429-4432) (which can be used to target cell types
specifically expressing the receptors), etc. In another embodiment, nucleic
acid-
ligand complexes can be formed in which the Iigand comprises a fusogenic viral
peptide to disrupt endosomes, allowing the nucleic acid to avoid Iysosomal
degradation. In yet another embodiment, the nucleic acid can be targeted in
vivo for
cell specific uptake and expression, by targeting a specific receptor (see,
e.g., PCT
Publications WO 92/06180 dated April 16, 1992 (Wu et al.); WO 92/22635 dated
December 23, 1992 (Wilson et al.); W092/20316 dated November 26, 1992 (Findeis
et al.); W093/14188 dated July 22, 1993 (Clarke et al.), WO 93/20221 dated
October
I4, 1993 (Young)). Alternatively, the nucleic acid can be introduced
intracellularly
and incorporated within host cell DNA for expression, by homologous
recombination
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(Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra
et al.,
1989, Nature 342:435-438).
In a specific embodiment, viral vectors that contains nucleic acid sequences
encoding an antibody of the invention are used. For example, a retroviral
vector can
be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). These
retroviral
vectors have been to delete retroviral sequences that are not necessary for
packaging
of the viral genome and integration into host cell DNA. The nucleic acid
sequences
encoding the antibody to be used in gene therapy are cloned into one or more
vectors,
which facilitates delivery of the gene into a patient. More detail about
retroviral
vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, which
describes
the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem
cells in
order to make the stem cells more resistant to chemotherapy. Other references
illustrating the use of retroviral vectors in gene therapy are: Clowes et al.,
1994, J.
Clin. Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and
Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993,
Curr. Opin. in Genetics and Devel. 3:110-114.
Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to
respiratory
epithelia. Adenoviruses naturally infect respiratory epithelia where they
cause a mild
disease. Other targets for adenovirus-based delivery cystems are liver. the
central
nervous system, endothelial cells, and muscle. Adenoviruses have the advantage
of
being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993,
Current
Opinion in Genetics and Development 3:499-503 present a review of adenovirus-
based gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated
the
use of adenovirus vectors to transfer genes to the respiratory epithelia of
rhesus
monkeys. Other instances of the use of adenoviruses in gene therapy can be
found in
Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell
68:143- 155;
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT Publication
W094/12649;
and Wang, et al., 1995, Gene Therapy 2:775-783. In a preferred embodiment,
adenovirus vectors are used.
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Adeno-associated virus (AAV) has also been proposed for use in gene therapy
(Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:2$9-300; U.S. Patent No.
5,436,146).
Another approach to gene therapy involves transferring a gene to cells in
tissue
culture by such methods as electroporation, lipofection, calcium phosphate
mediated
transfection, or viral infection. Usually, the method of transfer includes the
transfer of
a selectable marker to the cells. The cells are then placed under selection to
isolate
those cells that have taken up and are expressing the transferred gene. Those
cells are
then delivered to a patient.
In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be
carried out by any method known in the art, including but not limited to
transfection,
electroporation, microinjection, infection with a viral or bacteriophage
vector
containing the nucleic acid sequences, cell fusion, chromosome-mediated gene
transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous
techniques are known in the art for the introduction of foreign genes into
cells (see,
e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al., 1993,
Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be
used
in accordance with the present invention, provided that the necessary
developmental
and physiological functions of the recipient cells are not disrupted. The
technique
should provide for the stable transfer of the nucleic acid to the cell, so
that the nucleic
acid is expressible by the cell and preferably heritable and expressible by
its cell
progeny.
The resulting recombinant cells can be delivered to a patient by various
methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are preferably administered intravenously. The amount of
cells
a
envisioned for use depends on the desired effect, patient state, etc., and can
be
determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy
encompass any desired, available cell type, and include but are not limited to
epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes;
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blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages,
neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or
progenitor
cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained
from bone
marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
S In a preferred embodiment, the cell used for gene therapy is autologous to
the
patient.
In an embodiment in which recombinant cells are used in gene therapy, nucleic
acid sequences encoding an antibody are introduced into the cells such that
they are
expressible by the cells or their progeny, and the recombinant cells are then
administered in vivo for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which can be
isolated and
maintained in vitro can potentially be used in accordance with this embodiment
of the
present invention (see e.g. PCT Publication WO 94/08598, dated April 28, 1994;
Stemple and Anderson, 1992, Cell 71:973-985; Rheinwald, 1980, Meth. Cell Bio.
21 A:229; and Pittelkow and Scott, 1986, Mayo Clinic Proc. 61:771 ).
In a specific embodiment, the nucleic acid to be introduced for purposes of
gene therapy comprises an inducible promoter operably linked to the coding
region,
such that expression of the nucleic acid is controllable by controlling the
presence or
absence of the appropriate inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity.
The compounds or pharmaceutical compositions of the invention are preferably
tested in vitro, and then in vivo for the desired therapeutic or prophylactic
activity,
prior to use in humans. For example, in vitro assays to demonstrate the
therapeutic or
prophylactic utility of a compound or pharmaceutical composition include, the
effect
of a compound on a cell line or a patient tissue sample. The effect of the
compound or
composition on the cell line and/or tissue sample can be determined utilizing
f
techniques known to those of skill in the art including, but not limited to,
rosette
formation assays and cell lysis assays. In accordance with the invention, in
vitro
assays which can be used to determine whether administration of a specific
compound
is indicated, include in vitro cell culture assays in which a patient tissue
sample is
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grown in culture, and exposed to or otherwise administered a compound, and the
effect of such compound upon the tissue sample is observed.
TherapeuticlProphylactic Administration and Composition.
The invention provides methods of treatment, inhibition and prophylaxis by
administration to a subject of an effective amount of a compound or
pharnnaceutical
composition of the invention, preferably an antibody of the invention. In a
preferred
aspect, the compound is substantially purified (e.g., substantially free from
substances
that limit its effect or produce undesired side-effects). The subject is
preferably an
animal, including but not limited to animals such as cows, pigs, horses,
chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably human.
Formulations and methods of administration that can be employed when the
compound comprises a nucleic acid or an immunoglobulin are described above;
additional appropriate formulations and routes of administration can be
selected from
among those described herein below.
IS Various delivery systems are known and can be used to administer a compound
of the invention, e.g., encapsulation in liposomes, microparticles,
microcapsules,
recombinant cells capable of expressing the compound, receptor-mediated
endocytosis
(see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a
nucleic acid as part of a retroviral or other vector, etc. Methods of
introduction
include but are not limited to intradermal, intramuscular, intraperitoneal,
intravenous,
subcutaneous, intranasal, epidural, and oral routes. The compounds or
compositions
may be administered by any convenient route, for example by infusion or bolus
injection, by absorption through epithelial or mucocutaneous linings (e.g.,
oral
mucosa, rectal and intestinal mucosa, etc.) and rnay be administered together
with
other biologically active agents. Administration can be systemic or local. In
addition,
it may be desirable to introduce the pharmaceutical compounds or compositions
of the
invention into the central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular injection may be
facilitated
by an intraventricular catheter, for example, attached to a reservoir, such as
an
Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use
of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
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In a specific embodiment, it may be desirable to administer the pharmaceutical
compounds or compositions of the invention locally to the area in need of
treatment;
this may be achieved by, for example, and not by way of limitation, local
infusion
during surgery, topical application, e.g., in conjunction with a wound
dressing after
surgery, by injection, by means of a catheter, by means of a suppository, or
by means
of an implant, said implant being of a porous, non-porous, or gelatinous
material,
including membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention, care must be
taken to
use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a
vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533;
Treat et
al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-
Berestein
and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein,
ibid., pp.
317-327; see generally ibid.)
In yet another embodiment, the compound or composition can be delivered in a
controlled release system. In one embodiment, a pump may be used (see Langer,
supra; Sefton, 1987, CRC C'.rit. Ref. Biomed. Eng. 14:201; Buchwald et al.,
1980,
Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another
embodiment, polymeric materials can be used (see Medical Applications of
Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974);
Controlled
Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball
(eds.),
Wiley, New York ( 1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev.
Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et
al.,
1989, Ann. Neurol. 25:351; Howard et al., 1989, J.Neurosurg. 71:105). In yet
another
embodiment, a controlled release system can be placed in proximity of the
therapeutic
target, i.e., the brain, thus requiring only a fraction of the systemic dose
(see, e.g.,
Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-
138
( 1984)).
Other controlled release systems are discussed in the review by Langer (1990,
Science 249:1527-1533).
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In a specific embodiment where the compound of the invention is a nucleic
acid encoding a protein, the nucleic acid can be administered in vivo to
promote
expression of its encoded protein, by constructing it as part of an
appropriate nucleic
acid expression vector and administering it so that it becomes intracellular,
e.g., by
S use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct
injection, or by
use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating
with lipids or cell-surface receptors or transfecting agents, or by
administering it in
linkage to a homeobox- like peptide which is known to enter the nucleus (see
e.g.,
Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.
Alternatively, a
nucleic acid can be introduced intracellularly and incorporated within host
cell DNA
for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of a compound, and a
pharmaceutically acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency of the
Federal
or a state government or listed in the U.S. Pharmacopeia or other generally
recognized
pharmacopeia for use in animals, and more particularly in humans. The term
"carrier"
refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids, such as
water and
oils, including those of petroleum, animal, vegetable or synthetic origin,
such as
peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a
preferred
carrier when the pharmaceutical composition is administered intravenously.
Saline
solutions and aqueous dextrase and glycerol solutions can also be employed as
liquid
carriers, particularly for injectable solutions. Suitable pharmaceutical
excipients
include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk,
glycerol, propylene, glycol, water, ethanol and the like. The composition, if
desired,
can also contain minor amounts of wetting or emulsifying agents, or pH
buffering
agents. These compositions can take the form of solutions, suspensions,
emulsion,
tablets, pills, capsules, powders, sustained-release formulations and the
like. The
composition can be formulated as a suppository, with traditional binders and
carriers
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such as triglycerides. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of suitable
pharmaceutical
carriers are described in '°Remington's Pharmaceutical Sciences" by
E.W. Martin.
Such compositions will contain a therapeutically effective amount of the
compound,
preferably in purified form, together with a suitable amount of carrier so as
to provide
the form for proper administration to the patient. The formulation should suit
the
mode of administration.
In a preferred embodiment, the composition is formulated in accordance with
routine procedures as a pharmaceutical composition adapted for intravenous
administration to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer. Where
necessary, the
composition may also include a solubilizing agent and a local anesthetic such
as
lignocaine to ease pain at the site of the injection. Generally, the
ingredients are
supplied either separately or mixed together in unit dosage form, for example,
as a dry
lyophilized powder or water free concentrate in a hermetically sealed
container such
as an ampoule or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed with an
infusion
bottle containing sterile pharmaceutical grade water or saline. Where the
composition
is administered by injection, an ampoule of sterile water for injection or
saline can be
provided so that the ingredients may be mixed prior to administration.
The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as
those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc.,
and those
formed with cations such as those derived from sodium, potassium, ammonium,
calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino
ethanol,
histidine, procaine, etc.
The amount of the compound of the invention which will be effective in the
treatment, inhibition and prevention of a disease or disorder associated with
aberrant
expression and/or activity of a polypeptide of the invention can be determined
by
standard clinical techniques. In addition, in vitro assays may optionally be
employed
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to help identify optimal dosage ranges. The precise dose to be employed in the
formulation will also depend on the route of administration, and the
seriousness of the
disease or disorder, and should be decided according to the judgment of the
practitioner and each patient's circumstances. Effective doses may be
extrapolated
from dose-response curves derived from in vitro or animal model test systems.
For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to
100 mg/kg of the patient's body weight. Preferably, the dosage administered to
a
patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more
preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half life within the human body than antibodies from
other
species due to the immune response to the foreign polypeptides. Thus, lower
dosages
of human antibodies and less frequent administration is often possible.
Further, the
dosage and frequency of administration of antibodies of the invention may be
reduced
by enhancing uptake and tissue penetration (e.g., into the brain) of the
antibodies by
modifications such as, for example, lipidation.
The invention also provides a pharmaceutical pack or kit comprising one or
more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the invention. Optionally associated with such containers) can
be a
notice in the form prescribed by a governmental agency regulating the
manufacture,
use or sale of pharmaceuticals or biological products, which notice reflects
approval
by the agency of manufacture, use or sale for human administration. Diagnosis
and
Imaging
Labeled antibodies, and derivatives and analogs thereof, which specifically
bind to a polypeptide of interest can be used for diagnostic purposes to
detect,
diagnose, or monitor diseases and/or disorders associated with the aberrant
expression
and/or activity of a polypeptide of the invention. The invention provides for
the
detection of aberrant expression of a polypeptide of interest, comprising (a)
assaying
the expression of the polypeptide of interest in cells or body fluid of an
individual
using one or more antibodies specific to the polypeptide interest and (b)
comparing the
level of gene expression with a standard gene expression level, whereby an
increase
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or decrease in the assayed polypeptide gene expression level compared to the
standard
expression level is indicative of aberrant expression.
The invention provides a diagnostic assay for diagnosising a disorder,
comprising (a) assaying the expression of the polypeptide of interest in cells
or body
fluid of an individual using one or more antibodies specific to the
polypeptide interest
and (b) comparing the level of gene expression with a standard gene expression
level,
whereby an increase or decrease in the assayed polypeptide gene expression
level
compared to the standard expression level is indicative of a particular
disorder. With
respect to cancer, the presence of a relatively high amount of transcript 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.
Antibodies of the invention can be used to assay protein levels in a
biological
sample using classical immunohistological methods known to those of skill in
the art
(e.g., see Jalkanen, M., et al., J. Cell. Biol. 101:976-985 ( 1985); Jalkanen,
M., et al., J.
Cell . Biol. 105:3087-3096 ( 1987)). Other antibody-based methods useful for
detecting protein gene expression include immunoassays, such as the enzyme
linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody
assay labels are known in the art and include enzyme labels, such as, glucose
oxidase;
radioisotopes, such as.iodine (125I, 121I), carbon (14C), sulfur (355),
tritium (3H),
indium ( 1 l2In), and technetium (99Tc); luminescent labels, such as luminol;
and
fluorescent labels, such as fluorescein and rhodamine, and biotin.
One aspect of the invention is the detection and diagnosis of a disease or
disorder associated with aberrant expression of a polypeptide of the interest
in an
anima, preferably a mammal and most preferably a human. In one embodiment,
diagnosis comprises: a) administering (for example, parenterally,
subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled molecule
which
specifically binds to the polypeptide of interest; b) waiting for a time
interval
following the administering for permitting the labeled molecule to
preferentially
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concentrate at sites in the subject where the polypeptide is expressed (and
for
unbound labeled molecule to be cleared to background level); c) determining
background level; and d) detecting the labeled molecule in the subject, such
that
detection of labeled molecule above the background level indicates that the
subject has
a particular disease or disorder associated with aberrant expression of the
polypeptide
of interest. Background level can be determined by various methods including,
comparing the amount of libeled molecule detected to a standard value
previously
determined for a particular system.
It will be understood in the art that the size of the subject and the imaging
system used will determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a human subject,
the
quantity of radioactivity injected will normally range from about S to 20
millicuries of
99mTc. The labeled antibody or antibody fragment will then preferentially
accumulate at the location of cells which contain the specific protein. In
vivo tumor
imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging:
The
Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson
Publishing Inc. ( 1982).
Depending on several variables, including the type of label used and the mode
of administration, the time interval following the administration for
permitting the
labeled molecule to preferentially concentrate at sites in the subject and for
unbound
labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24
hours or
6 to 12 hours. In another embodiment the time interval following
administration is 5
to 20 days or 5 to 10 days.
In an embodiment, monitoring of the disease or disorder is carried out by
repeating the method for diagnosing the disease or disease, for example, one
month
after initial diagnosis, six months after initial diagnosis, one year after
initial diagnosis,
etc.
Presence of the labeled molecule can be detected in the patient using methods
known in the art for in vivo scanning. These methods depend upon the type of
label
used. Skilled artisans will be able to determine the appropriate method for
detecting a
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particular label. Methods and devices that may be used in the diagnostic
methods of
the invention include, but are not limited to, computed tomography (C'T),
whole body
scan such as position emission tomography (PET), magnetic resonance imaging
(MRI), and sonography.
In a specific embodiment, the molecule is labeled with a radioisotope and is
detected in the patient using a radiation responsive surgical instrument
(Thurston et al.,
U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled
with a
fluorescent compound and is detected in the patient using a fluorescence
responsive
scanning instrument. In another embodiment, the molecule is labeled with a
positron
emitting metal and is detected in the patent using positron emission-
tomography. In
yet another embodiment, the molecule is labeled with a paramagnetic label and
is
detected in a patient using magnetic resonance imaging (MRI).
Kits.
The present invention provides kits that can be used in the above methods. In
one embodiment, a kit comprises an antibody of the invention, preferably a
purified
antibody, in one or more containers. In a specific embodiment, the kits of the
present
invention contain a substantially isolated polypeptide comprising an epitope
which is
specifically immunoreactive with an antibody included in the kit. Preferably,
the kits
of the present invention further comprise a control antibody which does not
react with
the polypeptide of interest. In another specific embodiment, the kits of the
present
invention contain a means for detecting the binding of an antibody to a
polypeptide of
interest (e.g., the antibody may be conjugated to a detectable substrate such
as a
fluorescent compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the first antibody
may
be conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a
diagnostic
kit for use in screening serum containing antibodies specific against
proliferative
and/or cancerous polynucleotides and polypeptides. Such a kit may include a
control
antibody that does not react with the polypeptide of interest. Such a kit may
include a
substantially isolated polypeptide antigen comprising an epitope which is
specifically
immunoreactive with at least one anti-polypeptide antigen antibody. Further,
such a
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kit includes means for detecting the binding of said antibody to the antigen
(e.g., the
antibody may be conjugated to a fluorescent compound such as fluorescein or
rhodamine which can be detected by flow cytometry). In specific embodiments,
the
kit may include a recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to a solid
support.
In a more specific embodiment the detecting means of the above-described kit
includes a solid support to which said polypeptide antigen is attached. Such a
kit may
also include a non-attached reporter-labeled anti-human antibody. In this
embodiment,
binding of the antibody to the polypeptide antigen can be detected by binding
of the
said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use
in
screening serum containing antigens of the polypeptide of the invention. The
diagnostic kit includes a substantially isolated antibody specifically
immunoreactive
with polypeptide or polynucleotide antigens, and means for detecting the
binding of
the polynucleotide or polypeptide antigen to the antibody. In one embodiment,
the
antibody is attached to a solid support. In a specific embodiment, the
antibody may be
a monoclonal antibody. The: detecting means of the kit may include a second,
labeled
monoclonal antibody. Alternatively, or in addition, the detecting means may
include
a labeled, competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase
reagent
having a surface-bound antigen obtained by the methods of the present
invention.
After binding with specific antigen antibody to the reagent and removing
unbound
serum components by washing, the reagent is reacted with reporter-labeled anti-
human antibody to bind reporter to the reagent in proportion to the amount of
bound
anti-antigen antibody on the solid support. The reagent is again washed to
remove
unbound labeled antibody, and the amount of reporter associated with the
reagent is
determined. Typically, the reporter is an enzyme which is detected by
incubating the
solid phase in the presence of a suitable fluorometric, luminescent or
colorimetric
substrate (Sigma, St. Louis, :MO).
The solid surface reagent in the above assay is prepared by known techniques
for attaching protein material to solid support material, such as polymeric
beads, dip
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sticks, 96-well plate or filter material. These attachment methods generally
include
non-specific adsorption of the protein to the support or covalent attachment
of the
protein, typically through a free amine group, to a chemically reactive group
on the
solid support, such as an acaivated carboxyl, hydroxyl, or aldehyde group.
Alternatively, streptavidin coated plates can be used in conjunction with
biotinylated
antigen(s).
Thus, the invention provides an assay system or kit for carrying out this
diagnostic method. The kit generally includes a support with surface- bound
recombinant antigens, and a reporter-labeled anti-human antibody fox detecting
surface-bound anti-antigen antibody.
The invention further relates to antibodies which act as agonists or
antagonists
of the polypeptides of the present invention. For example, the present
invention
includes antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the invention either partially or fully. Included are both
receptor-
specific antibodies and ligand-specific antibodies. Included are receptor-
specific
antibodies which do not prevent ligand binding but prevent receptor
activation.
Receptor activation (i.e., signaling) may be determined by techniques
described herein
or otherwise known in the art. Also included are receptor-specific antibodies
which
both prevent ligand binding and receptor activation. Likewise, included are
neutralizing antibodies which bind the ligand and prevent binding of the
ligand to the
receptor, as well as antibodies which bind the ligand, thereby preventing
receptor
activation, but do not prevent the ligand from binding the receptor. Further
included
are antibodies which activate the receptor. These antibodies may act as
agonists for
either all or less than all of the biological activities affected by ligand-
mediated
receptor activation. The antibodies may be specified as agonists or
antagonists for
biological activities comprising specific activities disclosed herein. Further
included
are antibodies that bind to Brainiac-5 irrespective of whether Brainiac-5 is
bound to a
Brainiac-5 Receptor. These antibodies act as Brainiac-5 agonists as reflected
in an
increase in cellular proliferation in response to binding of Brainiac-5 to a
Brainiac-5
receptor in the presence of these antibodies. The above antibody agonists can
be made
using methods known in the art. See e.g., WO 96/40281; US Patent 5,811,097;
Deng,
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B. et al., Blood 92(6):1981-1988 (1998); Chen, Z. et al., Cancer Res.
58(16):3668-
3678 (1998); Harrop, J.A. et al., J. Immunol. 161(4):1786-1794 (1998); Zhu, Z.
et al.,
Cancer Res. 58(15):3209-3214 (1998); Yoon, D.Y. et al., J. Immunol.
160(7):3170-
3179 ( 1998); Prat, M. et al., J. Cell. Sci. 111 (Pt2):237-247 ( 1998);
Pitard, V. et ai., J.
Immunol. Methods 205(2)::177-190 (1997); Liautard, J. et al., Cytokinde
9(4):233-241
(1997); Carlson, N.G. et al., J. Biol. Chem. 272(17):11295-11301 (1997);
Taryman,
R.E. et al., Neuron 14(4):755-762 ( 1995); Muller, Y.A. et al., Structure
6(9): I 153-
1167 ( 1998); Bartunek, P. et al., Cytokine 8( 1 ):14-20 ( 1996) (said
references
incorporated by reference in their entireties).
In a specific embodiment, the antibodies of the invention fix complement. In
other specific embodiments, as further described herein, the antibodies of the
invention
(or fragments thereof] are associated with heterologous polypeptides or
nucleic acids
(e.g. toxins, such as, compounds that bind and activate endogenous cytotoxic
effecter
systems, and radioisotopes; and cytotoxic prodrugs).
As discussed above, antibodies to the Brainiac-5 polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that "mimic"
the Brainiac-
5, using techniques well known to those skilled in the art. (See, e.g.,
Greenspan &
Bona, FASEB J. 7(5):437-444 ( 1989), and Nissinoff, J. Immunol. 147(8):2429-
2438
( 1991 )). For example, antibodies which bind to Brainiac-5 and competitively
inhibit
the Brainiac-5 multimerization and/or binding to ligand can be used to
generate anti-
idiotypes that "mimic" the Brainiac-5 mutimerization and/or binding domain
and, as a
consequence, bind to and neutralize Brainiac-5 and/or its ligand. Such
neutralizing
anti-idiotypes or Fab fragments of such anti-idiotypes can be used in
therapeutic
regimens to neutralize Brainiac-5 ligand. For example, such anti-idiotypic
antibodies
can be used to bind Brainiac-5 on the surface of the cell, and thereby block
Brainiac-5-
mediated cellular activation, proliferation, and/or differentiation.
Disorders Related to the Immune and Nervous Systems
Diagnosis
The present inventors have discovered that Brainiac-5 is expressed not only in
ovarian tumor cells, but also (using BLAST analysis of the HGS EST database)
in
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bone marrow stromal cells and synovial sarcoma cells. For a number of immune
and/or nervous system-related and/or developmental disorders, substantially
altered
(increased or decreased) levels of Brainiac-5 gene expression can be detected
in
immune and/or nervous system tissue or other cells or bodily fluids (e.g.,
sera, plasma,
urine, synovial fluid or spinal fluid) taken from an individual having such a
disorder,
relative to a "standard" Brainiac-5 gene expression level, that is, the
Brainiac-5
expression levels in immune and/or nervous system tissues or bodily fluids
from an
individual not having the immune andlor nervous system disorder. Thus, the
invention
provides a diagnostic method useful during diagnosis of a immune and/or
nervous
system disorder, which involves measuring the expression level of the gene
encoding
the Brainiac-5 polypeptides in immune and/or nervous system tissue or other
cells or
body fluid from an individual and comparing the measured gene expression level
with
a standard Brainiac-5 gene expression level, whereby an increase or decrease
in the
gene expression level compared to the standard is indicative of an immune
and/or
nervous system disorder.
In particular, it is believed that certain tissues in mammals with cancer of
the
immune and nervous systems express significantly enhanced or reduced levels of
the
Brainiac-5 polypeptides and mRNA encoding the Brainiac-5 polypeptides when
compared to a corresponding "standard" level. Further, it is believed that
enhanced
levels of the Brainiac-5 polypeptides can be detected in certain body fluids
(e.g., sera,
plasma, urine, and spinal fluid) from mammals with such a cancer when compared
to
sera from mammals of the same species not having the cancer.
In specific embodiments, cancers that may be treated, prevented, and/or
diagnosed using Brainiac-5 polynucleotides, polypeptides, andlor agonists
and/or
antagonists of the invention include, but are not limited to, leukemia; acute
leukemia
(e.g., acute lymphocytic leukemia, acute rnyelocytic leukemia (specific
examples
thereof include myeloblastic, ;promyelocytic, myelomonocytic, monocytic, and
erythroleukemia)); chronic leukemia (e.g., chronic myelocytic (granulocytic)
leukemia, and chronic lymphocytic leukemia); polycythemia vera; lymphoma
(e.g.,
Hodgkin's disease, and non-Hodgkin's disease); multiple myeloma; Waldenstrom's
macroblogulinemia; heavy chain disease; and/or solid tumors (e.g., sarcomas
and
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carcinomas {specific examples thereof include fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcorna, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon
carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous
cell
carcinoma, basal cell carcinoma, adenomcarcinoma, sweat gland carcinoma,
sebaceous
gland carcinoma, papillary carcinoma, papillary adenocarcinoma,
cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatorna,
bile
duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,
cervical carcinoma, testicular cancer, lung carcinoma, small cell lung
carcinoma,
bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependyrnoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma).
Thus, the invention provides a diagnostic method useful during diagnosis of an
immune and/or nervous system disorder, including cancers of these systems,
which
involves measuring the expression level of the gene encoding the Brainiac-5
polypeptides in immune and/or nervous system tissue or other cells or body
fluid from
an individual and comparing the measured gene expression level with a standard
Brainiac-5 gene expression level, whereby an increase or decrease in the gene
expression level compared to the standard is indicative of an immune and/or
nervous
system disorder.
Where a diagnosis of a disorder in the immune and/or nervous systems
including diagnosis of a tumor, has already been made according to
conventional
methods, the present invention is useful as a prognostic indicator, whereby
patients
exhibiting enhanced or depressed Brainiac-5 gene expression will experience a
worse
clinical outcome relative to patients expressing the genes at a level nearer
the standard
level.
By "assaying the expression level of the genes encoding the Brainiac-5
polypeptides" is intended qualitatively or quantitatively measuring or
estimating the
level of the Brainiac-5 polypeptides or the level of the mRNA encoding the
Brainiac-S
polypeptides in a first biological sample either directly (e.g., by
determining or
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estimating absolute protein level or mRNA level) or relatively (e.g., by
comparing to
the Brainiac-5 polypeptide levels or mRNA levels in a second biological
sample).
Preferably, the Brainiac-5 polypeptides level or mRNA level in the first
biological
sample is measured or estimated and compared to a standard Brainiac-5
polypeptide
level or mRNA level, the standard being taken from a second biological sample
obtained from an individual not having the disorder or being determined by
averaging
levels from a population of individuals not having a disorder of the immune
and/or
nervous systems. As will be appreciated in the art, once a standard Brainiac-5
polypeptide level or mRNA level is known, it can be used repeatedly as a
standard for
comparison.
By "biological sample" is intended any biological sample obtained from an
individual, body fluid, cell line, tissue culture, or other source which
contains
Brainiac-5 polypeptides or mRNA. As indicated, biological samples include body
fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which
contain free
Brainiac-5 polypeptides, immune and/or nervous system tissue, and other tissue
sources found to express complete or mature Brainiac-5 polypeptides or a
Brainiac-5
receptor. Methods for obtaining tissue biopsies and body fluids from mammals
are
well known in the art. Where the biological sample is to include mRNA, a
tissue
biopsy is the preferred source.
The present invention is useful for treating, preventing, and/or diagnosing
various immune and/or nervous system-related disorders in mammals, preferably
humans. A nonexclusive list of preferred mammals includes monkeys, apes, cats,
dogs, cows, pigs, horses, rabbits, and humans. Humans are particularly
preferred
mammals. Such disorders include any disregulation of immune and/or nervous
system
cell and/or tissue function including, but not limited to Alzheimer's Disease,
Parkinson's Disease, Huntington's Disease, Tourette's Syndrome, epilepsy,
schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic
disorder, learning disabilities, ALS, psychoses , autism, and altered
behaviors,
including disorders in feeding, sleep patterns, balance, and perception,
neuronal
survival; synapse formation; conductance; neural differentiation,
autoimmunity,
arthritis, leukemias, lymphomas, immunosuppression, immunity, humoral
immunity,
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inflammatory bowel disease, myelosuppression, lymphoproliferative disorders,
in the
maintenance and differentiation of various hematopoietic lineages from early
hematopoietic stem and committed progenitor cells, anemia, pancytopenia,
leukopenia, thrombocytopenia or leukemia, bone marrow cell ex vivo culture,
bone
marrow transplantation, bone marrow reconstitution, radiotherapy or
chemotherapy of
neoplasia, asthma, immune deficiency diseases such as AIDS, rheumatoid
arthritis,
sepsis, acne, psoriasis, Grave's Disease, lymphocytic thyroiditis,
hyperthyroidism,
hypothyroidism, graft versus host reaction, graft versus host disease,
transplant
rejection, myelogenous leukemia, bone marrow fibrosis, and myeloproliferative
disease, Huntington's disease gene and other neurodegenerative diseases
including
spinocerebullar ataxia types I and III, dentatorubropallidoluysian and spinal
bulbar
muscular atrophy, and the like.
Total cellular RNA can be isolated from a biological sample using any suitable
technique such as the single-step guanidinium-thiocyanate-phenol-chloroform
method
described by Chomczynski and Sacehi (Anal. Biochem. 162:156-159 (1987)).
Levels
of mRNA encoding the Brainiac-5 polypeptides are then assayed using any
appropriate method. These include Northern blot analysis, S 1 nuclease
mapping, the
polymerase chain reaction {PCR), reverse transcription in combination with the
polymerase chain reaction (RT-PCR), and reverse transcription in combination
with
the ligase chain reaction (RT-LCR).
Assaying Brainiac-5 polypeptide levels in a biological sample can occur using
antibody-based techniques. For example, Brainiac-5 polypeptide expression in
tissues
can be studied with classical immunohistological methods (Jalkanen, M., et
al., J.
Cell. Biol. 101:976-985 ( 198:5); Jalkanen, M., et al., J. Cell. Biol.
105:3087-3096
{ 1987)). Other antibody-based methods useful for detecting Brainiac-5 gene
expression include immunoassays, such as the enzyme linked immunosorbent assay
a
(ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are
known
in the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such
as iodine (''SI, '2'I), carbon ('~C), sulfur (i5S), tritium (3H), indium (' ~'-
In), and
technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine,
and
biotin.
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In addition to assaying Brainiac-5 polypeptide levels in a biological sample
obtained from an individual, Brainiac-5 polypeptides can also be detected in
vivo by
imaging. Antibody labels or markers for in vivo imaging of Brainiac-5
polypeptides
include those detectable by X-radiography, NMR or ESR. For X-radiography,
suitable
S labels include radioisotopes such as barium or cesium, which emit detectable
radiation
but are not overtly harmful to the subject. Suitable markers for NMR and ESR
include
those with a detectable characteristic spin, such as deuterium, which may be
incorporated into the antibody by labeling of nutrients for the relevant
hybridoma.
A Brainiac-5-specific antibody or antibody fragment, which has been labeled
with an appropriate detectable imaging moiety, such as a radioisotope (for
example,
"'I, "zIn, ~'"'Tc), a radio-opaque substance, or a material detectable by
nuclear
magnetic resonance, is introduced (for example, parenterally, subcutaneously
or
intraperitoneally) into the mammal to be examined for immune system disorder.
It
will be understood in the art that the size of the subject and the imaging
system used
I S will determine the quantity of imaging moiety needed to produce diagnostic
images.
In the case of a radioisotope moiety, for a human subject, the quantity of
radioactivity
injected will normally range from about S to 20 millicuries of ~'"'Te. The
labeled
antibody or antibody fragment will then preferentially accumulate at the
location of
cells which contain Brainiac-5 polypeptides. In vivo tumor imaging is
described by
Burchiel and coworkers (Chapter 13 in Tumor Imaging: The Radiochemical
Detection
of Cancer, Burchiel, S. W. and Rhodes, B. A., eds., Masson Publishing Inc.
(1982)).
Treatment
As noted above, Brainiac-5 polynucleotides and polypeptides are useful for
diagnosis of conditions involving abnormally high or low expression of
Brainiac-5
activities. Given the cells and tissues where Brainiac-5 poIypeptides are
expressed as
well as the activities modulated by Brainiac-5 polypeptides, it is readily
apparent that a
substantially altered (increased or decreased) level of expression of Brainiac-
5
polypeptides in an individual compared to the standard or "normal" level
produces
pathological conditions related to the bodily systems) in which Brainiac-5
polypeptides are expressed and/or is active.
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It is well-known in the art that, in addition to a specific cellular function,
cellular receptor molecules :may also often be exploited by a virus as a means
of
initiating entry into a potential host cell. For example, it was recently
discovered by
Wu and colleagues (J. Exp. .Med. 185:1681-1691 ( 1997)) that the cellular
chemokine
receptor CCRS functions not only as a cellular chemokine receptor, but also as
a
receptor for macrophage-tropic human immunodeficiency virus (HIV)-1. In
addition,
RANTES, MIP-la, and MIP-lb, which are agonists for the cellular chemokine
receptor CCRS, inhibit entry of various strains of HIV-1 into susceptible cell
lines
(Cocchi, F., et al., Science 270:1811-1815 ( 1995)). Thus, the invention also
provides
a method of treating, preventing, and/or diagnosing an individual exposed to,
or
infected with, a virus through the prophylactic or therapeutic administration
of
Brainiac-5 polypeptides, or an agonist or antagonist thereof, to block or
disrupt the
interaction of a viral particle with the Brainiac-5 receptors and, as a
result, block the
initiation or continuation of viral infectivity.
The Brainiac-5 polypeptides of the present invention binds to the Brainiac-5
receptor and, as such, is likely to block immune-tropic viral infections.
Agonists and
antagonists of the Brainiac-SBrainiac-5 Receptor interaction are also likely
to
interfere with immune-tropic viral infection. As a result, such an interaction
is likely
to interfere with the infectious life cycle of one or more immune-tropic
viruses such as
HIV-l, HIV-2, HTLV-III, HSV-1, HSV-2, and the like.
The ability of Brainiac-5 polypeptides of the present invention, or agonists
or
antagonists thereof, to prophylactically or therapeutically block viral
infection may be
easily tested by the skilled artisan. For example, Simmons and coworkers
(Science
276:276-279 ( 1997)) and Arenzana-Seisdedos and colleagues (Nature 383:400 (
1996))
each outline a method of observing suppression of HIV-1 infection by an
antagonist of
the CCRS chemokine receptor and of the CC chemokine RANTES, respectively, in
cultured peripheral blood mononuclear cells. Cells are cultured and infected
with a
virus, HIV-1 in both cases noted above. An agonist or antagonist of the CC
chemokine or its receptor is then immediately added to the culture medium.
Evidence
of the ability of the agonist or antagonist of the chemokine or cellular
receptor is
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determined by evaluating the relative success of viral infection at 3, 6, and
9 days
postinfection.
Administration of a pharmaceutical composition comprising an amount of an
isolated Brainiac-5 polypeptide, or an agonist or antagonist thereof, of the
invention to
an individual either infected with a virus or at risk for infection with a
virus is
performed as described below.
It will also be appreciated by one of ordinary skill that, since the Brainiac-
5
polypeptides of the invention is a member of the Brainiac family, the mature
secreted
form of the polypeptide may be released in soluble form from the cells which
express
the Brainiac-5 polypeptides by proteolytic cleavage. Therefore, when the
mature form
of a Brainiac-5 polypeptide is added from an exogenous source to cells,
tissues or the
body of an individual, the polypeptide will exert its physiological activities
on its
target cells of that individual.
Therefore, it will be appreciated that conditions caused by a decrease in the
standard or normal level of Brainiac-5 activity in an individual, particularly
disorders
of the immune and/or nervous systems, can be treated, prevented, and/or
diagnosed by
administration of Brainiac-5 polypeptide (preferably in the form of the mature
form of
the polypeptide). Thus, the invention also provides a method of treatment,
prevention,
and/or diagnosis of an individual in need of an increased level of Brainiac-5
activity
comprising administering to such an individual a pharmaceutical composition
comprising an amount of an isolated Brainiac-5 polypeptide of the invention,
particularly a mature form of the Brainiac-5 polypeptides of the invention,
effective to
increase the Brainiac-5 polypeptide activity level in such an individual.
Brainiac-5 polypeptides are believed to elicit a potent cellular response
including any genotypic, phenotypic, and/or morphologic change to the cell,
cell line,
tissue, tissue culture or patient. As indicated, such cellular responses
include not only
normal physiological responses to Brainiac-5, but also diseases associated
with
increased apoptosis or the inhibition of apoptosis. Apoptosis-programmed cell
death-
is a physiological mechanism involved in the deletion of peripheral B and/or T
lymphocytes of the immune system, and its disregulation can lead to a number
of
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different pathogenic processes (J.C. Ameisen, AIDS 8:1197-1213 ( 1994); P.H.
Krammer et al., Curr. Opin. Immunol. 6:279-289 ( 1994)).
Diseases associated with increased cell survival, or the inhibition of
apoptosis,
and which may be treated or prevented with polynucleotides, polypeptides,
and/or
agonists or antagonists of the invention, include, but are not limited to,
cancers (such
as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent
tumors, including, but not limited to, colon cancer, cardiac tumors,
pancreatic cancer,
melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular
cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcorna, chondrosarcoma, adenoma, breast
cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune
disorders
(such as systemic lupus erythematosus and immune-related glomerulonephritis
rheumatoid arthritis); viral infections (such as herpes viruses, pox viruses
and
adenoviruses); inflammation; graft vs. host disease; acute graft rejection and
chronic
graft rejection. Thus, in preferred embodiments Brainiac-5 polynucleotides or
polypeptides of the inventian are used to treat, prevent, and/or diagnose
autoimmune
diseases and/or inhibit the growth, progression, and/or metastasis of cancers,
including, but not limited to, those cancers disclosed herein, such as, for
example,
lymphocytic leukemias (including, for example, MLL and chronic lymphocytic
leukemia (CLL)) and follicular lymphomas. In another embodiment Brainiac-5
polynucleotides or polypeptides of the invention are used to activate,
differentiate or
proliferate cancerous cells or tissue (e.g., B cell lineage related cancers
(e.g., CLL and
MLL), lymphocytic leukemia, or lymphoma) and thereby render the cells more
vulnerable to cancer therapy (e.g., chemotherapy or radiation therapy).
Moreover, in other embodiments, Brainiac-5 polynucleotides or polypeptides
of the invention are used to inhibit the growth, progression, and/or
metastases of
malignancies and related disorders such as leukemia (including acute leukemias
(e.g.,
acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,
promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic
leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic
lymphocytic
leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and
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non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia,
heavy
chain disease, and solid tumors including, but not limited to, sarcomas and
carcinomas
such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,
leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,
cervical
cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis include AIDS; neurodegenerative
disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic
lateral
sclerosis, Retinitis pigmentosa, Cerebellar degeneration); myelodysplastic
syndromes
(such as aplastic anemia), ischemic injury (such as that caused by myocardial
infarction, stroke and reperfusion injury), toxin-induced liver disease (such
as that
caused by alcohol), septic shock, cachexia and anorexia. Thus, in preferred
embodiments Brainiac-5 polynucleotides or polypeptides of the invention are
used to
treat, prevent, and/or diagnose the diseases and disorders listed above.
In preferred embodiments, Brainiac-5 polypeptides of the invention inhibit the
growth of human histiocytic lymphoma U-937 cells in a dose-dependent manner.
In
additional preferred embodiments, Brainiac-5 polypeptides of the invention
inhibit the
growth of PC-3 cells, HT-29 cells, HeLa cells, MCF-7 cells, and A293 cells. In
highly
preferred embodiments, Brainiac-5 polynucleotides or polypeptides of the
invention
are used to inhibit growth, progression, and/or metastasis of prostate cancer,
colon
cancer, cervical carcinoma, and breast carcinoma.
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Because Brainiac-5 belongs to the Brainiac family, the polypeptides should
also modulate angiogenesis. In addition, since Brainiac-5 inhibits immune cell
functions, the polypeptides will have a wide range of anti-inflammatory
activities.
Brainiac-5 may be employed as an anti-neovascularizing agent to treat,
prevent, and/or
diagnose solid tumors by stimulating the invasion and activation of host
defense cells,
e.g., cytotoxic T cells and macrophages and by inhibiting the angiogenesis of
tumors.
Those of skill in the art will recognize other non-cancer indications where
blood vessel
proliferation is not wanted. They may also be employed to enhance host
defenses
against resistant chronic and acute infections, for example, myobacterial
infections via
the attraction and activation of microbicidal leukocytes. Brainiac-S may also
be
employed to inhibit T-cell proliferation by the inhibition of IL-2
biosynthesis for the
treatment of T-cell mediated auto-immune diseases and lymphocytic leukemias
(including, for example, chronic lymphocytic leukemia (CLL)). Brainiac-5 may
also
be employed to stimulate wound healing, both via the recruitment of debris
clearing
and connective tissue promoting inflammatory cells. In this same manner,
Brainiac-5
may also be employed to treat, prevent, and/or diagnose other fibrotic
disorders,
including liver cirrhosis, osteoarthritis and pulmonary fibrosis. Brainiac-5
also
increases the presence of eosinophils that have the distinctive function of
killing the
larvae of parasites that invade tissues, as in schistosomiasis, trichinosis
and ascariasis.
It may also be employed to regulate hematopoiesis, by regulating the
activation and
differentiation of various hematopoietic progenitor cells, for example, to
release
mature leukocytes from the bone marrow following chemotherapy, i.e., in stem
cell
mobilization. Brainiac-5 may also be employed to treat, prevent, and/or
diagnose
sepsis.
Brainiac-5 polynucleotides or polypeptides, or agonists of Brainiac-5, can be
used in the treatment of infectious agents. For example, by increasing the
immune
response, particularly increasing the proliferation and differentiation of B
cells,
infectious diseases may be treated. The immune response may be increased by
either
enhancing an existing immune response, or by initiating a new immune response.
Alternatively, Brainiac-5 polynucleotides or polypeptides, or agonists or
antagonists of
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Brainiac-5, may also directly inhibit the infectious agent, without
necessarily eliciting
an immune response.
Viruses are one example of an infectious agent that can cause disease or
symptoms that can be treated by Brainiac-5 polynucleotides or polypeptides, or
agonists of Brainiac-5. Examples of viruses, include, but are not limited to
the
following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae,
Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,
Circoviridae,
Coronaviridae, Dengue, EB V, HIV, Flaviviridae, Hepadnaviridae (Hepatitis),
Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),
Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),
Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma
virus,
Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or
Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,
Lentivirus),
and Togaviridae (e.g., Rubivirus). Viruses falling within these families can
cause a
I S variety of diseases or symptoms, including, but not limited to: arthritis,
bronchiollitis,
respiratory syncytial virus, encephalitis, eye infections (e.g.,
conjunctivitis, keratitis),
chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),
Japanese B
encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis,
opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma,
chickenpox,
hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold,
Polio,
leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g.,
Kaposi's, warts),
and viremia. Brainiac-5 polynucleotides or polypeptides, or agonists or
antagonists of
Brainiac-5, can be used to treat, prevent, diagnose, and/or detect any of
these
symptoms or diseases. In specific embodiments, Brainiac-5 polynucleotides,
polypeptides, or agonists are used to treat, prevent, andlor diagnose:
meningitis,
Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific
embodiment Brainiac-5 polynucleotides, polypeptides, or agonists are used to
treat
patients nonresponsive to one or more other commercially available hepatitis
vaccines.
In a further specific embodiment, Brainiac-S polynucleotides, polypeptides, or
agonists are used to treat, prevent, and/or diagnose AIDS. In an additional
specific
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embodiment Brainiac-5 polypeptides, agonists, andlor antagonists are used to
treat,
prevent, and/or diagnose patients with cryptosporidiosis.
Similarly, bacterial or fungal agents that can cause disease or symptoms and
that can be treated by Brainiac-5 polynucleotides or polypeptides, or agonists
or
S antagonists of Brainiac-5, include, but not limited to, the following Gram-
Negative
and Gram-positive bacteria and bacterial families and fungi: Actinomycetales
(e.g.,
Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans,
Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae,
Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi, Brucellosis,
Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis,
Dermatocycoses,
E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),
Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, and
Salmonella
paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis,
Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio
cholerae,
Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria
meningitidis,
Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,
Heamophilus
influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae,
Syphilis, Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and
Streptococcal (e.g., Streptococcus pneumoniae and Group B Streptococcus).
These
bacterial or fungal families c:an cause the following diseases or symptoms,
including,
but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis,
tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS
related
infections), paronychia, prosthesis-related infections, Reiter's Disease,
respiratory tract
infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-
Scratch
Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia,
Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis,
Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism,
gangrene,
tetanus, impetigo, Rheumatic: Fever, Scarlet Fever, sexually transmitted
diseases, skin
diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract
infections, wound
infections. Brainiac-5 polynucleotides or polypeptides, or agonists or
antagonists of
Brainiac-S, can be used to treat, prevent, diagnose, and/or detect any of
these
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symptoms or diseases. In specific embodiments, Brainiac-5 polynucleotides,
polypeptides, or agonists thereof are used to treat, prevent, and/or diagnose:
tetanus,
Diptheria, botulism, and/or meningitis type B.
Moreover, parasitic agents causing disease or symptoms that can be treated by
Brainiac-5 polynucleotides or polypeptides, or agonists of Brainiac-5,
include, but not
limited to, the following families or class: Amebiasis, Babesiosis,
Coccidiosis,
Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis,
Helminthiasis,
Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas
and
Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium
malariae
and Plasmodium ovate). These parasites can cause a variety of diseases or
symptoms,
including, but not limited to: Scabies, Trombiculiasis, eye infections,
intestinal
disease (e.g., dysentery, giardiasis), liver disease, lung disease,
opportunistic infections
{e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis.
Brainiac-5
polynucleotides or polypeptides, or agonists or antagonists of Brainiac-5, can
be used
to treat, prevent, diagnose, and/or detect any of these symptoms or diseases.
In
specific embodiments, Brainiac-5 polynucleotides, polypeptides, or agonists
thereof
are used to treat, prevent, and/or diagnose malaria.
In another embodiment, the invention provides a method of delivering
compositions containing the polypeptides of the invention (e.g., compositions
containing Brainiac-5 polypeptides or anti-Brainiac-5 antibodies associated
with
heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to
targeted
cells, such as, for example, cells expressing Brainiac-5 receptor. Brainiac-5
polypeptides or anti-Brainiac-5 antibodies of the invention may be associated
with
heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via
hydrophobic, hydrophilic, ionic and/or covalent interactions.
In one embodiment, the invention provides a method for the specific delivery
of compositions of the invention to cells by administering polypeptides of the
invention (e.g., Brainiac-5 polypeptides or anti-Brainiac-S antibodies) that
are
associated with heterologous polypeptides or nucleic acids. In one example,
the
invention provides a method for delivering a therapeutic protein into the
targeted cell.
In another example, the invention provides a method for delivering a single
stranded
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nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid
(e.g., DNA
that can integrate .into the cell's genome or replicate episomally and that
can be
transcribed) into the targeted cell.
In another embodiment, the invention provides a method for the specific
S destruction of cells (e.g., the destruction of tumor cells) by administering
polypeptides
of the invention (e.g., Brainiac-5 polypeptides or anti-Brainiac-5 antibodies)
in
association with toxins or cytotoxic prodrugs.
By "toxin" is meant compounds that bind and activate endogenous cytotoxic
effector systems, radioisotopes, holotoxins, modified toxins, catalytic
subunits of
toxins, or any molecules or enzymes not normally present in or on the surface
of a cell
that under defined conditions cause the cell's death. Toxins that may be used
according to the methods of the invention include, but are not limited to,
radioisotopes
known in the art, compounds such as, for example, antibodies (or complement
fixing
containing portions thereof) that bind an inherent or induced endogenous
cytotoxic
effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin,
abrin,
Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed
antiviral protein, alpha-sarcin and cholera toxin. "Toxin" also includes a
cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-
emitters such
as, for example, z"Bi. A cytotoxin or cytotoxic agent includes any agent that
is
detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin
D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine,
colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs
thereof.
Therapeutic agents include, but are not limited to, antimetabolites (e.g.,
methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
alkylating
t
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine
(BSNU)
and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP)
cisplatin),
anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin),
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antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and
anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine).
By "cytotoxic prod~~ug" is meant a non-toxic compound that is converted by an
enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic
prodrugs
that may be used according to the methods of the invention include, but are
not limited
to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate
derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin,
and
phenoxyacetamide derivatives of doxorubicin.
An additional condition, disease or symptom that can be treated, prevented,
andlor diagnosed by Brainiac-5 polynucleotides or polypeptides, or agonists of
Brainiac-5, is osteomyelitis.
An additional condition, disease or symptom that can be treated, prevented,
and/or diagnosed by Brainiac-S polynucleotides or polypeptides, or agonists of
Brainiac-5, is endocarditis.
Preferably, treatment using Brainiac-5 polynucleotides or polypeptides, or
agonists of Brainiac-5, could either be by administering an effective amount
of
Brainiac-5 polypeptide to the patient, or by removing cells from the patient,
supplying
the cells with Brainiac-5 polynucleotide, and returning the engineered cells
to the
patient (ex vivo therapy). Moreover, as further discussed herein, the Brainiac-
5
polypeptide or polynucleotide can be used as an adjuvant in a vaccine to raise
an
immune response against infectious disease.
Additional preferred embodiments of the invention include, but are not limited
to, the use of Brainiac-5 and functional agonists thereof, in the following
applications:
Administration to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig,
pigs,
micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human
primate, and
human, most preferably human) to boost the immune system to produce increased
s
quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce
higher
affinity antibody production (e.g., IgG, IgA, IgM, and IgEj, and/or to
increase an
immune response.
Administration to an animal (including, but not limited to, those listed
above,
and also including transgenic animals) incapable of producing functional
endogenous
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antibody molecules or having an otherwise compromised endogenous immune
system,
but which is capable of producing human immunoglobulin molecules by means of a
reconstituted or partially reconstituted immune system from another animal
(see, e.g.,
published PCT Application Nos. W098/24893, WO/9634096, WO/9633735, and
WO/9110741.
The antagonists may be employed for instance to inhibit Brainiac-S-mediated
chemotaxis and activation raf macrophages and their precursors, and of
neutrophils,
basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8
cytotoxic T
cells and natural killer cells, in certain auto-immune and chronic
inflammatory and
infective diseases. Examples of auto-immune diseases include multiple
sclerosis, and
insulin-dependent diabetes. The antagonists may also be employed to treat,
prevent,
and/or diagnose infectious diseases including silicosis, sarcoidosis,
idiopathic
pulmonary fibrosis by preventing the recruitment and activation of mononuclear
phagocytes. They may also be employed to treat, prevent, and/or diagnose
idiopathic
hyper-eosinophilic syndrome by preventing eosinophil production and migration.
Endotoxic shock may also be treated by the antagonists by preventing the
migration of
macrophages and their production of the Brainiac-5 polypeptides of the present
invention. The antagonists may also be employed for treating atherosclerosis,
by
preventing monocyte infiltration in the artery wall. The antagonists may also
be
employed to treat, prevent, andlor diagnose histamine-mediated allergic
reactions and
immunological disorders including late phase allergic reactions, chronic
urticaria, and
atopic dermatitis by inhibiting chemokine-induced mast cell and basophil
degranulation and release of histamine. IgE-mediated allergic reactions such
as
allergic asthma, rhinitis, and eczema may also be treated. The antagonists may
also be
employed to treat, prevent, and/or diagnose chronic and acute inflammation by
preventing the attraction of monocytes to a wound area. They may also be
employed
to regulate normal pulmonary macrophage populations, since chronic and acute
inflammatory pulmonary diseases are associated with sequestration of
mononuclear
phagocytes in the lung. Antagonists may also be employed to treat, prevent,
and/or
diagnose rheumatoid arthritis by preventing the attraction of monocytes into
synovial
fluid in the joints of patients. Monocyte influx and activation plays a
significant role
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in the pathogenesis of both degenerative and inflammatory arthropathies. The
antagonists may be employed to interfere with the deleterious cascades
attributed
primarily to IL-1 and TNF, which prevents the biosynthesis of other
inflammatory
cytokines. In this way, the antagonists may be employed to prevent
inflammation.
S The antagonists may also be employed to inhibit prostaglandin-independent
fever
induced by Brainiac-S. Thf; antagonists may also be employed to treat,
prevent, and/or
diagnose cases of bone marrow failure, for example, aplastic anemia and
myelodysplast'ic syndrome. The antagonists may also be employed to treat,
prevent,
and/or diagnose asthma and allergy by preventing eosinophil accumulation in
the lung.
The antagonists may also be employed to treat, prevent, and/or diagnose
subepithelial
basement membrane fibrosis which is a prominent feature of the asthmatic lung.
The
antagonists may also be employed to treat, prevent, and/or diagnose lymphomas
(e.g.,
one or more of the extensive, but not limiting, list of lymphomas provided
herein).
All of the above described applications as they may apply to veterinary
1 S medicine. Moreover, all applications described herein may also apply to
veterinary
medicine.
Antibodies against Brainiac-S may be employed to bind to and inhibit
Brainiac-S activity to treat, prevent, and/or diagnose ARDS, by preventing
infiltration
of neutrophils into the lung after injury. The antagonists and antagonists of
the instant
may be employed in a composition with a pharmaceutically acceptable carrier,
e.g., as
described hereinafter.
Brainiac-S polynucleotides or polypeptides of the invention and/or agonists
and/or antagonists thereof, are used to treat, prevent, and/or diagnose
various immune
system-related disorders andlor conditions associated with these disorders, in
2S mammals, preferably humans. Many autoimmune disorders result from
inappropriate
recognition of self as foreign material by immune cells. This inappropriate
recognition
results in an immune response leading to the destruction of the host tissue.
Therefore,
the administration of Brainiac-5 polynucleotides or polypeptides of the
invention
and/or agonists and/or antagonists thereof that can inhibit an immune
response,
particularly the proliferation, differentiation, or chemotaxis of T cells, may
be an
effective therapy in treating and/or preventing autoimmune disorders. Thus, in
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preferred embodiments, Brainiac-5 antagonists of the invention (e.g.,
polypeptide
fragments of Brainiac-5 and anti-Brainiac-5 antibodies) are used to treat,
prevent,
and/or diagnose an autoimmune disorder.
Such autoimmune disorders include, but are not limited to, autoimmune
diseases such as, for example, autoimmune hemolytic anemia, autoimmune
neonatal
thrombocytopenia, autoimrnunocytopenia, hemolytic anemia, antiphospholipid
syndrome, dermatitis, allergic encephalomyelitis, glomerulonephritis, Multiple
Sclerosis, Neuritis, Ophthalmia, Polyendocrinopathies, Purpura, Reiter's
Disease,
Stiff Man Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Bane
Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye
disease.
Additional autoimmune disorders (that are highly probable) include, but are
not
limited to, autoimmune thyraiditis (i.e., Hashimoto's thyroiditis) (often
characterized,
e.g., by cell-mediated and humoral thyroid cytotoxicity), systemic lupus
erhthematosus
(often characterized, e.g., by circulating and locally generated immune
complexes),
Goodpasture's syndrome (often charac~.erized, e.g., by anti-basement membrane
antibodies), Pemphigus (often characterized, e.g., by epidermal acantholytic
antibodies), Receptor autoimmunities such as, for example, (a) Graves' Disease
(often
characterized, e.g., by TSH receptor antibodies), (b) Myasthenia Gravis (often
characterized, e.g., by acetylc:holine receptor antibodies). and (c) insulin
resistance
(often characterized, e.g., by insulin receptor antibodies), autoimmune
hemolytic
anemia (often characterized, e.g., by phagocytosis of antibody-sensitized
RBCs),
autoimmune thrombocytopenic purpura (often characterized, e.g., by
phagocytosis of
antibody-sensitized platelets.
Additional autoimmune disorders (that are probable) include, but are not
limited to, rheumatoid arthritis (often characterized, e.g., by immune
complexes in
joints), scleroderma with anti-collagen antibodies (often characterized, e.g.,
by
nucleolar and other nuclear antibodies), mixed connective tissue disease
(often
characterized, e.g., by antibodies to extractable nuclear antigens (e.g.,
ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone
ANA),
pernicious anemia (often characterized, e.g., by antiparietal cell,
microsomes, and
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intrinsic factor antibodies).. idiopathic Addison's disease (often
characterized, e.g., by
humoral and cell-mediated adrenal cytotoxicity, infertility (often
characterized, e.g., by
antispermatozoal antibodies), glomerulonephritis (often characterized, e.g.,
by
glomerular basement membrane antibodies or immune complexes), bullous
pemphigoid (often characterized, e.g., by IgG and complement in basement
membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue
antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes millitus (often
characterized, e.g., by cell-mediated and humoral islet cell antibodies), and
adrenergic
drug resistance (including adrenergic drug resistance with asthma or cystic
fibrosis)
(often characterized, e.g., by beta-adrenergic receptor antibodies).
Additional autoimmune disorders (that are possible) include, but are not
limited to, chronic active hepatitis (often characterized, e.g., by smooth
muscle
antibodies), primary biliary cirrhosis (often characterized, e.g., by
mitchondrial
antibodies), other endocrine gland failure (often characterized, e.g., by
specific tissue
antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte
antibodies), vasculitis (often characterized, e.g., by Ig and complement in
vessel walls
and/or low serum complement), post-MI (often characterized, e.g., by
myocardial
antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial
antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies
to IgE),
atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to
IgE), asthma
(often characterized, e.g., by IgG and IgM antibodies to IgE), and many other
inflammatory, granulamatous, degenerative, and atrophic disorders.
In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions associated with the diseases and disorders recited above are
treated,
prevented, and/or diagnosed using anti-Brainiac-5 antibodies.
Similarly, allergic reactions and conditions, such as asthma (particularly
allergic asthma) or other respiratory problems, may also be treated by
Brainiac-5
polynucleotides or polypeptides of the invention and/or agonists and/or
antagonists
thereof. Moreover, these molecules can be used to treat, prevent, and/or
diagnose
anaphylaxis, hypersensitivity to an antigenic molecule, or blood group
incompatibility.
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Brainiac-5 polynucleotides or polypeptides of the invention and/or agonists
and/or antagonists thereof, may also be used to treat, prevent, and/or
diagnose organ
rejection or graft-versus-host disease (GVHD) and/or conditions associated
therewith.
Organ rejection occurs by host immune cell destruction of the transplanted
tissue
through an immune response. Similarly, an immune response is also involved in
GVHD, but, in this case, the foreign transplanted immune cells destroy the
host
tissues. The administration of Brainiac-5 polynucleotides or polypeptides of
the
invention and/or agonists and/or antagonists thereof, that inhibits an immune
response,
particularly the proliferation, differentiation, or chemotaxis of T-cells, may
be an
effective therapy in preventing organ rejection or GVHD.
Similarly, Brainiac-5 polynucleotides or polypeptides of the invention and/or
agonists and/or antagonists thereof, may also be used to modulate
inflammation. For
example, Brainiac-5 polynucleotides or polypeptides of the invention and/or
agonists
and/or antagonists thereof, may inhibit the proliferation and differentiation
of cells
involved in an inflammatory response. These molecules can be used to treat,
prevent,
and/or diagnose inflammatory conditions, both chronic and acute conditions,
including
chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation
associated with infection (e.g., septic shock, sepsis, or systemic
inflammatory response
syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,
complement-mediated hyperacute rejection, nephritis, cytokine or chemokine
induced
lung injury, inflammatory bowel disease, Crohn's disease, or resulting from
over
production of cytokines (e.g., TNF or IL-1.)
In additional preferred embodiments, the present invention is directed to a
method for enhancing apoptosis induced by a Brainiac-5 polypeptide, which
involves
administering to a cell which expresses a Brainiac-5 receptor an effective
amount of
Brainiac-5, analog or an agonist capable of increasing Brainiac-5-mediated
signaling.
Preferably, Brainiac-S-mediated signaling is increased to treat, prevent,
and/or
diagnose a disease wherein decreased apoptosis or decreased cytokine and
adhesion
molecule expression is exhibited. An agonist can include soluble forms of
Brainiac-5
and monoclonal antibodies directed against the Brainiac-5 polypeptide.
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In a further embodiment, the present invention is directed to a method for
inhibiting apoptosis induced by a Brainiac-5 polypeptide, which involves
administering to a cell which expresses the Brainiac-5 receptor an effective
amount of
an antagonist capable of decreasing Brainiac-5-mediated signaling. Preferably,
Brainiac-5-mediated signaling is decreased to treat, prevent, and/or diagnose
a disease
wherein increased apoptosis or NF-kappaB expression is exhibited. An
antagonist can
include soluble forms of Brainiac-5 and monoclonal antibodies directed against
the
Brainiac-5 polypeptide.
Polynucleotides and/or polypeptides of the invention and/or agonists and/or
antagonists thereof are useful in the diagnosis and treatment or prevention of
a wide
range of diseases and/or conditions. Such diseases and conditions include, but
are not
limited to, cancer (e.g., immune cell related cancers, breast cancer, prostate
cancer,
ovarian cancer, follicular lymphoma, cancer associated with mutation or
alteration of
p53, brain tumor, bladder cancer, uterocervical cancer, colon cancer,
colorectal cancer,
non-small cell carcinoma of the lung, small cell carcinoma of the lung,
stomach
cancer, etc.), lymphoproliferative disorders (e.g., lymphadenopathy),
microbial (e.g.,
viral, bacterial, etc.) infection (e.g., HIV-1 infection, HIV-2 infection,
herpesvirus
infection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-
7,
EBV), adenovirus infection, hoxvirus infection, human papilloma virus
infection,
hepatitis infection (e.g., HAV, HBV, HCV, etc.), Helicobacter pylori
infection,
invasive Staphylococcia, etc.), parasitic infection, nephritis, bone disease
(e.g.,
osteoporosis), atherosclerosis, pain, cardiovascular disorders (e.g.,
neovascularization,
hypovascularization or reduced circulation (e.g., ischemic disease (e.g.,
myocardial
infarction, stroke, etc.))), AIDS, allergy, inflammation, neurodegenerative
disease
(e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral
sclerosis,
pigmentary retinitis, cerebellar degeneration, etc.), graft rejection (acute
and chronic),
graft vs. host disease, diseases due to osteomyelodysplasia (e.g., aplastic
anemia, etc.),
joint tissue destruction in rheumatism, liver disease (e.g., acute and chronic
hepatitis,
liver injury, and cirrhosis), autoimmune disease (e.g., multiple sclerosis,
rheumatoid
arthritis, systemic lupus erythematosus, immune complex glomerulonephritis,
autoimmune diabetes, autoimmune thrombocytopenic purpura, Grave's disease,
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Hashimoto's thyroiditis, etc.), cardiomyopathy (e.g., dilated cardiomyopathy),
diabetes, diabetic complications (e.g., diabetic nephropathy, diabetic
neuropathy,
diabetic retinopathy), influenza, asthma, psoriasis, glomerulonephritis,
septic shock,
and ulcerative colitis.
Polynucleotides and/or polypeptides of the invention and/or agonists and/or
antagonists thereof are useful in promoting angiogenesis, wound healing (e.g.,
wounds, burns, and bone fractures). Polynucleotides and/or polypeptides of the
invention and/or agonists and/or antagonists thereof are also useful as an
adjuvant to
enhance immune responsiveness to specific antigen, anti-viral immune
responses,.
More generally, polynucleotides and/or polypeptides of the invention and/or
agonists and/or antagonists thereof are useful in regulating (i.e., elevating
or reducing)
immune response. For example, polynucleotides and/or polypeptides of the
invention
may be useful in preparation or recovery from surgery, trauma, radiation
therapy,
chemotherapy, and transplantation, or may be used to boost immune response
and/or
recovery in the elderly and immunocompromised individuals. Alternatively,
polynucleotides and/or polypeptides of the invention and/or agonists andlor
antagonists thereof are useful as immunosuppressive agents, for example in the
treatment or prevention of autoimmune disorders. In specific embodiments,
polynucleotides andlor polypeptides of the invention are used to treat or
prevent
chronic inflammatory, allergic or autoimmune conditions, such as those
described
herein or are otherwise known in the art.
Formulations
The Brainiac-5 polypeptide composition will be formulated and dosed in a
fashion consistent with good medical practice, taking into account the
clinical
condition of the individual patient (especially the side effects of treatment,
prevention,
and/or diagnosis with Brainiac-5 polypeptides alone), the site of delivery of
the
Brainiac-5 polypeptide composition, the method of administration, the
scheduling of
administration, and other factors known to practitioners. The "effective
amount" of
Brainiac-5 polypeptide for purposes herein is thus determined by such
considerations.
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As a general proposition, the total pharmaceutically effective amount of
Brainiac-5 polypeptide administered parenterally per dose will be in the range
of about
1 Ng/kg/day to 10 mg/kg/day of patient body weight, although, as noted above,
this
will be subject to therapeutic discretion. More preferably, this dose is at
least 0.01
mg/kg/day, and most preferably for humans between about 0.01 and I mg/kg/day
for
the hormone. If given continuously, the Brainiac-5 polypeptide is typically
administered at a dose rate of about 1 ~g/kg/hour to about 50 ug/kg/hour,
either by I-4
injections per day or by continuous subcutaneous infusions, for example, using
a mini-
pump. An intravenous bag solution may also be employed. The length of
treatment
needed to observe changes and the interval following treatment for responses
to occur
appears to vary depending on the desired effect.
Effective dosages of the compositions of the present invention to be
administered may be determined through procedures well known to those in the
art
which address such parameters as biological half life, bioavailability, and
toxicity.
Such determination is well within the capability of those skilled in the art,
especially in
light of the detailed disclosure provided herein.
Bioexposure of an organism to Brainiac-5 polypeptide during therapy may also
play an important role in determining a therapeutically and/or
pharmacologically
effective dosing regime. Variations of dosing such as repeated administrations
of a
relatively low dose of Brainiac-5 polypeptide for a relatively long period of
time may
have an effect which is therapeutically and/or pharmacologically
distinguishable from
that achieved with repeated administrations of a relatively high dose of
Brainiac-5 for
a relatively short period of time. See, for instance, the serum immunoglobulin
level
experiments presented in Example 6.
Using the equivalent surface area dosage conversion factors supplied by
Freireich, E. J., et al. (Cancer Chemotherapy Reports 50(4):219-44 ( 1966)),
one of
ordinary skill in the art is able to conveniently convert data obtained from
the use of
Brainiac-5 in a given experimental system into an accurate estimation of a
pharmaceutically effective amount of Brainiac-5 polypeptide to be administered
per
dose in another experimental system. Experimental data obtained through the
administration of Brainiac-5 in mice may be converted through the conversion
factors
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supplied by Freireich, et al., to accurate estimates of pharmaceutically
effective doses
of Brainiac-5 in rat, monkey, dog, and human. The following conversion table
(Table
III) is a summary of the data provided by Freireich, et al. Table III gives
approximate
factors for converting doses expressed in terms of mg/kg from one species to
an
equivalent surface area dose expressed as mg/kg in another species tabulated.
Table III. Equivalent Surface Area Dosage Conversion Factors.
--TO--
Mouse Rat Monkey Dog Human
--FROM-- (20g~ (150g) (3.Skg~gZ (60kg)
Mouse 1 1/2 1/4 1/6 1/12
Rat ? 1 1/2 I/4 1/7
Monkey 4 2 1 3/5 1/3
Dog 6 4 5/3 1 1/2
Human 12 7 3 2 1
Thus, for example, using the conversion factors provided in Table III, a dose
of
50 mg/kg in the mouse converts to an appropriate dose of 12.5 mg/kg in the
monkey
because (SO mg/kg) x (I/4) = 12.5 mg/kg. As an additional example, doses of
0.02,
0.08, 0.8, 2, and 8 mg/kg in the mouse equate to effect doses of 1.667
micrograms/kg,
6.67 micrograms/kg, 66.7 micrograms/kg, 166.7 micrograms/kg, and 0.667 mg/kg,
respectively, in the human.
Pharmaceutical compositions containing the Brainiac-5 polypeptides of the
invention may be administered orally, rectally, parenterally, intracistemally,
intravaginally, intraperitoneally, topically (as by powders, ointments, drops
or
transdermal patch), bucally, or as an oral or nasal spray. By
"pharmaceutically
s
acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler,
diluent,
encapsulating material or formulation auxiliary of any type. In one
embodiment,
"pharmaceutically acceptable carrier" means a non-toxic solid, semisolid or
liquid
filler, diluent, encapsulating material or formulation auxiliary of any type.
In a
specific embodiment, "pharmaceutically acceptable" means approved by a
regulatory
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agency of the federal or a state government or listed in the U.S. Pharmacopeia
or other
generally recognized pharmacopeia for use in animals, and more particularly
humans.
Nonlimiting examples of suitable pharmaceutical carriers according to this
embodiment are provided in "Remington's Pharmaceutical Sciences" by E.W.
Martin,
and include sterile liquids, such as water and oils, including those of
petroleum,
animal, vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil,
sesame oil and the like. Water is a preferred carrier when the pharmaceutical
composition is administered intravenously. Saline solutions and aqueous
dextrose and
glycerol solutions can be employed as liquid carriers, particularly for
injectable
solutions. The composition, if desired, can also contain minor amounts of
wetting or
emulsifying agents, or pH buffering agents. These compositions can take the
form of
solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-
release
formulations and the like. The term "parenteral" as used herein refers to
modes of
administration which include intravenous, intramuscular, intraperitoneal,
intrasternal,
subcutaneous and intraarticular injection and infusion.
The Brainiac-5 polyf~eptide is also suitably administered by sustained-release
systems. Suitable examples of sustained-release compositions include semi-
permeable
polymer matrices in the form of shaped articles, e.g., films, or mirocapsules.
Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP
58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, LT,, et
al.,
Biopolymers 22:547-556 ( 1983)), poly (2- hydroxyethyl methacrylate; Langer,
R., et
al., J. Biomed. Mater. Res. 1.5:167-277 ( 1981 ), and Langer, R., Chem. Tech.
12:98-105
(1982)), ethylene vinyl acetate (Langer, R., et al., Id.) or poly-D- (-)-3-
hydroxybutyric
acid (EP 133,988). Sustained-release Brainiac-5 polypeptide compositions also
include liposomally entrapped Brainiac-5 polypeptide. Liposornes containing
Brainiac-5 polypeptides are prepared by methods known in the art (DE
3,218,121;
a
Epstein, et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 ( 1985); Hwang, et
al., Proc.
Natl. Acad. Sci. (USA) 77:4030-4034 ( 1980); EP 52,322; EP 36,676; EP 88,046;
EP
143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045
and
4,544,545; and EP 102,324). Ordinarily, the liposomes are of the small (about
200-
800 Angstroms) unilamellar type in which the lipid content is greater than
about 30
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mol. percent cholesterol, the selected proportion being adjusted for the
optimal
Brainiac-5 polypeptide therapy.
For parenteral administration, in one embodiment, the Brainiac-5 polypeptide
is formulated generally by mixing it at the desired degree of purity, in a
unit dosage
S injectable form (solution, suspension, or emulsion), with a pharmaceutically
acceptable carrier, i.e., one that is non-toxic to recipients at the dosages
and
concentrations employed and is compatible with other ingredients of the
formulation.
For example, the formulation preferably does not include oxidizing agents and
other
compounds that are known to be deleterious to polypeptides.
Generally, the formulations are prepared by contacting the Brainiac-5
polypeptide uniformly and intimately with liquid carriers or finely divided
solid
carriers or both. Then, if necessary, the product is shaped into the desired
formulation.
Preferably the carrier is a parenteral carrier, more preferably a solution
that is isotonic
with the blood of the recipient. Examples of such carrier vehicles include
water,
saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as
fixed
oils and ethyl oleate are also useful herein, as well as liposomes.
The carrier suitably contains minor amounts of additives such as substances
that enhance isotonicity and chemical stability. Such materials are non-toxic
to
recipients at the dosages and concentrations employed, and include buffers
such as
phosphate, citrate, succinate, acetic acid, and other organic acids or their
salts;
antioxidants such as ascorbic acid; low molecular weight (less than about ten
residues)
polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum
albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or arginine;
monosaccharides, disaccharides, and other carbohydrates including cellulose or
its
derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA;
sugar
alcohols such as mannitol or sorbitol; counterions such as sodium; and/or
nonionic
surfactants such as polysorbates, poloxamers, or PEG.
The Brainiac-5 polypeptide is typically formulated in such vehicles at a
concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH
of
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about 3 to 8. It will be understood that the use of certain of the foregoing
excipients,
carriers, or stabilizers will result in the formation of Brainiac-5
polypeptide salts.
Brainiac-5 polypeptide to be used for therapeutic administration must be
sterile. Sterility is readily accomplished by filtration through sterile
filtration
membranes (e.g., 0.2 micron membranes). Therapeutic Brainiac-5 polypeptide
compositions generally are placed into a container having a sterile access
port, for
example, an intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
Brainiac-5 polypeptide ordinarily will be stored in unit or mufti-dose
containers, for example, sealed ampoules or vials, as an aqueous solution or
as a
lyophilized formulation for reconstitution. As an example of a lyophilized
formulation, 10-ml vials are filled with 5 ml of sterile-filtered I % (w/v)
aqueous
Brainiac-5 polypeptide solution, and the resulting mixture is lyophilized. The
infusion
solution is prepared by reconstituting the lyophilized Brainiac-5 polypeptide
using
bacteriostatic water-for-injection (WFI).
The invention also provides a pharmaceutical pack or kit comprising one or
more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the inventian. Associated with such containers) can be a
notice in the
form prescribed by a governmental agency regulating the manufacture, use or
sale of
pharmaceuticals or biological products, which notice reflects approval by the
agency
of manufacture, use or sale for human administration. In addition, the
poIypeptides of
the present invention may be employed in conjunction with other therapeutic
compounds.
The compositions of the invention may be administered alone or in
combination with other adjuvants. Adjuvants that may be administered with the
compositions of the inventian include, but are not limited to, alum, alum plus
deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG,
and MPL. In a specific embodiment, compositions of the invention are
administered
in combination with alum. In another specific embodiment, compositions of the
invention are administered in combination with QS-21. Further adjuvants that
may be
administered with the compositions of the invention include, but are not
limited to,
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Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005,
Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be
administered with the compositions of the invention include, but are not
limited to,
vaccines directed toward protection against MMR (measles, mumps, rubella),
polio,
varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae
B,
whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera,
yellow
fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and
pertussis.
Combinations may be administered either concomitantly, e.g., as an admixture,
separately but simultaneously or concurrently; or sequentially. This includes
presentations in which the combined agents are administered together as a
therapeutic
mixture, and also procedures in which the combined agents are administered
separately but simultaneously, e.g., as through separate intravenous lines
into the same
individual. Administration "in combination" further includes the separate
administration of one of the compounds or agents given first, followed by the
second.
The compositions of the invention may be administered alone or in
combination with other therapeutic agents, including but not limited to,
chemotherapeutic agents, antibiotics, antivirals, steroidal and non-steroidal
and-
inflammatories, conventional immunotherapeutic agents and cytokines.
Combinations
may be administered either concomitantly, e.g., as an admixture, separately
but
simultaneously or concurrently; or sequentially. This includes presentations
in which
the combined agents are administered together as a therapeutic mixture, and
also
procedures in which the combined agents are administered separately but
simultaneously, e.g., as through separate intravenous lines into the same
individual.
Administration "in combination" further includes the separate administration
of one of
the compounds or agents given first, followed by the second.
In one embodiment, the compositions of the invention are administered in
combination with members of the TNF family. TNF, TNF-related or TNF-like
molecules that may be administered with the compositions of the invention
include,
but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-
alpha, also
known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta),
OPGL, Fast, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma
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(International Publication No. WO 96/14328), AIM-I (International Publication
No.
WO 97/33899), AIM-II (International Publication No. WO 97/34911), APRIL (J.
Exp.
Med. 188(6):1185-1 I90), endokine-alpha (International Publication No. WO
98/07880), TR6 (International Publication No. WO 98/30694), OPG, and
neutrolcine-
alpha (International Publication No. WO 98/18921, OX40, and nerve growth
factor
(NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2
(International
Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904),
DR4 (International Publication No. WO 98/32856), TR5 (International
Publication
No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7
(International Publication No. WO 98/41629), TRANK, TR9 (International
Publication No. WO 98/56892), TR10 (International Publication No. WO
98/54202),
312C2 (International Publication No. WO 98/06842), and TR12.
In certain embodiments, compositions of the invention are administered in
combination with antiretroviral agents, nucleoside reverse transcriptase
inhibitors,
non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors.
Nucleoside
reverse transcriptase inhibitors that may be administered in combination with
the
compositions of the invention, include, but are not limited to, RETROVIRTM
(zidovudine/AZT), VIDEXT"' (didanosine/ddI), HIVIDT"" (zalcitabine/ddC),
ZERITT""
(stavudine/d4T), EPIVIRT"" (lamivudine/3TC), and COMBIVIRT""
(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitors that
may be
administered in combination with the compositions of the invention, include,
but are
not limited to, VIRAMUNET~' (nevirapine), RESCRIPTORT"" (delavirdine), and
SUSTIVAT"' (efavirenz). Protease inhibitors that may be administered in
combination
with the compositions of the :invention, include, but are not limited to,
CRIXIVANT""
(indinavir), NORVIRT"" (ritonavir), INVIRASET"" (saquinavir), and VIRACEPTT""
(nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside
reverse
transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors,
and/or
protease inhibitors may be used in any combination.with compositions of the
invention
to treat, prevent, and/or diagnose AIDS and/or to treat, prevent, and/or
diagnose HIV
infection.
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In other embodiments, compositions of the invention may be administered in
combination with anti-opportunistic infection agents. Anti-opportunistic
agents that
may be administered in combination with the compositions of the invention,
include,
but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLET"", DAPSONET"",
PENTAMIDINET"", ATOVAQUONET"", ISONIAZIDT"", RIFAMPINT"",
PYRAZINAMIDET"", ETHAMBUTOLT"", RIFABUTINTM, CLARITHROMYCINT"",
AZITHROMYCINT"", GANCICLOVIRT"", FOSCARNETT"", CIDOFOVIRT"",
FLUCONAZOLET"", ITRACONAZOLET"", KE'TOCONAZOLET"", ACYCLOVIRT"",
FAMCICOLVIRT"", PYRIMETHAMINET"", LEUCOVORINr"", NEUPOGENT"'
(filgrastim/G-CSF), and LEUKINET"" (sargramostim/GM-CSF). In a specific
embodiment, compositions of the invention are used in any combination with
TRIMETHOPRIM-SULFAMETHOXAZOLET"", DAPSONET"", PENTAMIDINET"",
and/or ATOVAQUONET"' to prophylactically treat, prevent, and/or diagnose an
opportunistic Pneumocystis c-arinii pneumonia infection. In another specific
embodiment, compositions of the invention are used in any combination with
ISONIAZIDT"~, RIFAMPINT"", PYRAZINAMIDETM, and/or ETHAMBUTOLT"" to
prophylactically treat, prevent, and/or diagnose an opportunistic
Mycobacterium avium
complex infection. In another specific embodiment, compositions of the
invention are
used in any combination with RIFABUTINT"', CLARITHROMYCINT"~, and/or
AZITHROMYCINT"" to prophylactically treat, prevent, and/or diagnose an
opportunistic Mycobacterium tuberculosis infection. In another specific
embodiment,
compositions of the invention are used in any combination with GANCICLOVIRTM,
FOSCARNETT"", and/or CIDOFOVIRT"" to prophylactically treat, prevent, and/or
diagnose an opportunistic cytomegalovirus infection. In another specific
embodiment,
compositions of the invention are used in any combination with FLUCONAZOLET"",
ITRAC~ONAZOLET"", and/or KETOCONAZOLET"" to prophylactically treat, prevent,
and/or diagnose an opportunistic fungal infection. In another specific
embodiment,
compositions of the invention are used in any combination with ACYCLOVIRT""
and/or FAMCICOLVIRT"" to prophylactically treat, prevent, and/or diagnose an
opportunistic herpes simplex virus type I and/or type II infection. In another
specific
embodiment, compositions of the invention are used in any combination with
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PYRIMETHAMINET"" andl'or LEUCOVORINT"' to prophylactically treat, prevent,
and/or diagnose an opportunistic Toxoplasma gondii infection. In another
specific
embodiment, compositions of the invention are used in any combination with
LEUCOVORINT"" and/or NEUPOGENT"" to prophylactically treat, prevent, and/or
diagnose an opportunistic bacterial infection.
In a further embodiment, the compositions of the invention are administered in
combination with an antiviral agent. Antiviral agents that may be administered
with
the compositions of the invention include, but are not limited to, acyelovir,
ribavirin,
amantadine, and remantidine.
In a further embodiment, the compositions of the invention are administered in
combination with an antibiotic agent. Antibiotic agents that may be
administered with
the compositions of the invention include, but axe not limited to,
amoxicillin,
aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin,
chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,
fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,
rifampin,
streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-
sulfamthoxazole, and vancomycin.
Conventional nonspecific imrnunosuppressive agents, that may be
administered in combination with the compositions of the invention include,
but are
not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide
methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and
other
immunosuppressive agents that act by suppressing the function of responding T
cells.
In specific embodiments, compositions of the invention are administered in
combination with immunosuppressants. Immunosuppressants preparations that may
be administered with the compositions of the invention include, but are not
limited to,
ORTHOCLONET"" (OKT3), SANDIMMUNET""/NEORALT""/SANGDYATM
(cyclosporin), PROGRAFT"~ (tacrolimus), CELLCEPTT"" (mycophenolate),
Azathioprine, glucorticosteroids, and RAPAMUNET"" (sirolimus). In a specific
embodiment, immunosuppressants may be used to prevent rejection of organ or
bone
marrow transplantation.
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In an additional embodiment, compositions of the invention are administered
alone or in combination with one or more intravenous immune globulin
preparations.
Intravenous immune globulin preparations that may be administered with the
compositions of the invention include, but riot limited to, GAMMART"",
IVEEGAMT"",
SANDOGLOBULINT"", GAMMAGARD S/DT"", and GAMIMUNET"". In a specific
embodiment, compositions of the invention are administered in combination with
intravenous immune globulin preparations in transplantation therapy (e.g.,
bone
marrow transplant).
In an additional embodiment, the compositions of the invention are
administered alone or in combination with an anti-inflammatory agent. Anti-
inflammatory agents that may be administered with the compositions of the
invention
include, but are not limited to, glucocorticoids and the nonsteroidal anti-
inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid
derivatives,
arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid
derivatives,
pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-
acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid,
amixetrine, bendazac, benzydanune, bucolome, difenpiramide, ditazol,
emorfazone,
guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline,
perisoxal,
pifoxime, proquazone, proxazole, and tenidap.
In another embodiment, compostions of the invention are administered in
combination with a chemotherapeutic agent. Chemotherapeutic agents that may be
administered with the compositions of the invention include, but are not
limited to,
antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and
dactinomycin);
antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU,
methotrexate,
floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine,
and 6-
thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU,
cytosine
arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine,
mitomycin,
busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,
medroxyprogesterone,
estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol
acetate,
methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and
testolactone);
nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine
(nitrogen
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mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium
phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine
sulfate,
vinblastine sulfate, and etoposide).
In a specific embodiment, compositions of the invention are administered in
combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and
prednisone) or any combination of the components of CHOP. In another
embodiment,
compositions of the invention are administered in combination with Rituximab.
In a
further embodiment, compositions of the invention are administered with
Rituxmab
and CHOP, or Rituxmab and any combination of the components of CHOP.
In an additional embodiment, the compositions of the invention are
adnunistered in combination with cytokines. Cytokines that may be administered
with
the compositions of the invention include, but are not limited to, GM-CSF, G-
CSF,
IL2, IL3, IL4, ILS, IL6, IL7, IL 10, IL 12, IL 13, IL 15, anti-CD40, CD40L,
IFN-alpha,
IFN-beta, IFN-gamma, TNF-alpha, and TNF-beta. In another embodiment,
compositions of the invention may be administered with any interleukin,
including, but
not limited to, IL-1 alpha, IL-~ 1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL,-
10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-
21, and
IL-22.
In an additional embodiment, the compositions of the invention are
administered with a chemokine. In another embodiment, the compositions of the
invention are administered with chemokine beta-8, chemokine beta-1, andlor
macrophage inflammatory protein-4. In a preferred embodiment, the compositions
of
the invention are administered with chemokine beta-8.
In an additional embodiment, the compositions of the invention are
administered in combination with an IL-4 antagonist. IL-4 antagonists that may
be
administered with the compositions of the invention include, but are not
limited to:
soluble IL-4 receptor polypeptides, multimeric forms of soluble IL-4 receptor
polypeptides; anti-IL-4 receptor antibodies that bind the IL-4 receptor
without
transducing the biological signal elicited by IL-4, anti-IL4 antibodies that
block
binding of IL-4 to one or more IL-4 receptors, and muteins of IL-4 that bind
IL-4
receptors but do not transducer the biological signal elicited by IL-4.
Preferably, the
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antibodies employed according to this method are monoclonal antibodies
(including
antibody fragments, such as, for example, those described herein).
In an additional embodiment, the compositions of the invention are
administered in combination with hematopoietic growth factors. Hematopoietic
growth factors that may be administered with the compositions of the invention
include, but are not limited to, LEUKINET"~ (SARGRAMOSTIMTM) and
NEUPOGENT"" (FILGRASTIMT"").
In an additional embodiment, the compositions of the invention are
administered in combination with fibroblast growth factors. Fibroblast growth
factors
IO that may be administered with the compositions of the invention include,
but are not
limited to, FGF-l, FGF-2, F'GF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9,
FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.
Additionally, the compositions of the invention may be administered alone or
in combination with other therapeutic regimens, including but not limited to,
radiation
15 therapy. Such combinatorial therapy may be administered sequentially and/or
concomitantly.
Agonists and Antagonists - Assays and Molecules
The invention also provides a method of screening compounds to identify those
20 which enhance or block the action of Brainiac-5 polypeptides on cells, such
as its
interaction with Brainiac-5 polypeptide-binding molecules such as receptor
molecules.
An agonist is a compound which increases the natural biological functions of
Brainiac-5 polypeptides or which functions in a manner similar to Brainiac-5
polypeptides, while antagonists decrease or eliminate such functions.
25 In another aspect of this embodiment the invention provides a method for
identifying a receptor protein or other ligand-binding protein which binds
specifically
to a Brainiac-5 polypeptide. For example, a cellular compartment, such as a
membrane or a preparation thereof, may be prepared from a cell that expresses
a
molecule that binds Brainiac-5 polypeptides. The preparation is incubated with
30 labeled Brainiac-5 polypeptide and complexes of Brainiac-5 polypeptide
bound to the
receptor or other binding pratein are isolated and characterized according to
routine
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methods known in the art. Alternatively, the Brainiac-5 polypeptide may be
bound to
a solid support so that binding molecules solubilized from cells are bound to
the
column and then eluted and characterized according to routine methods.
In the assay of the invention for agonists or antagonists, a cellular
compartment, such as a membrane or a preparation thereof, may be prepared from
a
cell that expresses a molecule that binds Brainiac-5 polypeptide, such as a
molecule of
a signaling or regulatory pathway modulated by Brainiac-5 polypeptide. The
preparation is incubated with labeled Brainiac-S polypeptide in the absence or
the
presence of a candidate molecule which may be a Brainiac-5 polypeptide agonist
or
antagonist. The ability of the candidate molecule to bind the binding molecule
is
reflected in decreased binding of the labeled Iigand. Molecules which bind
gratuitously, i.e., without inducing the effects of Brainiac-5 polypeptide on
binding the
Brainiac-5 polypeptide-binding molecule, are most likely to be good
antagonists.
Molecules that bind well and elicit effects that are the same as or closely
related to
Brainiac-5 polypeptide are agonists.
Brainiac-5 polypeptide-like effects of potential agonists and antagonists may
by measured, for instance, by determining activity of a second messenger
system
following interaction of the candidate molecule with a cell or appropriate
cell
preparation, and comparing the effect with that of Brainiac-5 polypeptides or
molecules that elicit the same effects as Brainiac-5 polypeptide. Second
messenger
systems that may be useful in this regard include but are not limited to AMP
guanylate
cyclase, ion channel or phosphoinositide hydrolysis second messenger systems.
Another example of an assay for Brainiac-S polypeptide antagonists is a
competitive assay that combines Brainiac-S polypeptides and a potential
antagonist
with membrane-bound Brainiac-S polypeptide receptor molecules or recombinant
Brainiac-5 polypeptide receptor molecules under appropriate conditions for a
i
competitive inhibition assay. Brainiac-5 polypeptides can be labeled, such as
by
radioactivity, such that the number of Brainiac-5 polypeptide molecules bound
to a
receptor molecule can be determined accurately to assess the effectiveness of
the
potential antagonist.
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Potential antagonists include small organic molecules, peptides, polypeptides
and antibodies that bind to a polypeptide of the invention and thereby inhibit
or
extinguish its activity. Potential antagonists also may be small organic
molecules, a
peptide, a polypeptide such as a closely related protein or antibody that
binds the same
sites on a binding molecule, such as a receptor molecule, without inducing
Brainiac-5
polypeptide-induced activities, thereby preventing the action of Brainiac-5
polypeptides by excluding Brainiac-5 polypeptides from binding.
Other potential antagonists include antisense molecules. Antisense technology
can be used to control gene expression through antisense DNA or RNA or through
triple-helix formation. Antisense techniques are discussed in a number of
studies (for
example, Okano, J. Neurochem. 56:560 ( 1991 ); "Oligodeoxynucleotides as
Antisense
Inhibitors of Gene Expression." CRC Press, Boca Raton, FL (1988)). Triple
helix
formation is discussed in a number of studies, as well (for instance, Lee, et
al., Nucleic
Acids Research 6:3073 ( 1979); Cooney, et al., Science 241;456 ( 1988);
Dervan, et al.,
Science 251:1360 ( 1991 )). The methods are based on binding of a
polynucleotide to a
complementary DNA or RNA. For example, the 5' coding portion of a
polynucleotide
that encodes the mature polypeptide of the present invention may be used to
design an
antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A
DNA
oligonucleotide is designed to be complementary to a region of the gene
involved in
transcription thereby preventing transcription and the production of a
Brainiac-5
polypeptide. The antisense R.NA oligonucleotide hybridizes to the mRNA in vivo
and
blocks translation of the mRNA molecule into a Brainiac-5 polypeptide. The
oligonucleotides described above can also be delivered to cells such that the
antisense
RNA or DNA may be expressed in vivo to inhibit production of Brainiac-5
polypeptides.
In one embodiment, the Brainiac-5 antisense nucleic acid of the invention is
produced intracellularly by transcription from an exogenous sequence. For
example, a
vector or a portion thereof, is transcribed, producing an antisense nucleic
acid (RNA)
of the invention. Such a vector would contain a sequence encoding the Brainiac-
5
antisense nucleic acid. Such a vector can remain episomal or become
chromosomally
integrated, as long as it can be transcribed to produce the desired antisense
RNA.
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Such vectors can be constructed by recombinant DNA technology methods standard
in
the art. Vectors can be plasmid, viral, or others know in the art, used for
replication
and expression in vertebrate cells. Expression of the sequence encoding
Brainiac-5, or
fragments thereof, can be by any promoter known in the art to act in
vertebrate,
preferably human cells. Such promoters can be inducible or constitutive. Such
promoters include, but are not limited to, the SV40 early promoter region
(Bernoist
and Chambon, Nature 29:3()4-310 ( 1981 ), the promoter contained in the 3'
long
terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (
1980), the
herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.
78:1441-1445
I O ( 1981 ), the regulatory sequences of the metallothionein gene (Brinster,
et al., Nature
296:39-42 ( 1982)), etc.
The antisense nucleic acids of the invention comprise a sequence
complementary to at least a portion of an RNA transcript of a Brainiac-5 gene.
However, absolute complementarity, although preferred, is not required. A
sequence
"complementary to at least a portion of an RNA," referred to herein, means a
sequence
having sufficient cornplernentarity to be able to hybridize with the RNA,
forming a
stable duplex; in the case of double stranded Brainiac-S antisense nucleic
acids, a
single strand of the duplex DNA may thus be tested, or triplex formation may
be
assayed. The ability to hybridize will depend on both the degree of
complementarity
and the length of the antisense nucleic acid Generally, the larger the
hybridizing
nucleic acid, the more base mismatches with a Brainiac-5 RNA it may contain
and still
form a stable duplex (or triplex as the case may be). One skilled in the art
can
ascertain a tolerable degree of mismatch by use of standard procedures to
determine
the melting point of the hybridized complex.
Oligonucleotides that are complementary to the 5' end of the message, e.g.,
the
5' untranslated sequence up to and including the AUG initiation codon, should
work
a
most efficiently at inhibiting translation. However, sequences complementary
to the 3'
untranslated sequences of mRNAs have been shown to be effective at inhibiting
translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-
335.
Thus, oligonucleotides complementary to either the 5'- or 3'- non- translated,
non-
coding regions of Brainiac-5 shown in Figures lA-B, could be used in an
antisense
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approach to inhibit translation of endogenous Brainiac-5 mRNA.
Oligonucleotides
complementary to the 5' untranslated region of the mRNA should include the
complement of the AUG start codon. Antisense oligonucleotides complementary to
mRNA coding regions are less efficient inhibitors of translation but could be
used in
accordance with the invention. Whether designed to hybridize to the 5'-, 3'-
or coding
region of Brainiac-5 mRNA, antisense nucleic acids should be at least six
nucleotides
in length, and are preferably oligonucleotides ranging from 6 to about 50
nucleotides
in length. In specific aspects the oligonucleotide is at least 10 nucleotides,
at least 17
nucleotides, at least 25 nucleotides or at least 50 nucleotides.
The polynucleotides of the invention can be DNA or RNA or chimeric
mixtures or derivatives or modified versions thereof, single-stranded or
double-
stranded. The oligonucleotide can be modified at the base moiety, sugar
moiety, or
phosphate backbone, for example, to improve stability of the molecule,
hybridization,
etc. The oligonucleotide may include other appended groups such as peptides
(e.g.,
for targeting host cell receptors in vivo), or agents facilitating transport
across the cell
membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.
86:6553-
6556; Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652 ( 1987); PCT
Publication No.
W088/09810, published December 15, 1988) or the blood-brain barrier (see,
e.g., PCT
Publication No. W089/10134, published April 25, 1988), hybridization-triggered
cleavage agents. (See, e.g., Krol et al., BioTechniques 6:958-976 ( 1988)) or
intercalating agents. (See, e.g., Zon, Pharm. Res. 5:539-549 ( 1988)). To this
end, the
oligonucleotide may be conjugated to another molecule, e.g., a peptide,
hybridization
triggered cross-linking agent, transport agent, hybridization-triggered
cleavage agent,
etc.
The antisense oligonucleotide may comprise at least one modified base moiety
which is selected from the group including, but not limited to, 5-
fluorouracil, 5-
bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-
acetylcytosine, 5-
(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-
carboxyrnethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine,
N6-isopentenyladenine, 1-rnethylguanine, 1-methylinosine, 2,2-
dirnethylguanine, 2-
methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine, 7-
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methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5-methoxycarboxymethyluracil, 5-methoxyuraeil, 2-
methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine,
pseudouracil, queosine, 2-thiocytosine, S-methyl-2-thiouracil, 2-thiouracil, 4-
thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-
oxyacetic acid
(v), 5-methyl-2-thiouracil, :3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,
and 2,6-
diaminopurine.
The antisense oligonucleotide may also comprise at least one modified sugar
moiety selected from the group including, but not limited to, arabinose,
2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at least
one modified phosphate backbone selected from the group including, but not
limited
to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, and a formacetal or analog thereof.
In yet another embodiment, the antisense oligonucleotide is an a-anomeric
oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded
hybrids with complementary RNA in which, contrary to the usual b-units, the
strands
run parallel to each other (Gautier et al., Nucl. Acids Res. 15:6625-6641 (
1987)). The
oligonucleotide is a 2-0-methylribonucleotide (moue et al., Nucl. Acids Res.
15:6131-
6148 ( 1987)), or a chimeric RNA-DNA analogue (moue et al., FEBS Lett. 215:327-
330 ( 1997)).
Polynucleotides of the invention may be synthesized by standard methods
known in the art, e.g. by use of an automated DNA synthesizer (such as are
commercially available from Biosearch, Applied Biosystems, etc.). As examples,
phosphorothioate oligonucleotides may be synthesized by the method of Stein et
al.
(Nucl ~ Acids Res. 16:3209 ( 1988)), methylphosphonate oligonucleotides can be
prepared by use of controlled pore glass polymer supports (Sarin et al., Proc.
Natl.
Acad. Sci. U.S.A. 85:7448-7451 ( 1988)), etc.
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While antisense nucleotides complementary to the Brainiac-5 coding region
sequence could be used, those complementary to the transcribed untranslated
region
are most preferred.
Potential antagonists according to the invention also include catalytic RNA,
or
a ribozyme (See, e.g., PCT International Publication WO 90/11364, published
October
4, 1990; Sarver et al, Science 247:1222-1225 (1990). While ribozymes that
cleave
mRNA at site specific recognition sequences can be used to destroy Brainiac-5
mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozyrnes
cleave mRNAs at locations dictated by flanking regions that form complementary
base
pairs with the target mRNA. The sole requirement is that the target mRNA have
the
following sequence of two bases: 5'-UG-3'. The construction and production of
hammerhead ribozymes is well known in the art and is described more fully in
Haseloff and Gerlach, Nature 334:585-591 ( 1988). There are numerous potential
hammerhead ribozyme cleavage sites within the nucleotide sequence of Brainiac-
5
(Figures lA-B). Preferably, the ribozyme is engineered so that the cleavage
recognition site is located near the 5' end of the Brainiac-5 mRNA; i.e., to
increase
efficiency and minimize the intracellular accumulation of non-functional mRNA
transcripts.
As in the antisense approach, the ribozymes of the invention can be composed
of modified oligonucleotides (e.g. for improved stability, targeting, etc.)
and should be
delivered to cells which express Brainiac-5 in vivo. DNA constructs encoding
the
ribozyme may be introduced into the cell in the same manner as described above
for
the introduction of antisense encoding DNA. A preferred method of delivery
involves
using a DNA construct "encoding" the ribozyme under the control of a strong
constitutive promoter, such as, for example, pol III or pol II promoter, so
that
transfected cells will produce sufficient quantities of the ribozyme to
destroy
endogenous Brainiac-5 messages and inhibit translation. Since ribozymes unlike
antisense molecules, are catalytic, a lower intracellular concentration is
required for
efficiency.
Endogenous gene expression can also be reduced by inactivating or "knocking
out" the Brainiac-5 gene and/or its promoter using targeted homologous
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recombination. (E.g., see Smithies et al., Nature 317:230-234 ( 1985); Thomas
&
Capecchi, Cell 51:503-512 ( 1987); Thompson et al., Cell 5:313-321 ( 1989);
each of
which is incorporated by reference herein in its entirety). For example, a
mutant, non-
functional polynucleotide of the invention (or a completely unrelated DNA
sequence)
flanked by DNA homologous to the endogenous polynucleotide sequence (either
the
coding regions or regulatory regions of the gene) can be used, with or without
a
selectable marker andlor a negative selectable marker, to transfect cells that
express
polypeptides of the invention in vivo. In another embodiment, techniques known
in
the art are used to generate knockouts in cells that contain, but do not
express the gene
of interest. Insertion of the DNA construct, via targeted homologous
recombination,
results in inactivation of the targeted gene. Such approaches are particularly
suited in
research and agricultural fields where modifications to embryonic stem cells
can be
used to generate animal offspring with an inactive targeted gene (e.g., see
Thomas &
Capecchi 1987 and Thompsan 1989, supra). However this approach can be
routinely
adapted for use in humans provided the recombinant DNA constructs are directly
administered or targeted to the required site in vivo using appropriate viral
vectors that
will be apparent to those of skill in the art. The contents of each of the
documents
recited in this paragraph is herein incorporated by reference in its entirety.
In other embodiments, antagonists according to the present invention include
soluble forms of Brainiac-5. Such soluble forms of the Brainiac-5, which may
be
naturally occurring or synthetic, antagonize Brainiac-5-mediated signaling by
competing with native Brainiac-5 for binding to Brainiac-5 receptors and/or by
forming a multimer that may or may not be capable of binding the receptor, but
which
is incapable of inducing signal transduction. Preferably, these antagonists
inhibit
Brainiac-5-mediated stimulation of lymphocyte (e.g., Dendritic cell, monocyte,
macrophage, T cell, and/or B-cell) proliferation, differentiation, and/or
activation.
Antagonists of the present invention also include antibodies specific for TNF-
family
ligands (e.g., CD30) and Brainiac-5-Fc fusion proteins.
Polyclonal and monoclonal antibody agonists or antagonists according to the
present invention can be raised according to the methods disclosed in
Tartaglia and
Goeddel, J. Biol. Chem. 267( 7):4304-4307( 1992)); Tartaglia et al., Cell
73:213-216
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(1993)), and PCT Application WO 94/09137 and are preferably specific to (i.e.,
bind
uniquely to polypeptides of the invention having the amino acid sequence of
SEQ ID
N0:2. The term "antibody" (Ab) or "monoclonal antibody" (mAb) as used herein
is
meant to include intact molecules as well as fragments thereof (such as, for
example,
Fab and F(ab') fragments) which are capable of binding an antigen. Fab, Fab'
and
F(ab') fragments lack the F'c fragment intact antibody, clear more rapidly
from the
circulation, and may have less non-specific tissue binding of an intact
antibody (Wahl
et al., J. Nucl. Med., 24:316-325 ( 1983)).
In a preferred method, antibodies according to the present invention are mAbs.
Such mAbs can be prepared using hybridoma technology (Kohler and Millstein,
Nature 256:495-497 ( 1975) and U.S. Patent No. 4,376,110; Harlow et al.,
Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
1988; Monoclonal Antibodies and Hybridomas: A New Dimension in Biological
Analyses, Plenum Press, New York, NY, 1980; Campbell, "Monoclonal Antibody
Technology," In: Laboratory Techniques in Biochemistry and Molecular Biology,
Volume 13 (Burdon et al., eds.), Elsevier, Amsterdam (198;)).
Proteins and other compounds which bind the Brainiac-5 domains are also
candidate agonists and antagonists according to the present invention. Such
binding
compounds can be "captured" using the yeast two-hybrid system (Fields and
Song,
Nature 340:245-246 ( 1989)). A modified version of the yeast two- hybrid
system has
been described by Roger Brent and his colleagues (Gyuris, Cell 75:791-803 (
1993);
Zervos et al., Cell 72:223-232 (1993)). Such compounds are good candidate
agonists
and antagonists of the present invention.
Other screening techniques include the use of cells which express the
polypeptide of the present invention (for example, transfected CHO cells) in a
system
which measures extracellular pH changes caused by receptor activation, for
example,
as described in Science, 246:181-296 (1989). In another example, potential
agonists or
antagonists may be contacted with a cell which expresses the polypeptide of
the
present invention and a second messenger response, e.g., signal transduction
may be
measured to determine whether the potential antagonist or agonist is
effective.
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Agonists according to the present invention include naturally occurring and
synthetic compounds such as, for example, TNF family ligand peptide fragments,
transforming growth factor, neurotransmitters (such as glutamate, dopamine, N
methyl-D-aspartate), tumor suppressors (p53), cytolytic T cells and
antimetabolites.
Preferred agonists include chemotherapeutic drugs such as, for example,
cisplatin,
doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotxexate
and
vincristine. Others include ethanol and -amyloid peptide. (Science 267:1457-
1458
( 1995)).
Preferred agonists are fragments of Brainiac-5 polypeptides of the invention
which stimulate lymphocyte (e.g., B cell) proliferation, differentiation
and/or
activation. Further preferred agonists include polyclonal and monoclonal
antibodies
raised against the Brainiac-5 poIypeptides of the invention, or a fragment
thereof.
Such agonist antibodies raised against a TNF-family receptor are disclosed in
Tartaglia
et aL, Proc. Natl. Acad Sci. USA 88:9292-9296 ( 1991 ); and Tartaglia et al.,
J. Biol.
Chem. 267:4304- 4307( 1992). See, also, PCT Application WO 94/09137.
In an additional embodiment, immunoregulatory molecules such as, for
example, IL2, IL3, IL4, ILS, IL6, IL7, IL 10, IL 12, IL 13, IL 15, anti-CD40,
CD40L,
IFN-gamma and TNF-alpha, may be used as agonists of Brainiac-5 polypeptides of
the
invention which stimulate lymphocyte proliferation, differentiation and/or
activation.
In further embodiments of the invention, cells that are genetically engineered
to express the polypeptides of the invention, or alternatively, that are
genetically
engineered not to express the polypeptides of the invention (e.g., knockouts)
are
administered to a patient in vivo. Such cells may be obtained from the patient
(i.e.,
animal, including human) or an MHC compatible donor and can include, but are
not
limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes),
adipocytes,
muscle cells, endothelial cells etc. The cells are genetically engineered in
vitro using
recombinant DNA techniques to introduce the coding sequence of polypeptides of
the
invention into the cells, or alternatively, to disrupt the coding sequence
and/or
endogenous regulatory sequence associated with the polypeptides of the
invention,
e.g., by transduction (using viral vectors, and preferably vectors that
integrate the
transgene into the cell genome) or transfection procedures, including, but not
limited
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to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes,
etc.
The coding sequence of the polypeptides of the invention can be placed under
the
control of a strong constitutive or inducible promoter or promoter/enhancer to
achieve
expression, and preferably secretion, of the polypeptides of the invention.
The
S engineered cells which express and preferably secrete the polypeptides of
the
invention can be introduced into the patient systemically, e.g., in the
circulation, or
intraperitoneally.
Alternatively, the cells can be incorporated into a matrix and implanted in
the
body, e.g., genetically engineered fibroblasts can be implanted as part of a
skin graft;
genetically engineered endothelial cells can be implanted as part of a
lymphatic or
vascular graft. (See, for example, Anderson et al. U.S. Patent No. 5,399,349;
and
Mulligan & Wilson, U.S. Patent No. 5,460,959 each of which is incorporated by
reference herein in its entirety).
When the cells to be administered are non-autologous or non-MHC compatible
1S cells, they can be administered using well known techniques which prevent
the
development of a host immune response against the introduced cells. For
example, the
cells may be introduced in an encapsulated form which, while allowing for an
exchange of components with the immediate extracellular environment, does not
allow
the introduced cells to be recognized by the host immune system.
, In yet another embodiment of the invention, the activity of Brainiac-S
polypeptide can be reduced using a "dominant negative." To this end,
constructs
which encode defective Brainiac-S polypeptide, such as, for example, mutants
lacking
all or a portion of a conserved domain, can be used in gene therapy approaches
to
diminish the activity of Brainiac-S on appropriate target cells. For example,
2S nucleotide sequences that direct host cell expression of Brainiac-S
polypeptide in
which all or a portion of a conserved domain is altered or missing can be
introduced
f
into monocytic cells or other cells or tissues (either by in vivo or ex vivo
gene therapy
methods described herein or otherwise known in the art). Alternatively,
targeted
homologous recombination can be utilized to introduce such deletions or
mutations
into the subject's endogenous Brainiac-5 gene in monocytes. The engineered
cells will
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express non-functional Brainiac-5 polypeptides (i.e., a ligand (e.g.,
multimer) that may
be capable of binding, but which is incapable of inducing signal
transduction).
The agonists and antagonists may be employed in a composition with a
pharmaceutically acceptable carrier, e.g., as described above.
Gene Mapping
The nucleic acid molecules of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to and can
hybridize
with a particular location on an individual human chromosome. Moreover, there
is a
current need for identifying particular sites on the chromosome. Few
chromosome
marking reagents based on actual sequence data (repeat polymorphisms) are
presently
available for marking chromosomal location. The mapping of DNAs to chromosomes
according to the present invention is an important first step in correlating
those
sequences with genes associai:ed with disease.
In certain preferred embodiments in this regard, the cDNA herein disclosed is
used to clone genomic DNA of a Brainiac-5 gene. This can be accomplished using
a
variety of well known techniques and libraries, which generally are available
commercially. The genomic DNA then is used for in situ chromosome mapping
using
well known techniques for this purpose.
In addition, in some cases, sequences can be mapped to chromosomes by
preparing PCR primers (preferably 1 S-25 bp) from the cDNA. Computer analysis
of
the 3' untranslated region of the gene is used to rapidly select primers that
do not span
more than one exon in the genomic DNA, thus complicating the amplification
process.
These primers are then used for PCR screening of somatic cell hybrids
containing
individual human chromosomes. Fluorescence in situ hybridization ("FISH") of a
cDNA clone to a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with probes from
the
cDNA as short as 50 or 60 by (for a review of this technique, see Verma, et
al.,
Human Chromosomes: A Manual Of Basic Techniques, Pergamon Press, New York
( 1988)).
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Once a sequence has been mapped to a precise chromosomal location, the
physical position of the sequence on the chromosome can be correlated with
genetic
map data. Such data are found, for example, on the World Wide Web (McKusick,
V.
Mendelian Inheritance In Man, available on-line through Johns Hopkins
University,
Welch Medical Library). The relationship between genes and diseases that have
been
mapped to the same chromosomal region are then identified through linkage
analysis
(coinheritance of physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic
sequence between affected and unaffected individuals. If a mutation is
observed in
some or all of the affected individuals but not in any normal individuals,
then the
mutation is likely to be the causative agent of the disease.
Having generally described the invention, the same will be more readily
understood by reference to the following examples, which are provided by way
of
illustration and are not intended as limiting.
Examples
Example I: Expression and Purification of "His-tagged" Brainiac-5 in E. eoli
The novel pHE4 series of bacterial expression vectors, in particular, the pHE4-
S vector may be used for bacterial expression in this example. (QIAGEN, Inc.,
9259
Eton Avenue, Chatsworth, CA, 91311 ). pHE4-5/MPIFD23 vector plasmid DNA
contains an insert which encodes another ORF. The construct was deposited with
the
American Type Culture Collection, 10801 University Boulevard, Manassas,
Virginia
20110-2209, on September 30, 1997 and given Accession No. 209311. Using the
Nde
I and Asp 718 restriction sites flanking the irrelevant MPIF ORF insert, one
of
ordinary skill in the art could easily use current molecular biological
techniques to
replace the irrelevant ORF in the pHE4-S vector with the Brainiac-5 ORF of the
present invention.
The pHE4-5 bacterial expression vector includes a neomycin
Phosphotransferase gene for selection, an E. coli origin of replication, a TS
phage
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promoter sequence, two lac operator sequences, a Shine-Delgarno sequence, and
the
lactose operon repressor gene (lacIq). These elements are arranged such that
an
inserted DNA fragment encoding a polypeptide expresses that polypeptide with
the six
His residues (i.e., a "6 X His tag") covalently linked to the amino terminus
of that
polypeptide. The promoter and operator sequences of the pHE4-5 vector were
made
synthetically. Synthetic production of nucleic acid sequences is well known in
the art
(CLONETECH 95/96 Catalog, pages 215-216, CLONETECH, 1020 East Meadow
Circle, Palo Alto, CA 94303).
The DNA sequence encoding the desired portion of the Brainiac-5 polypeptide
is amplified from the deposited cDNA clone using PCR oligonucleotide primers
which
anneal to the amino terminal sequences of the desired portion of the Brainiac-
5
polypeptide and to sequences in the deposited construct 3' to the cDNA coding
sequence. Additional nucleotides containing restriction sites to facilitate
cloning in the
pHE4-5 vector are added to the S' and 3' primer sequences, respectively.
For cloning the Brainiac-5 polypeptide, the 5' primer has the sequence 5' CAA
TTG GAT CCG TGG CAG AGG ACT TCG AGC 3' (SEQ ID NO:11 ) containing the
underlined Bam HI restriction site followed by 18 nucleotides of the amino
terminal
coding sequence of the Brainiac-5 sequence in SEQ ID N0:2. One of ordinary
skill in
the art would appreciate, of ecrurse, that the point in the protein coding
sequence where
the 5' primer begins may be varied to amplify a DNA segment encoding any
desired
portion of the complete Brainiac-5 polypeptide shorter or longer than the
complete
sequence of the polypeptide shown in Figures lA and 1B or in SEQ ID N0:2. The
3'
primer has the sequence 5' GTA CGC AAG CTT GGA GTC CCA TTG GAA GGG 3'
(SEQ ID N0:12) containing the underlined Hin dIII restriction site followed by
18
nucleotides complementary to the 3' end of the coding sequence of the Brainiac-
5
DNA sequence shown in Figures 1 A and 1 B (SEQ ID NO:1 ).
The amplified Brainiac-5 DNA fragment and the vector pHE4-S are digested
with Bam HI and Hin dIII and the digested DNAs are then ligated together.
Insertion
of the Brainiac-5 DNA into the restricted pHE4-5 vector places the Brainiac-5
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polypeptide coding region downstream from the IPTG-inducible promoter and in-
frame with an initiating AUG and the six histidine codons.
The ligation mixture is transformed into competent E. coli cells using
standard
procedures such as those described by Sambrook and colleagues (Molecular
Cloning:
a Laboratory Manual, 2nd ~:'d.; Cold Spring Harbor Laboratory Press, Cold
Spring
Harbor, NY ( 1989)). E. cola strain M 15/rep4, containing multiple copies of
the
plasmid pREP4, which expresses the lac repressor and confers kanamycin
resistance
("Kanr"), is used in carrying out the illustrative example described herein.
This strain,
which is only one of many that are suitable for expressing Brainiac-5
polypeptide, is
available commercially (QIAGEN, Inc., supra). Transformants are identified by
their
ability to grow on LB plates in the presence of ampicillin and kanamycin.
Plasmid
DNA is isolated from resistant colonies and the identity of the cloned DNA
confirmed
by restriction analysis, PCR and DNA sequencing.
Clones containing the desired constructs are grown overnight {"O/N") in liquid
culture in LB media supplemented with both ampicillin ( 100 ug/ml) and
kanamycin
(25 ug/ml). The O/N culture is used to inoculate a large culture, at a
dilution of
approximately 1:25 to 1:250.. The cells are grown to an optical density at 600
nm
("OD600") of between 0.4 and 0.6. Isopropyl-beta-D-thiogalactopyranoside
("IPTG")
is then added to a final concentration of 1 mM to induce transcription from
the lac
repressor sensitive promoter, by inactivating the lacI repressor. Cells
subsequently are
incubated further for 3 to 4 hours. Cells then are harvested by
centrifugation.
The cells are then stirred for 3-4 hours at 4°C in 6M guanidine-HCI, pH
8. The
cell debris is removed by centrifugation, and the supernatant containing the
Brainiac-5
polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA")
affinity resin
column (QIAGEN, Inc., supra). Proteins with a 6 x His tag bind to the Ni-NTA
resin
with high affinity and can be purified in a simple one-step procedure (for
details see:
The QIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly the supernatant is
loaded
onto the column in 6 M guanidine-HCI, pH 8, the column is first washed with 10
volumes of 6 M guanidine-HCI, pH 8, then washed with 10 volumes of 6 M
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guanidine-HCl pH 6, and finally the Brainiac-S polypeptide is eluted with 6 M
guanidine-HCI, pH S.
The purified polypeptide is then renatured by dialyzing it against
phosphate-buffered saline (PBS) or SO mM Na-acetate, pH 6 buffer plus 200 mM
S NaCI. Alternatively, the protein can be successfully refolded while
immobilized on
the Ni-NTA column. The recommended conditions are as follows: renature using a
linear 6M-1M urea gradient in S00 mM NaCI, 20% glycerol, 20 mM Tris/HCl pH
7.4,
containing protease inhibitors. The renaturation should be performed over a
period of
1.S hours or more. After renaturation the proteins can be eluted by the
addition of 2S0
mM i~idazole. Immidazole is removed by a final dialyzing step against PBS or
SO
mM sodium acetate pH 6 buffer plus 200 mM NaCI. The purified protein is stored
at
4° C or frozen at -80° C.
The following alternative method may be used to purify Brainiac-S polypeptide
expressed in E coli when it is present in the form of inclusion bodies. Unless
1S otherwise specified, all of the following steps are conducted at 4-
10°C.
Upon completion of the production phase of the E. coli fermentation, the cell
culture is cooled to 4-10°C and the cells are harvested by continuous
centrifugation at
15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein
per unit
weight of cell paste and the amount of purified protein required, an
appropriate
amount of cell paste, by weight, is suspended in a buffer solution containing
100 mM
Tris, SO mM EDTA, pH 7.4. 'The cells are dispersed to a homogeneous suspension
using a high shear mixer.
The cells ware then lysed by passing the solution through a microfluidizer
(Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-60U0 psi. The
homogenate is
2S then mixed with NaCI solution to a final concentration of O.S M NaCI,
followed by
centrifugation at 7000 x g for 1S min. The resultant pellet is washed again
using O.SM
NaCI, 100 mM Tris, SO mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.S M guanidine
hydrochloride (GuHCI) for 2-4 hours. After 7000 x g centrifugation for 1S
min., the
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pellet is discarded and the Brainiac-5 polypeptide-containing supernatant is
incubated
at 4°C overnight to allow further GuHCI extraction.
Following high speed centrifugation (30,000 x g) to remove insoluble
particles,
the GuHCI solubilized protein is refolded by quickly mixing the GuHCI extract
with
20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCI, 2 mM EDTA
by vigorous stirring. The refolded diluted protein solution is kept at
4°C without
mixing for 12 hours prior to further purification steps.
To clarify the refolded Brainiac-5 polypeptide solution, a previously prepared
tangential filtration unit equipped with 0.16 micrometer membrane filter with
aPPropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium
acetate, pH
6.0 is employed. The filtered sample is loaded onto a cation exchange resin
(e.g.,
Poros HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium
acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and IS00 mM NaCI in
the same buffer, in a stepwise manner. The absorbance at 280 mm of the
effluent is
continuously monitored. Fractions are collected and further analyzed by SDS-
PAGE
Fractions containing the Brainiac-5 polypeptide are then pooled and mixed
with 4 volumes of water. The diluted sample is then loaded onto a previously
prepared set of tandem columns of strong anion (Poros HQ-50, Perseptive
Biosystems)
and weak anion (Poros CM-20, Perseptive Biosystems) exchange resins. The
columns
~'e equilibrated with 40 mM sodium acetate, pH 6Ø Both columns are washed
with
40 mM sodium acetate, pH 6.0, 200 mM NaCI. The CM-20 column is then eluted
using a 10 column volume linear gradient ranging from 0.2 M NaCI, 50 nnM
sodium
acetate, pH 6.0 to 1.0 M NaCI, 50 mM sodium acetate, pH 6.5. Fractions are
collected
under constant Azgo monitoring of the effluent. Fractions containing the
Brainiac-5
Polypeptide (determined, for instance, by 16%~ SDS-PAGE) are then pooled.
The resultant Brainiac-5 polypeptide exhibits greater than 95% purity after
the
above refolding and purification steps. No major contaminant bands are
observed
from Commassie blue stained 16% SDS-PAGE gel when 5 micrograms of purified
protein is loaded. The purified protein is also tested for endotoxin/LPS
contamination,
~d typically the LPS content is less than 0.1 ng/ml according to LAL assays.
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Example 2: Cloning and Expression of Brainiac-5 polypeptide in a Baculovirus
Expression System
In this illustrative example, the plasmid shuttle vector pA2 GP is used to
insert
the cloned DNA encoding the mature protein, lacking its naturally associated
secretory
signal (leader) sequence, into a baculovirus to express a Brainiac-5
polypeptide, using
a baculovirus leader and standard methods as described by Summers and
colleagues (A
Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,
Texas Agricultural Experimental Station Bulletin No. 1555 ( 1987)). This
expression
vector contains the strong polyhedrin promoter of the Autographa californica
nuclear
polyhedrosis virus (AcMNPV) followed by the secretory signal peptide (leader)
of the
baculovirus gp67 protein and convenient restriction sites such as Bam HI, Xba
I and
Asp 718. The polyadenylation site of the simian virus 40 ("SV40") is used for
efficient
polyadenylation. For easy selection of recombinant virus, the plasmid contains
the
Vita-galactosidase gene from E. coli under control of a weak Drosophila
promoter in
the same orientation, followed by the polyadenylation signal of the polyhedrin
gene.
The inserted genes are flanked on both sides by viral sequences for cell-
mediated
homologous recombination with wild-type viral DNA to generate viable virus
that
expresses the cloned polynucleotide.
Many other baculovirus vectors could be used in place of the vector above,
such as pA2, pAc373, pVL941 and pAcIMl, as one skilled in the art would
readily
appreciate, as long as the construct provides appropriately located signals
for
transcription, translation, secretion and the like, including a signal peptide
and an in
frame AUG as required. Such vectors are described, for instance, by Luckow and
colleagues (Virology 174:31-39 (1989)).
The cDNA sequence encoding the Brainiac-5 polypeptide in the deposited
clone, is amplified using PCR oligonucleotide primers corresponding to the 5'
and 3'
sequences of the gene. The 5' primer has the sequence 5'-CGC GGA TCC GCC ATC
ATG GTG GCA GAG GAC TTC GAG C-3' (SEQ ID N0:13) containing the
underlined Bam HI restriction enzyme site followed by 19 nucleotides of the
sequence
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of the Brainiac-5 polypeptide shown in SEQ ID N0:2, beginning with the
currently
known N-terminus. The 3' primer has the sequence 5'-CAC TTA GGT ACC GGA
GTC CCA TTG GAA GG(.J-3' (SEQ ID N0:14) containing the underlined Asp 718
restriction site followed by 18 nucleotides complementary to the carboxy-
terminal
sequence in Figures lA and 1B.
The amplified fragment is isolated from a 1 % agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.). The
fragment
then is digested with Bam HI and Asp 718 and again is purified on a 1 %
agarose gel.
This fragment is designated herein Fl.
The plasmid is digested with the restriction enzymes Bam HI and Asp 718 and
optionally, can be dephosphorylated using calf intestinal phosphatase, using
routine
procedures known in the art. The DNA is then isolated from a 1 % agarose gel
using a
commercially available kit ('"Geneclean" BIO 101 Inc., La Jolla, Ca.). This
vector
DNA is designated herein "V 1 ".
Fragment Fl and the dephosphorylated plasmid V 1 are ligated together with
T4 DNA ligase. E. coli HB 101 or other suitable E. coli hosts such as XL-1
Blue
(Statagene Cloning Systems, La Jolla, CA) cells are transformed with the
ligation
mixture and spread on culture plates. Bacteria are identified that contain the
plasmid
with the human Brainiac-5 gene by digesting DNA from individual colonies using
Bam HI and Asp 718 and then analyzing the digestion product by gel
electrophoresis.
The sequence of the cloned fragment is confirmed by DNA sequencing. This
plasmid
is designated herein pA2GPBrainiac-5.
Five pg of the plasmid pA2GPBrainiac-5 is co-transfected with 1.0 pg of a
commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus
DNA', Pharmingen, San Diego, CA}, using the lipofection method described by
Felgner and colleagues (Pros. Natl. Acad. Sci. USA 84:7413-7417 ( 1987)). One
pg of
BaculoGoldTM virus DNA and 5 lrg of the plasmid pA2GPBrainiac-5 are mixed in a
sterile well of a microtiter plate containing 50 ul of serum-free Grace's
medium (Life
Technologies Inc., Gaithersburg, MD). Afterwards, 10 pl Lipofectin plus 90 pl
Grace's medium are added, mixed and incubated for 15 minutes at room
temperature.
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Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL
1711 ) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without
serum. The plate is then incubated for 5 hours at 27°C. The
transfection solution is
then removed from the plate and 1 ml of Grace's insect medium supplemented
with
10°lo fetal calf serum is added. Cultivation is then continued at
27°C for four days.
After four days the supernatant is collected and a plague assay is performed,
as
described by Summers and Smith (supra). An agarose gel with "Blue Gal" (Life
Technologies Inc., Gaithersburg) is used to allow easy identification and
isolation of
gal-expressing clones, which produce blue-stained plaques. (A detailed
description of
a "plaque assay" of this type can also be found in the user's guide for insect
cell culture
and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-
10).
After appropriate incubation, blue stained plaques are picked with the tip of
a
micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses
is then
resuspended in a microcentrifuge tube containing 200 pl of Grace's medium and
the
suspension containing the recombinant baculovirus is used to infect Sf9 cells
seeded in
35 mm dishes. Four days later the supernatants of these culture dishes are
harvested
and then they are stored at 4°('. The recombinant virus is called V-
Brainiac-5.
To verify the expression of the Brainiac-5 gene Sf9 cells are grown in Grace's
medium supplemented with 10% heat-inactivated FBS. The cells are infected with
the
recombinant baculovirus V-Brainiac-5 at a multiplicity of infection ("MOI") of
about
2. If radiolabeled polypeptides are desired, 6 hours later the medium is
removed and is
replaced with SF900 II medium minus methionine and cysteine (available from
Life
Technologies Inc., Rockville, MD). After 42 hours, 5 pCi of ;SS-methionine and
5 NCi
'SS-cysteine (available from Amersham) are added. The cells are further
incubated for
16 hours and then are harvested by centrifugation. The polypeptides in the
supernatant
as well as the intracellular polypeptides are analyzed by SDS-PAGE followed by
autoradiography (if radiolabeled).
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Example 3: Cloning and Expression of Brainiac-5 in Mammalian Cells
A typical mammalian expression vector contains the promoter element, which
mediates the initiation of transcription of mRNA, the polypeptide coding
sequence,
and signals required fox the termination of transcription and polyadenylation
of the
transcript. Additional elements include enhancers, Kozak sequences and
intervening
sequences flanked by donor and acceptor sites for RNA splicing. Highly
efficient
transcription can be achieved with the early and late promoters from SV40, the
long
terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLV-I, HIV-1 and the
early
promoter of the cytomegalovirus (CMV). However, cellular elements can also be
used
(e-g~~ the human actin promoter). Suitable expression vectors for use in
practicing the
present invention include, for example, vectors such as pSVL and pMSG
(Pharmacia,
Uppsala, Sweden), pRSVcat {ATCC 37152), pSV2dhfr (ATCC 37146) and pBCI2MI
(ATCC 67109). Mammalian host cells that could be used include, human Hela,
293,
H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail
QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, the gene can be expressed in stable cell lines that contain the
gene integrated into a chromosome. The co-transfection with a selectable
marker such
as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of
the
transfected cells.
The transfected gene can also be amplified to express large amounts of the
encoded polypeptide. The DHFR (dihydrofolate reductase) marker is useful to
develop cell lines that carry several hundred or even several thousand copies
of the
gene of interest. Another useful selection marker is the enzyme glutamine
synthase
(GS; Murphy, et al., Bioehem J. 227:277-279 ( 1991 ); Bebbington, et al.,
Bio/lechnology 10:169-175 ( 1992)). Using these markers, the mammalian cells
are
grown in selective medium and the cells with the highest resistance are
selected.
These cell lines contain the amplified genes) integrated into a chromosome.
Chinese
hamster ovary (CHO) and NSO cells are often used for the production of
polypeptides.
The expression vectors pC 1 and pC4 contain the strong promoter (LTR) of the
Rous Sarcoma Virus (Cullen, et al., Mol. Cel. Biol. 5:438-447 { 1985)) plus a
fragment
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of the CMV-enhancer (Boshart, et al., Cell 41:521-530 ( 1985)). Multiple
cloning
sites, e.g., with the restriction enzyme cleavage sites Bam HI, Xba I and Asp
718,
facilitate the cloning of the gene of interest. The vectors contain in
addition the 3'
intron, the polyadenylation and termination signal of the rat preproinsulin
gene.
Example 3(a): Cloning and Expression in COS Cells
The expression plasmid, pBrainiac-SHA, is made by cloning a portion of the
cDNA encoding the mature form of the Brainiac-5 polypeptide into the
expression
vector pcDNAI/Amp or pcDNAIII (which can be obtained from Invitrogen, Inc.).
The expression vector pcDNAI/amp contains: ( 1 ) an E. coli origin of
replication effective for propagation in E. coli and other prokaryotic cells;
(2) an
ampicillin resistance gene for selection of plasmid-containing prokaryotic
cells; (3) an
SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV
promoter, a
polylinker, an SV40 intron; (5) several codons encoding a hemagglutinin
fragment
G.e., an "HA" tag to facilitate purification) followed by a termination codon
and
polyadenylation signal arranged so that a cDNA can be conveniently placed
under
expression control of the CMV promoter and operably linked to the SV40 intron
and
the polyadenylation signal by means of restriction sites in the polylinker.
The HA tag
corresponds to an epitope derived from the influenza hemagglutinin protein
described
bY Wilson and colleagues (Cel137:767 {1984)). The fusion of the HA tag to the
target
polypeptide allows easy detection and recovery of the recombinant polypeptide
with
an antibody that recognizes the HA epitope. pcDNAIII contains, in addition,
the
selectable neomycin marker.
A DNA fragment encoding the Brainiac-5 polypeptide is cloned into the
polylinker region of the vector so that recombinant polypeptide expression is
directed
by the CMV promoter. The plasmid construction strategy is as follows. The
Brainiac-5 cDNA of the deposited clone is amplified using primers that contain
convenient restriction sites, much as described above for construction of
vectors for
expression of Brainiac-5 in E. coli. Suitable primers include the following,
which are
used in this example. The 5' primer, containing the underlined Bam HI site, a
Kozak
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sequence (in italics), an AUG start codon, and 16 nucleotides of the 5' coding
region of
the Brainiac-5 polypeptide, has the following sequence: 5' CGC GGA TCC GCCATC
ATG GTG GCA GAG GAC TTC GAG C 3' (SEQ ID N0:15). The 3' primer,
containing the underlined Asp 718 and 17 of nucleotides complementary to the
3'
coding sequence immediately before the stop codon, has the following sequence:
5'-CAC TTA GT A C GGA GTC CCA TTG GAA GGG-3' (SEQ ID N0:16).
The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested
with Bam HI and Asp 718 and then ligated. The ligation mixture is transformed
into
E. coli strain SURE (Stratagene Cloning Systems, La Jolla, CA 92037), and the
transformed culture is plated on ampicillin media plates which then are
incubated to
allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from
resistant
colonies and examined by restriction analysis or other means for the presence
of the
fragment encoding the complete Brainiac-5 polypeptide
For expression of recombinant Brainiac-5, COS cells are transfected with an
expression vector, as described above, using DEAF-dextran, as described, for
instance,
by Sambrook and coworkers (Molecular Cloning: a Laboratory Manual, Cold Spring
Laboratory Press, Cold Spring Harbor, New York ( 1989)). Cells are incubated
under
conditions for expression of Brainiac-5 by the vector.
Expression of the Brainiac-5-HA fusion polypeptide is detected by
radiolabeling and immunoprecipitation, using methods described in, for example
Harlow and colleagues (Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, New York ( 1988)). To this end,
two
days after transfection, the cells are labeled by incubation in media
containing 35S-
cysteine for 8 hours. The cells and the media are collected, and the cells are
washed
and the lysed with detergent-containing RIPA buffer: 150 mM NaCI, 1 % NP-40,
0.1 %
SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson and
colleagues (supra). Polypeptides are precipitated from the cell lysate and
from the
culture media using an HA-specific monoclonal antibody. The precipitated
polypeptides then are analyzed by SDS-PAGE and autoradiography. An expression
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product of the expected size is seen in the cell lysate, which is not seen in
negative
controls.
Example 3(b): Cloning and Expression in CHD Cells
The vector pC4 is used for the expression of Brainiac-5 polypeptide. Plasmid
S pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). The
plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
Chinese hamster ovary- or other cells lacking dihydrofolate activity that are
transfected with these plasmids can be selected by growing the cells in a
selective
medium (alpha minus MEM, Life Technologies) supplemented with the
chemotherapeutic agent methotrexate. The amplification of the DHFR genes in
cells
resistant to methotrexate (MTX) has been well documented (see, e.g., Alt, F.
W., et
al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C. Biochem. et
Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.
Biotechnology
9:64-68 ( 1991 )). Cells grown in increasing concentrations of MTX develop
resistance
to the drug by overproducing the target enzyme, DHFR, as a result of
amplification of
the DHFR gene. If a second gene is linked to the DHFR gene, it is usually co-
amplified and over-expressed. It is known in the art that this approach may be
used to
develop cell lines carrying more than 1,000 copies of the amplified gene(s).
Subsequently, when the methotrexate is withdrawn, cell lines are obtained
which
contain the amplified gene integrated into one or more chrornosome(s) of the
host cell.
Plasmid pC4 contains for expressing the gene of interest the strong promoter
of
the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al.,
Mol. Cell.
Binl. 5:438-447 ( 1985)) plus a fragment isolated from the enhancer of the
immediate
early gene of human cytomegalovirus (CMV; Boshart, et al., Cell 41:521-530
(1985)).
2S Downstream of the promoter are the following single restriction enzyme
cleavage sites
that allow the integration of the genes: Bam HI, Xba I, and Asp 718. Behind
these
cloning sites the plasmid contains the 3' intron and polyadenylation site of
the rat
preproinsulin gene. Other high efficiency promoters can also be used for the
expression, e.g., the human 13-actin promoter, the SV40 early or late
promoters or the
long terminal repeats from other retroviruses, e.g., HIV and HTLVI. Clontech's
Tet-
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Off and Tet-On gene expression systems and similar systems can be used to
express
the Brainiac-5 polypeptide in a regulated way in mammalian cells (Gossen, M.,
and
Bujard, H. Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992)). For the
polyadenylation
of the mRNA other signals, e.g., from the human growth hormone or globin genes
can
be used as well. Stable cell lines carrying a gene of interest integrated into
the
chromosomes can also be selected upon co-transfection with a selectable marker
such
as gpt, 6418 or hygromycin. It is advantageous to use more than one selectable
marker in the beginning, e.g., G418 plus methotrexate.
The plasmid pC4 is digested with the restriction enzymes Bam HI and Asp 718
and then dephosphorylated using calf intestinal phosphates by procedures known
in
the art. The vector is then isolated from a 1 % agarose gel.
The DNA sequence encoding the complete Brainiac-5 polypeptide is amplified
using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of
the
desired portion of the gene. The 5' primer contains a Bam HI restriction site,
a Kozak
se9uence, an AUG start colon, and 16 nucleotides of the 5' coding region of
the
Brainiac-5 polypeptide, and is shown in SEQ ID NO:15. The 3' primer, contains
an
Asp 718 restriction site, and 17 of nucleotides complementary to the 3' coding
sequence immediately before the stop colon, and is shown in SEQ ID N0:16.
The amplified fragment is digested with the endonucleases Bam HI and Asp
718 and then purified again on a 1 % agarose gel. The isolated fragment and
the
dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB 101 or
XL-1
Blue cells are then transformed and bacteria are identified that contain the
fragment
inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene are used for
transfection. Five pg of the expression plasmid pC4 is cotransfected with 0.5
~tg of
the plasmid pSVneo using lipofectin (Felgner, et al., supra). The plasmid pSV2-
neo
contains a dominant selectable marker, the neo gene from Tn5 encoding an
enzyme
that confers resistance to a group of antibiotics including 6418. The cells
are seeded
in alpha minus MEM supplemented with 1 mg/ml G4I8. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha
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minus MEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/ml
6418. After about 10-14 days single clones are trypsinized and then seeded in
6-well
petri dishes or 10 ml flasks using different concentrations of methotrexate
(50 nM, 100
nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of
S methotrexate are then transferred to new 6-well plates containing even
higher
concentrations of methotrexate ( 1 pM, 2 E.~M, 5 pM, 10 mM, 20 mM). The same
procedure is repeated until clones are obtained which grow at a concentration
of
100-200 NM. Expression of the desired gene product is analyzed, for instance,
by
SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 4: Tissue distribution of Brainiac-5 mRNA expression
Northern blot analysis is carried out to examine Brainiac-5 gene expression in
human tissues, using methods described by, among others, Sambrook and
colleagues
(supra). A cDNA probe containing the entire nucleotide sequence of the
Brainiac-5
polypeptide (SEQ ID NO:1 ) is labeled with 3'-P using the rediprimeTM DNA
labeling
system (Amersham Life Science), according to manufacturer's instructions.
After
labeling, the probe is purified using a CHROMA SPIN-100TM column (Clontech
Laboratories, Inc.), according to manufacturer's protocol number PT1200-1.
'The
purified labeled probe is then used to examine various human tissues for
Brainiac-S
mRNA.
Multiple Tissue Northern (MTN) blots containing various human tissues (H) or
human immune system tissues (IM) are obtained from Clontech and are examined
with the labeled probe using ExpressHybTM hybridization solution (Ciontech)
according to manufacturer's protocol number PTl 190-1. Following hybridization
and
washing, the blots are mounted and exposed to film at -70°C overnight,
and films
developed according to standard procedures.
Example 5: Production of an Antibody
(a) Hybridoma Technology
The antibodies of the present invention can be prepared by a variety of
methods. (See, Current Protocols, Chapter 2.) As one example of such methods,
cells
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expressing Brainiac-5 are administered to an animal to induce the production
of sera
containing polyclonal antibodies. In a preferred method, a preparation of
Brainiac-5
protein is prepared and purified to render it substantially free of natural
contaminants.
Such a preparation is then introduced into an animal in order to produce
polyclonal
antisera of greater specific activity.
Monoclonal antibodies specific far protein Brainiac-5 are prepared using
hybridoma technology. (Kohler et al., Nature 256:495 ( 1975); Kohler et al.,
Eur. J.
Immunol. 6:5 I 1 ( 1976); Kohler et al., Eur. J. Immunol. 6:292 ( 1976);
Hammerling et
al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-
681
( 1981 )). In general, an animal (preferably a mouse) is immunized with
Brainiac-5
polypeptide or, more preferably, with a secreted Brainiac-5 polypeptide-
expressing
cell. Such polypeptide-expressing cells are cultured in any suitable tissue
culture
medium, preferably in Earle's modified Eagle's medium supplemented with 10%
fetal
bovine serum (inactivated at about 56°C), and supplemented with about
10 gll of
nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 pg/ml
of
streptomycin.
The splenocytes of such mice are extracted and fused with a suitable myeloma
cell line. Any suitable myeloma cell line may be employed in accordance with
the
present invention; however, it is preferable to employ the parent myeloma cell
line
(SP20), available from the A'TCC. After fusion, the resulting hybridoma cells
are
selectively maintained in HAT medium, and then cloned by limiting dilution as
described by Wands et al. (Gastroenterology 80:225-232 ( 1981 ). The hybridoma
cells
obtained through such a selection are then assayed to identify clones which
secrete
antibodies capable of binding; the Brainiac-5 polypeptide.
Alternatively, additional antibodies capable of binding to Brainiac-5
polypeptide can be produced in a two-step procedure using anti-idiotypic
antibodies.
Such a method makes use of the fact that antibodies are themselves antigens,
and
therefore, it is possible to obtain an antibody which binds to a second
antibody. In
accordance with this method, protein specific antibodies are used to immunize
an
animal, preferably a mouse. T'he splenocytes of such an animal are then used
to
produce hybridoma cells, and the hybridoma cells are screened to identify
clones
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which produce an antibody whose ability to bind to the Brainiac-5 protein-
specific
antibody can be blocked by Brainiac-S. Such antibodies comprise anti-idiotypic
antibodies to the Brainiac-S protein-specific antibody and are used to
immunize an
animal to induce formation of further Brainiac-5 protein-specific antibodies.
For in vivo use of antibodies in humans, an antibody is "humanized". Such
antibodies can be produced using genetic constructs derived from hybridoma
cells
producing the monoclonal antibodies described above. Methods for producing
chimeric and humanized antibodies are known in the art and are discussed
infra. (See,
for review, Morrison, Science 229:1202 ( 1985); Oi et al., BioTechniques 4:214
( 1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP
171496;
Morrison et al., EP 173494; '~'Veuberger et al.> WO 8601533; Robinson et al.,
WO
8702671; Bouiianne et al., Nature 312:643 (1984); Neuberger et al.> Nature
314:268
( 1985).)
(b) Isolation Of Antibody Fragments Directed Against Brainiac-S From A
Library Of scFvs
Naturally occurring V-genes isolated from human PBLs are constructed into a
library of antibody fragments, which contain reactivities against Brainiac-5
to which
the donor may or may not have been exposed (see e.g., U.S. Patent 5,885,793
incorporated herein by reference in its entirety).
Rescue of the Library. A library of scFvs is constructed from the RNA of
human PBLs as described in PCT publication WO 92/01047. To rescue phage
displaying antibody fragments, approximately 109 E. coli harboring the
phagemid are
used to inoculate 50 ml of 2xTY containing 1% glucose and 100 pg/ml of
ampicillin
(2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five rnl of this
culture
is used to innoculate 50 ml of 2xTY-AMP-GLU, 2 x 108 TU of delta gene 3 helper
(M 13 delta gene III, see PCT publication WO 92/01047) are added and the
culture
incubated at 37°C for 45 minutes without shaking and then at
37°C for 45 minutes
with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the
pellet
resuspended in 2 liters of 2xTY containing 100 pg/ml ampicillin and 50 ug/ml
kanamycin and grown overnight. Phage are prepared as described in PCT
publication
WO 92/01047.
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-I86-
M 13 delta gene III i s prepared as follows: M I 3 delta gene III helper phage
does not encode gene III protein, hence the phage(mid) displaying antibody
fragments
have a greater avidity of binding to antigen. Infectious M I3 delta gene III
particles are
made by growing the helper phage in cells harboring a pUC 19 derivative
supplying
the wild type gene III protein during phage morphogenesis. The culture is
incubated
for 1 hour at 37° C without shaking and then for a further hour at
37°C with shaking.
Cells are spun down (IEC-(:entra 8,400 r.p.m. for 10 min), resuspended in 300
ml
2xTY broth containing I00 pg arnpicillin/ml and 25 pg kanamycin/ml (2xTY-AMP-
KAN) and grown overnight, shaking at 37°C. Phage particles are
purified and
I0 concentrated from the culture medium by two PEG-precipitations (Sambrook et
al.,
1990), resuspended in 2 ml PBS and passed through a 0.45 pm filter (Minisart
NML;
Sartorius) to give a final concentration of approximately 1013 transducing
units/ml
(ampicillin-resistant clones).
Panning of the Library. Immunotubes (Nuns) are coated overnight in PBS
with 4 ml of either 100 ug/ml or 10 pg/ml of a polypeptide of the present
invention.
Tubes are blocked with 2% Marvel-PBS for 2 hours at 37°C and then
washed 3 times
in PBS. Approximately 1013 TU of phage is applied to the tube and incubated
for 30
minutes at room temperature tumbling on an over and under turntable and then
left to
stand for another I .5 hours. Tubes are washed I O times with PBS 0.1 % Tween-
20
and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine
and
rotating 1 S minutes on an under and over turntable after which the solution
is
immediately neutralized with 0.5 ml of I.OM Tris-HCI, pH 7.4. Phage are then
used to
infect IO ml of mid-log E. coli TGI by incubating eluted phage with bacteria
for 30
minutes at 37°C. The E. coli are then plated on TYE plates containing
1% glucose
and 100 Ng/ml ampicillin. 7'he resulting bacterial library is then rescued
with delta
gene 3 helper phage as described above to prepare phage for a subsequent round
of
a
selection. This process is then repeated for a total of 4 rounds of affinity
purification
with tube-washing increased to 20 times with PBS, 0.1 % Tween-20 and 20 times
with
PBS for rounds 3 and 4.
Characterization of Bin ers. Eluted phage from the 3rd and 4th rounds of
selection are used to infect E. coli HB 2151 and soluble scFv is produced
(Marks, et
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al., 1991) from single colonies for assay. ELISAs are performed with
microtitre plates
coated with either 10 pg/ml of the polypeptide of the present invention in 50
mM
bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR
fingerprinting (see, e.g., PCT publication WO 92/01047) and then by
sequencing.
It will be clear that the invention may be practiced otherwise than as
particularly described in the foregoing description and examples. Numerous
modifications and variations of the present invention are possible in light of
the above
teachings and, therefore, are within the scope of the appended claims.
The entire disclosure of all publications (including patents, patent
applications,
journal articles, laboratory manuals, books, or other documents) cited herein
are
hereby incorporated by reference.
Further, the Sequence Listing submitted herewith, and the Sequence Listing
submitted in copending application Serial No. 60/113,804, filed December 23,
1998,
in both computer-readable and paper formats (in each case), are hereby
incorporated
by reference in their entireties.
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188
INDICATIONS RELATING TO A DEPOSItTED MICROORGANISM
(PCT Rule l3bis)
A. The indications made below relate
to themic:roorganismreferredtointhedescription
on page 3 ,1 inc 27
B. IDENTIFICATIONOFDEPOS1T Further
deposits are identified on an addi
tional sheet
Nameofdepositaryinstitution American
Type Culture COIIeCtfOn
Address of depositary institution
(including postal code and country)
10801 University Boulevard
Manassas, Virginia 20110-2209
United States of America
Date of deposit Accession Number
11 January 1999 203572
C. ADDITIONAL INDICATIONS (leave
blank ijnot applicable) This information
is continued on an additional sheet
D. DESIGNATED STATES FOR WHICH INDICATIONS
ARE MADE (if the indications are
not forall designated States)
Europe
In respect to those designations
in which a European Patent is sought
a sample of the deposited
microorganism will be made available
until the publication of the mention
of the grant of the European patent
or until the date on which application
has been refused or withdrawn or
is deemed to be withdrawn, only
by
the issue of such a sample to an
expert nominated by the person
requesting the sample (Rule 28
(4) EPC).
E. SEPARATE FURNISHING OFINDICATIONS(leaveblankifnotapplicable)
The indications listed below will
be submitted to the International
Bureau later (sped the general
natureoftheindicationse.g., 'Accession
Number of Deposit ")
rte- ForreceivingOfficeuseonly ForIntemationalBureauuseonly
This sheet was received with the international application ~ This sheet was
received by the International Bureau on:
Authorized officer
Form PCT/RO/134 (July 1992)
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WO 00/39136 PCTNS99/30452
189
ATCC Deposit No. 203572
CANADA
The applicant requests that, until either a Canadian patent has been issued on
the basis of an
application or the application has been refused, or is abandoned and no longer
subject to
reinstatement, or is withdrawn, the Commissioner of Patents only authorizes
the furnishing of
a sample of the deposited biological material referred to in the application
to an independent
expert nominated by the Commissioner, the applicant must, by a written
statement, inform the
International Bureau accordingly before completion of technical preparations
for publication
of the international application.
NORWAY
The applicant hereby requests that the application has been laid open to
public inspection (by
the Norwegian Patent Office), or lhas been finally decided upon by the
Norwegian Patent
Office without having been laid open inspection, the furnishing of a sample
shall only be
effected to an expert in the art. The request to this effect shall be filed by
the applicant with
the Norwegian Patent Office not later than at the time when the application is
made available
to the public under Sections 22 and 33(3) of the Norwegian Patents Act. If
such a request has
been filed by the applicant, any request made by a third party for the
furnishing of a sample
shall indicate the expert to be used. That expert may be any person entered on
the list of
recognized experts drawn up by the Norwegian Patent Office or any person
approved by the
applicant in the individual case.
AUSTRALIA
The applicant hereby gives notice that the furnishing of a sample of a
microorganism shall
only be effected prior to the grant of a patent, or prior to the lapsing,
refusal or withdrawal of
the application, to a person who is a skilled addressee without an interest in
the invention
(Regulation 3.25(3) of the Australian Patents Regulations).
FINLAND
The applicant hereby requests that, until the application has been laid open
to public
inspection (by the National Board of Patents and Regulations), or has been
finally decided
upon by the National Board of Patents and Registration without having been
laid open to
public inspection, the furnishing o:f a sample shall only be effected to an
expert in the art.
UNITED KINGDOM
The applicant hereby requests that the furnishing of a sample of a
microorganism shall only
be made available to an expert. The request to this effect must be filed by
the applicant with
the International Bureau before the completion of the technical preparations
for the
international publication of the application.
CA 02356548 2001-06-21
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190
ATCC Deposit No. 203572
DENMARK
The applicant hereby requests that, until the application has been laid open
to public
inspection (by the Danish Patent Office), or has been finally decided upon by
the Danish
Patent office without having been laid open to public inspection, the
furnishing of a sample
shall only be effected to an expert in the art. The request to this effect
shall be filed by the
applicant with the Danish Patent tJffice not later that at the time when the
application is made
available to the public under Sections 22 and 33(3) ofthe Danish Patents Act.
If such a
request has been f led by the applicant, any request made by a third party for
the furnishing of
a sample shall indicate the expert to be used. That expert may be any person
entered on a list
of recognized experts drawn up by the Danish Patent Office or any person by
the applicant in
the individual case.
SWEDEN
The applicant hereby requests that, until the application has been laid open
to public
inspection (by the Swedish Patent Office), or has been finally decided upon by
the Swedish
Patent Office without having been laid open to public inspection, the
furnishing of a sample
shall only be effected to an expert in the art. The request to this effect
shall be filed by the
applicant with the International Bureau before the expiration of 16 months
from the priority
date (preferably on the Form PCTiRO/134 reproduced in annex Z of Volume I of
the PCT
Applicant's Guide). If such a request has been filed by the applicant any
request made by a
third party for the furnishing of a sample shall indicate the expert to be
used. That expert may
be any person entered on a list of recognized experts drawn up by the Swedish
Patent Office
or any person approved by a applicant in the individual case.
NETHERLANDS
The applicant hereby requests that until the date of a grant of a Netherlands
patent or until the
date on which the application is refused or withdrawn or lapsed, the
microorganism shall be
made available as provided in the :31 F{1 ) of the Patent Rules only by the
issue of a sample to
an expert. The request to this effect must be furnished by the applicant with
the Netherlands
Industrial Property Office before the date on which the application is made
available to the
public under Section 22C or Section 25 of the Patents Act of the Kingdom of
the Netherlands,
whichever of the two dates occurs earlier.
CA 02356548 2001-06-21
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1
SEQUENCE LISTING
<110> Human Genome Sciences, Inc.
<120> Human Brainiac-5
<130> PF503PCT
<140> Unassigned
<141> 1999-12-20
<150> 60/113,804
<151> 1998-12-23
<160> 16
<170> PatentIn Ver. 2.0
<210> 1
<211> 977
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(834)
<400> 1
gtg gca gag gac ttc gag cgg cgc caa gcc gtg cgc cag acg tgg ggc 48
Val Ala Glu Asp Phe Glu Arg Arg Gln Ala Val Arg Gln Thr Trp Gly
1 5 10 15
gcg gag ggt cgc gtg cag ggg gcg ctg gtg cgc cgc gtg ttc ttg ctg 96
Ala Glu Gly Arg Val Gln Gly Ala Leu Val Arg Arg Val Phe Leu Leu
20 25 30
ggc gtg ccc agg ggc gca ggc tcg ggc ggg gcc gac gaa gtt ggg gag 144
Gly Val Pro Arg Gly Ala Gly Ser Gly Gly Ala Asp Glu Val Gly Glu
35 40 45
ggc gcg cga acc cac tgg cgc gcc ctg ctg cgg gcc gag agc ctt gcg 192
Gly Ala Arg Thr His Trp Arg Ala Leu Leu Arg Ala Glu Ser Leu Ala
50 55 60
tat gcg gac atc ctg ctc tgg gcc ttc gac gac acc ttt ttt aac cta 240
Tyr Ala Asp Ile Leu Leu Trp Ala Phe Asp Asp Thr Phe Phe Asn Leu
65 70 75 80
acg ctc aag gag atc cac ttt cta gcc tgg gcc tca get ttc tgc ccc 288
Thr Leu Lys Glu Ile His Phe Leu Ala Trp Ala Ser Ala Phe Cys Pro
85 90 95
gac gtg cgc ttc gtt ttt aag ggc gac gca gat gtg ttc gtg aac gtg 336
Asp Val Arg Phe Val Phe Lys Gly Asp Ala Asp Val Phe Val Asn Val
100 105 110
gga aat ctc ctg gag ttc ctg gcg ccg cgg gac ccg gcg caa gac ctg 384
Gly Asn Leu Leu Glu Phe Leu Ala Pro Arg Asp Pro Ala Gln Asp Leu
CA 02356548 2001-06-21
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2
115 120 125
ctt get ggt gac gta att gtg cat gcg cgg ccc atc cgc acg cgg get 432
Leu Ala Gly Asp Val Ile Val His Ala Arg Pro Ile Arg Thr Arg Ala
130 135 140
agc aag tac tac atc ccc gag gcc gtg tac ggc ctg ccc gcc tat ccg 480
Ser Lys Tyr Tyr Ile Pro Glu Ala Val Tyr Gly Leu Pro Ala Tyr Pro
145 150 155 160
gcc tac gcg ggc ggc ggt ggc ttt gtg ctt tcc ggg gcc acg ctg cac 528
Ala Tyr Ala Gly Gly Gly Gly Phe Val Leu Ser Gly Ala Thr Leu His
165 170 175
cgc ctg get ggc gcc tgt gcg cag gtc gag ctc ttc ccc atc gac gac 576
Arg Leu Ala Gly Ala Cys Aia Gln Val Glu Leu Phe Pro Ile Asp Asp
180 185 190
gtc ttt ctg ggc atg tgt ctg cag cgc ctg cgg ctc acg ccc gag cct 624
Val Phe Leu Gly Met Cys Leu Gln Arg Leu Arg Leu Thr Pro Glu Pro
195 200 205
cac cct gcc ttc cgc acc t~tt ggc atc ccc cag cct tca gcc gcg ccg 672
His Pro Ala Phe Arg Thr F?he Gly Ile Pro Gln Pro Ser Ala Ala Pro
210 <'?15 220
cat ttg agc acc ttc gac c:cc tgc ttt tac cgt gag ctg gtt gta gtg 720
His Leu Ser Thr Phe Asp Pro Cys Phe Tyr Arg Glu Leu Val Val Val
225 230 235 240
cac ggg ctc tcg gcc get gac atc tgg ctt atg tgg cgc ctg ctg cac 768
His Gly Leu Ser Ala Ala P,sp Ile Trp Leu Met Trp Arg Leu Leu His
245 250 255
ggg ccg cat ggg cca gcc tgt gcg cat cca cag cct gtc get gca ggc 816
Gly Pro His Gly Pro Ala Cys Ala His Pro Gln Pro Val Ala Ala Gly
260 265 270
ccc ttc caa tgg gac tcc tagctcccca ctacagcccc aagctcctaa 864
Pro Phe Gln Trp Asp Ser
275
ctcagaccca gaatggagcc ggtttcccag attattgccg tgtatgtggt tcttccctga 924
tcaccagggt gcctgtctcc acaggatccc agggggatgg gggttaagct tgg 977
<210> 2
<211> 278
<212> PRT
<213> Homo Sapiens
<400> 2
Val Ala Glu Asp Phe Glu A:rg Arg Gln Ala Val Arg Gln Thr Trp Gly
1 5 10 15
Ala Glu Gly Arg Val Gln G:Ly Ala Leu Va1 Arg Arg Val Phe Leu Leu
20 25 30
CA 02356548 2001-06-21
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3
Gly Val Pro Arg Gly Ala Gly Ser Gly Gly Ala Asp Glu Val Gly Glu
35 40 45
Gly Ala Arg Thr His Trp Arg Ala Leu Leu Arg Ala Glu Ser Leu Ala
50 55 60
Tyr Ala Asp Ile Leu Leu Trp Ala Phe Asp Asp Thr Phe Phe Asn Leu
65 70 75 80
Thr Leu Lys Glu Ile His Phe Leu Ala Trp Ala Ser Ala Phe Cys Pro
85 90 95
Asp Val Arg Phe Val Phe :Lys Gly Asp Ala Asp Val Phe Val Asn Val
100 105 110
Gly Asn Leu Leu Glu Phe Leu Ala Pro Arg Asp Pro Ala Gln Asp Leu
115 120 125
Leu Ala Gly Asp Val Ile Val His Ala Arg Pro Ile Arg Thr Arg Ala
130 _L35 140
Ser Lys Tyr Tyr Ile Pro Glu Ala Val Tyr Gly Leu Pro Ala Tyr Pro
145 150 :155 160
Ala Tyr Ala Gly Gly Gly C>ly Phe Val Leu Ser Gly Ala Thr Leu His
165 I'70 275
Arg Leu Ala Gly Ala Cys Ala Gln Val Glu Leu Phe Pro Ile Asp Asp
180 185 190
Val Phe Leu Gly Met Cys L~eu Gln Arg Leu Arg Leu Thr Pro Glu Pro
195 200 205
His Pro Ala Phe Arg Thr Phe Gly Ile Pro Gln Pro Ser Ala Ala Pro
210 215 220
His Leu Ser Thr Phe Asp Pro Cys Phe Tyr Arg Glu Leu Val Val Val
225 230 235 240
His Gly Leu Ser Ala Ala Asp Ile Trp Leu Met Trp Arg Leu Leu His
245 250 255
Gly Pro His Gly Pro Ala Cys Ala His Pro Gln Pro Val Ala Ala Gly
260 265 270
Pro Phe Gln Trp Asp Ser
275
<210> 3
<211> 325
<212> PRT
<213> Drosophila melanogaster
<400> 3
Met Gln Ser Lys His Arg L~_Js Leu Leu Leu Arg Cys Leu Leu Val Leu
10 15
Pro Leu Ile Leu Leu Val Asp Tyr Cys Gly Leu Leu Thr His Leu His
CA 02356548 2001-06-21
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4
20 25 30
Glu Leu Asn Phe Glu Arg His Phe His Tyr Pro Leu Asn Asp Asp Thr
35 40 45
Gly Ser Gly Ser Ala Ser Ser Gly Leu Asp Lys Phe Ala Tyr Leu Arg
50 55 60
Val Pro Ser Phe Thr Ala Glu Val Pro Val Asp Gln Pro Ala Arg Leu
65 70 75 80
Thr Met Leu Ile Lys Ser Ala Val Gly Asn Ser Arg Arg Arg Glu Ala
85 90 95
Ile Arg Arg Thr Trp Gly Tyr Glu Gly Arg Phe Ser Asp Val His Leu
100 105 110
Arg Arg Val Phe Leu Leu Gly Thr Ala Glu Asp Ser Glu Lys Asp Val
115 120 125
Ala Trp Glu Ser Arg Glu His Gly Asp Ile Leu Gln Ala Asp Phe Thr
130 135 140
Asp Ala Tyr Phe Asn Asn Thr Leu Lys Thr Met Leu Gly Met Arg Trp
145 150 155 160
Ala Ser Glu Gln Phe Asn Arg Ser Glu Phe Tyr Leu Phe Val Asp Asp
165 170 175
Asp Tyr Tyr Val Ser Ala Lys Asn Val Leu Lys Phe Leu Gly Arg Gly
180 185 190
Arg Gln Ser His Gln Pro Glu Leu Leu Phe Ala Gly His Val Phe Gln
195 200 205
Thr Ser Pro Leu Arg His Lys Phe Ser Lys Trp Tyr Val Ser Leu Glu
210 215 220
Glu Tyr Pro Phe Asp Arg Trp Pro Pro Tyr Val Thr Ala Gly Ala Phe
225 230 235 240
Ile Leu Ser Gln Lys Ala Leu Arg Gln Leu Tyr Ala Ala Ser Val His
245 250 255
Leu Pro Leu Phe Arg Phe Asp Asp Val Tyr Leu Gly Ile Val Ala Leu
260 265 270
Lys Ala Gly Ile Ser Leu Gln His Cys Asp Asp Phe Arg Phe His Arg
' 275 280 285
Pro Ala Tyr Lys Gly Pro Asp Ser Tyr Ser Ser Val Ile Ala Ser His
290 295 300
Glu Phe Gly Asp Pro Glu Glu Met Thr Arg Val Trp Asn Glu Cys Arg
305 310 315 320
Ser Ala Asn Tyr Ala
325
CA 02356548 2001-06-21
WO 00/39136 PCT/US99/30452
<210> 4
<211> 422
<212> PRT
<213> Homo Sapiens
<400> 4
Met Leu Gln Trp Arg Arg Arg His Cys Cys Phe Ala Lys Met Thr Trp
1 5 10 15
Asn Ala Lys Arg Ser Leu Phe Arg Thr His Leu Ile Gly Val Leu Ser
20 25 30
Leu Val Phe Leu Phe Ala Met Phe Leu Phe Phe Asn His His Asp Trp
35 40 45
Leu Pro Gly Arg Ala Gly Phe Lys Glu Asn Pro Val Thr Tyr Thr Phe
50 55 60
Arg Gly Phe Arg Ser Thr Lys Ser Glu Thr Asn His Ser Ser Leu Arg
65 70 75 80
Asn Ile Trp Lys Glu Thr Val Pro Gln Thr Leu Arg Pro Gln Thr Ala
85 90 95
Thr Asn Ser Asn Asn Thr Asp Leu Ser Pro Gln Gly Val Thr Gly Leu
100 105 110
Glu Asn Thr Leu Ser Ala Asn Gly Ser Ile Tyr Asn Glu Lys Gly Thr
115 120 125
Gly His Pro Asn Ser Tyr His Phe Lys Tyr Ile Ile Asn Glu Pro Glu
130 135 140
Lys Cys Gln Glu Lys Ser P:ro Phe Leu Ile Leu Leu Ile Ala Ala Glu
145 150 155 160
Pro Gly Gln Ile Glu Ala A:rg Arg Ala Ile Arg Gln Thr Trp Gly Asn
165 170 175
Glu Ser Leu Ala Pro Gly I:Le Gln I1e Thr Arg Ile Phe Leu Leu Gly
180 185 190
Leu Ser Ile Lys Leu Asn G:Ly Tyr Leu Gln Arg Ala Ile Leu Glu Glu
195 200 205
Ser Arg Gln Tyr His Asp I:Le Ile Gln Gln Glu Tyr Leu Asp Thr Tyr
210 2:L5 220
a
Tyr Asn Leu Thr Ile Lys Thr Leu Met Gly Met Asn Trp Val Ala Thr
225 230 235 240
Tyr Cys Pro His Ile Pro Tyr Val Met Lys Thr Asp Ser Asp Met Phe
245 250 255
Val Asn Thr Glu Tyr Leu Ile Asn Lys Leu Leu Lys Pro Asp Leu Pro
260 265 270
Pro Arg His Asn Tyr Phe Thr Gly Tyr Leu Met Arg Gly Tyr Ala Pro
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6
z75 280 285
Asn Arg Asn Lys Asp Ser Lys Trp Tyr Met Pro Pro Asp Leu Tyr Pro
290 295 300
Ser Glu Arg Tyr Pro Val Phe Cys Ser Gly Thr Gly Tyr Val Phe Ser
305 310 315 320
Gly Asp Leu Ala Glu Lys Ile Phe Lys Val Ser Leu Gly Ile Arg Arg
325 330 335
Leu His Leu Glu Asp Val 'ryr Val Gly Ile Cys Leu Ala Lys Leu Arg
340 345 350
Ile Asp Pro Val Pro Pro Pro Asn Glu Phe Val Phe Asn His Trp Arg
355 360 365
Val Ser Tyr Ser Ser Cys Lys Tyr Ser His Leu Ile Thr Ser His Gln
370 375 380
Phe Gln Pro Ser Glu Leu Il.e Lys Tyr Trp Asn His Leu Gln Gln Asn
385 390 395 400
Lys His Asn Ala Cys Ala Asn Ala Ala Lys Glu Lys Ala Gly Arg Tyr
405 410 415
Arg His Arg Lys Leu His
420
<210> 5
<211> 361
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<222> (31)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (156)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (158)
<223> r~ equals a, t, g or c
<220>
<221> misc_feature
<222> (184)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (223)
<223> n equals a, t, g or c
CA 02356548 2001-06-21
WO 00/39136 PCT/US99/30452
7
<220>
<221> misc_feature
<222> (223)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (260)
<223> n equals a, t, g ox- c
<220>
<221> misc_feature
<222> (279)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (319)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (322)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (345y
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (360)
<223> n equals a, t, g or c
<400> 5
gtggcagagg acttcgagcc ggcgccaagc ngtgcgccag acgtggggcg cggagggtcg 60
cgtgcagggg gcgctggtgc gccgacgtgt tcttgctggg cgtgcccagg ggcgcaggct 120
cgggcggggc cgaacgaagt tggggggggc gcgcgnancc cactggacgc gccctgctgc 180
gggncgagag ccttgcgtgt gcgggcatcc tgctctgggc ctncgacgaa cacctttttt 240
aaccttaacg ctcaaggagn tccactttct agcctgggnc tcagactttc tgccccgacg 300
tgcgcttacg ttttttaang gncgacgcga tgtgttcgtg aacgngggaa atctcctggn 360
g ' 361
<210> 6
<211> 505
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
CA 02356548 2001-06-21
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8
<222>(10)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(21)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(34)
<223>n equals g o:r
a, t, c
<220>
<221>misc_feature
<222>(106)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(194)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(216)
<223>n equals g oz-
a, t, c
<220>
<221>misc_feature
<222>(231)
<223>n equals g oz-
a, t, c
<220>
<221>misc_feature
<222>(253)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(266)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(293)
<223>n equals g or
a, t, c
<220>,
<221>misc_feature
<222>(314)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(327)
<223>n equals g or
a, t, c
<220>
CA 02356548 2001-06-21
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9
<221> misc_feature
<222> (329)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (342)
<223> n equals a, t, g o~r c
<220>
<221> misc_feature
<222> (350)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (353)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (356)
<223> n equals a, t, g ar c
<220>
<221> misc_feature
<222> (365)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (380)
<223> n equals a, t, g o:r c
<220>
<221> misc_feature
<222> (382)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (384)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (386)
<223> n equals a, t, g on c
<220>
<221> misc_feature
<222> (393)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (396)
<223> n equals a, t, g ox- c
CA 02356548 2001-06-21
WO 00/39136 PCT/US99/30452
<220>
<221>misc_feature
<222>(398)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(402)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(406)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(408)
<223>n equals g o:r
a, t, c
<220>
<221>misc_feature
<222>(414)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(416)
<223>n .equals g oz-
a, t, c
<220>
<221>misc_feature
<222>(430)
<223>n equals g or
a, t, c
<220>
<221>misc_feature
<222>(434)
<223>n equals g ox'
a, t, c
<220>
<221>
misc_feature
<222>(436)
<223> g or
n c
equals
a,
t,
<220>
<221>
misc_feature
<222>(439)
<223> g or
p c
equals
a,
t,
<220>
<221>
misc_feature
<222>(446)
<223> g or
n c
equals
a,
t,
<220>
<221>
misc_feature
<222>(453)
<223> g or
n c
equals
a,
t,
CA 02356548 2001-06-21
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11
<220>
<221>misc_feature
<222>(457)
<223>n equals g or c
a, t,
<220>
<221>misc_feature
<222>(460)
<223>n equals g or c
a, t,
<220>
<221>misc_feature
<222>(475)
<223>n equals g or c
a, t,
<220>
<221>misc_feature
<222>(478)
<223>n equals g or c
a, t,
<220>
<221>misc_feature
<222>(478)
<223>n equals g or c
a, t,
<220>
<221>misc_feature
<222>(490)
<223>n equals g or c
a, t,
<220>
<221>misc_feature
<222>(493)
<223>n equals g o:r c
a, t,
<220>
<221>misc_feature
<222>(496)
<223>n equals g or c
a, t,
<400> 6
aattcggcan agtgacattt ngc:ttatgtg gcgnctactg cacgggacgc atgggccagc 60
actgtgcgca tccacagact gtc:gctgcag gccccttcca atgggnctcc tagctcccca 120
ctacagcccc aagctcctaa ctc:agaccca gaatggagcc ggtttcccag attattgccg 180
tgtatg~ggt tctnacctga tcaccaggtg cctgt:ntcca cagggtccca ngggatgggg 240
ggttaaggtt ggnttcctgg gcg~gtncacc ctgctggagc cagtttgaaa ccngtgtaat 300
ggtggaccct ttgnggcgag ccaaggntng ggtggtaggt gnaccatttn ttngtnccaa 360
gaggnnccca gggcaggggn nnnngnntgg gtnctncnta gnaggnanag gggnnntttt 420
tgggggtggn nggnnnggnt tagggntttt tannaanngn ttgggtttgg aaccnctngg 480
ttaagggggn ngnntnaaag gtttt
505
CA 02356548 2001-06-21
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<210> 7
<211> 216
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<222> (26)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (57)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (129)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (139)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (143)
<223> n equals a, t, g o:r c
<220>
<221> misc_feature
<222> (180)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (193)
<223> n equals a, t, g on c
<400> 7
ggcacagagg agatccactt tct.agnctgg gcctcagctt tctgcccgga cgtgcgnttc 60
gtttttaagg gcgacgcaga tgtgttcgta aacgtgggaa atctcctggg agttattggc 120
gccgcgggna cacgggttng tanctgcttg ctggtgaacg taattgtgca tgcgcggccn 180
4
atttgcacgg cgngctagct agttactaca tgtccg 216
<210> 8
<211> 291
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
CA 02356548 2001-06-21
WO 00/39136 PCTNS99/30452
13
<222> (102)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (110)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (131)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (167)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (171)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (223)
<223> n equals a, t, g o:r c
<220>
<221> misc_feature
<222> (238)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (245)
<223> n equals a, t, g or- c
<220>
<221> misc_feature
<222> (260)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (275)
<223> n equals a, t, g or c
<400>
gcgacggcgc acccggtggc cgcccggacc tgcttattgc tgtcaagtcg gtggcagagg 60
acttcgagcg gcgccaagcc gtgcgccaga cgtggggcgc gnaaggtccn tgcaaggggc 120
gctggtgccc ncatgttctt gctgggcgtt cccaagggcc aagctcnggc ngactcttcc 180
catcgacaaa tctttctggg cattttctca acgctgcggt cancccaacc tcacctgnct 240
tccgnacttt ggatccccan ct.caccgccc gattnaacac tccaacctgt t 291
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<210> 9
<211> 483
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<222> (37)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (79)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (374)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (386)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (423)
<223> n equals a, t, g or c
<400> 9
tgcgcttcac gcccgagcct caccctgcct tccgcanctt tggcatcccc cagccttcag 60
ccgcgccgca tttgagcanc ttcgac:ccct gcttttaccg tgagctggtt gtagtgcacg 120
ggctctcggc cgctgacatc tggcttatgt ggcgcctgct gcacgggccg catgggcaga 180
cctgtgcgca tccacagcct gtcgctgcag gccccttcca atgggactcc tagctcccca 240
ctacagcccc aagctcctaa ctcagaccca gaatggagcc ggtttcccag attattgccg 300
tgtatgtggt tcttccctga tc:accagggt gcctgtctcc acaggatccc agggggatgg 360
gggttaagct tggntcctgg gcggtncacc ctgctgggaa ccagttgaaa acccgtgtaa 420
atnggtgaac ccttttgaag gcgagccaag gttggggtgg tagatgaacc atttttttgt 480
tca c
483
<210> 10
<211> 431
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<222> (24)
CA 02356548 2001-06-21
WO 00/39136 PCTIUS99/30452
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (333)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (383)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (396)
<223> n equals a, t, g or c
<220>
<221> misc_feature
<222> (416)
<223> n equals a, t, g or c
<400> 10
gggcatcccc cagccttcag c:cgngccgca tttgagcacc ttcgacccct gcttttaccg 60
tgagctggtt gtagtgcacg ggctctcggc cgctgacatc tggcttatgt ggcgcctgct 120
gcacgggccg catgggcagc ctgtgcgcat ccacagcctg tcgctgcagg ccccttccaa 180
tgggactcct agctccccac tacagcccca agctcctaac tcagacccag aatggagccg 240
gtttcccaga ttattgccgt gtatgtgggt tcttccctga tcaccagggt gcctgtctc:c 300
acaggatccc aggggatggg ggt~taagctt ggntcctggg gggttccacc ctgctgggaa 360
accagtttga aaaccgtgta atnggtgacc cttttnaggc gagccaaggt tgggtngtag 420
atgaccattt t 431
<210> 11
<211> 30
<212> DNA
<213> Homo sapiens
<400> 11
caattggatc cgtggcagag gacttcgagc
<210> 12
<211> 30
<212> DNA
<213> Homo sapiens
<400> 12
gtacgcaagc ttggagtccc attggaaggg
<210> 13
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16
<211> 37
<212> DNA
<213> Homo Sapiens
<400> 13
cgcggatccg ccatcatggt ggcagaggac ttcgagc 37
<210> 14
<211> 30
<212> DNA
<213> Homo Sapiens
<400> 14
cacttaggta ccggagtccc ataggaaggg
<210> 15
<211> 37
<212> DNA
<213> Homo Sapiens
<400> 15
cgcggatccg ccatcatggt ggcagaggac ttcgagc 37
<210> 16
<211> 30
<212> DNA
<213> Homo Sapiens
<400> 16
cacttaggta ccggagtccc attggaaggg 30
