Note: Descriptions are shown in the official language in which they were submitted.
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NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
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GLIOMA-SPECIFIC ANTIBODIES AGAINST BEHAB/BREVICAN FOR
DIAGNOSTIC AND THERAPEUTIC APPLICATIONS
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR
DEVELOPMENT
[0001] This invention was supported in part by funds obtained from the
U.S. Government (National Institutes of Health Grant Numbers RO1
NS5228) and the U.S. Government may therefore have certain rights in the
invention.
BACKGROUND OF THE INVENTION
[0002] Gliomas, the most common primary tumors of the central nervous
system (CNS) are notoriously difficult to control due in large measures to
their highly invasive behavior. Invasion into the surrounding normal brain
tissue is essentially a unique property of primary glioma cells; it is a
feature
not seen even in highly malignant tumor cells that metastasize to the brain,
which grow as circumscribed masses despite their high invasiveness in
other tissues.
[0003] The most dangerous property of malignant gliomas is their highly
invasive phenotype, which makes these primary brain tumors difficult to
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control and impossible to completely remove by surgery, thus accounting
for the high lethality of gliomas (Pilkington, 1996, Braz J Med Biol Res, 29:
1159-72; Giese and Westphal, 1996, Neurosurgery 39: 235-252).
Glioblastomas, the most common and most aggressive class of gliomas,
result in patient's death typically within one year of diagnosis, due to the
inevitable recurrence even after extensive resection (Bernstein and
Woodard, 1995, Neurosurgery, 36: 124-132, 19955). Despite considerable
advances in the understanding of these tumors, the survival rates for
patients with gliomas have remained essentially unchanged for 25 years
(Berens and Giese, 1999, Neoplasia, 1:208-19).
[0004] The composition of the extracellular matrix (ECM) in the CNS and
cell surface adhesion molecules that interact with the matrix are critical
factors in determining the invasive potential of glioma cells. The invasive
behavior of glioma cells in the central nervous system (CNS) is quite
unusual, in that the adult CNS is highly restrictive to cell movement even
for non-glial tumors that metastasize to the brain (Pilkington, 1997,
Anticancer Res. 17: 4103-4105; Subramanian et al., 2002, Lancet Oncol. 3:
498-507). The unique ability of gliomas to infiltrate and invade the
surrounding normal neural tissue indicates that these cells are able to
overcome the normal barriers to cell movement in the CNS (Giese and
Westphal, 1996, Neurosurgery 39: 235-252). One explanation for the
unusual behavior of glioma cells is that specific and unique matrix elements
or matrix binding molecules mediate glioma cell invasion.
[0005] A role for ECM components in brain tumor invasion has been
suggested by several lines of research. The ECM of the brain is quite
unique, in that it lacks the typical fibrous proteins found in the ECM of
other
tissues (Sanes, J.R., 1989, Annual Review of Neuroscience, 12: 491-516).
In the CNS, the polysaccharide hyaluronan (HA) is the backbone of the
ECM (Bignami et al., 1993, Anat Embryol, (Berl) 188: 419-433. The high
charge density of HA creates hydrated spaces, which are permissive for
the structural reorganization of tissues and cell movement (Toole, B.P.,
2001, Semin Cell Dev Biol 12: 79-87). Both the synthesis and degradation
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of HA are upregulated in gliomas and have been implicated in cellular
proliferation, differentiation and migration, and may be key facilitators of
glioma cell motility and invasion (Delpech et al., 1993, European J. Cancer,
29A: 1012-1017; Heldin, P., 2003, Braz J Med Biol Res, 36: 967-973;
Toole, B. P., 2002, Glycobiology, 12: 37R-42R).
[0006] HA mediates cell functions through its interactions with HA-binding
proteins. Because HA is a ubiquitous constituent of the ECM, any cell- or
tissue-specific functions attributed to HA must be mediated by cell- or
tissue-specific HA-binding proteins. Several HA-binding proteins have
been implicated in glioma proliferation and motility, including CD44
(Bouterfa et al., 1997, Neuropathology & Applied Neurobiology, 23: 373-
379; Goldbrunner et al., 1999, Acta Neurochirurgica, 141: 295-305
[discussion 304-295]), RHAMM (Turley et al., 1994, GLIA, 12: 68-80)
(receptor for hyaluronan-mediated motility), and BEHAB/brevican.
[0007] BEHAB (Brain Enriched HyAluronan-Binding)/brevican has been
shown to be involved in glioma invasion. Unlike many other HA-binding
proteins, it is expressed exclusively in the central nervous system (Jaworski
at el., 1994, J. of Cell Biology 125: 495-509) and it is markedly upregulated
in human primary gliomas and in experimental models of glioma (Gary et
al., 1998, Current Opinion in Neurobiology 8: 576-581; Gary et al., 2000,
Gene, 256, 139-147; Goetz et al., 2003, J Neurooncol. 62: 321-328;
Jaworski et al., 1996, Cancer Research, 56: 2293-2298; Nutt et al., 2000,
The Neuroscientist; Zhang et al., 1998, J. of Neuroscience, 18: 2370-2376).
Previous work has provided evidence that regulation of BEHAB/brevican
expression and proteolytic processing play a role in glioma invasion
(Matthews et al., 2000, J. of Biological Chemistry 275: 22695-22703).
[0008] A recently identified isoform of BEHAB/brevican is expressed
specifically and exclusively on the cell surface of high-grade and malignant
low-grade gliomas (Viapiano et al., 2003, J. Biol Chem, 278: 33239-33247;
Viapiano et al., 2005, Cancer Research 65(15): 6726-6733). This isoform
(B/bo9) contains some N-linked sugars but lacks the 0-linked sugars
detected in other isoforms of BEHAB/brevican. Analyses of a large number
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of surgical samples from patients with high-grade gliomas (n=20) and age-
matched cortical controls (n=1 8) have shown that B/bog is not detectable in
the normal adult human brain but is highly expressed in every high-grade
glioma sample analyzed to date. Not only is B/bog the major upregulated
BEHAB/brevican isoform in glioma, but in the adult human brain, it has
been found exclusively in glioma. Importantly, B/bog has not been detected
in samples from other neuropathologies, such as non-glial intracranial
tumors (n=5) and Alzheimer's Disease (n=5). B/bog is expressed on the
extracellular surface of glioma cells.
[0009] Considerable research over the past decade has made great
progress in demonstrating the utility of antibody immunotherapy in the
treatment of many tumor types (see Carter, 2001, Nat. Rev Cancer, 1: 118-
129), including glioma (Kurpad et al., 1995, Glia 15: 244-256; Kuan et al.,
2001, Endocr. Relat Cancer, 8 : 83-96; Goetz et al., 2003, J. Neurooncol.
62: 321- 328). However, a hurdle in using this approach as a therapy for
glioma has been the lack of good cellular targets that are both restricted to
the tumor cells and available at the cell surface for targeting (Yang et al.,
2003, Cancer Control. 10: 138-147).
[0010] Given the high mortality associated with malignant gliomas and
the paucity of effective therapies, there exists a long felt need, for novel
diagnostics and therapeutics that may aid in the treatment of these
neoplasias.
SUMMARY OF THE INVENTION
[0011] The present invention provides novel antibodies that specifically
bind to glycosylation-variant BEHAB isoforms, particularly human BEHAB,
compositions and methods for producing such antibodies, methods and
compositions comprising said antibodies for detecting malignant glioma in a
mammal, for differentially diagnosing malignant glioma from benign glioma,
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for monitoring malignant glioma tumor progression or regression and for
treating malignant glioma; and kits for detecting, diagnosing, monitoring
and treating a glioma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] For the purpose of illustrating the invention, there are depicted in
the drawings certain embodiments of the invention. However, the invention
is not limited to the precise arrangements and instrumentalities of the
embodiments depicted in the drawings.
[0013] Figure 1 is the amino acid sequence of human BEHAB (SEQ ID
NO : 1).
[0014] Figure 2 is the amino acid sequence of the Bg1 peptide (SEQ ID
NO : 2).
[0015] Figure 3 is the amino acid sequence of the Bg2 peptide (SEQ ID
NO : 3).
[0016] Figure 4 illustrates that site-directed mutagenesis identifies amino
acids that are normally glycosylated in BEHAB/brevican. U87 cells were
transiently transfected with constructs encoding for normal
BEHAB/brevican (B/b) or a mutated form (B/bm). It was noted that B/b runs
at a lower apparent size than the normal protein. After deglycosylation with
sialidase and 0-glycanase (deglyc), protein from both constructs runs at an
identical size indicating that differences were due solely to glycosylation.
[0017] Figure 5 illustrates that Bgl and Bg2 antibodies specifically detect
B/bog. U87MG cells were transfected with either a BEHAB/brevican cDNA
(B) or a mutated cDNA sequence. Culture media (CM) and cell
membranes (mb) were harvested and processed by SDS-PAGE and
Western blotting. CM was additionally deglycosylated with sialidase and 0-
glycosydase (CM deglyc). The results show that, in comparison to a pan-
BEHAB/brevican antibody (pan-B/b), antisera Bgl and Bg2 specifically
detect B/bog in the membrane and only detect the secreted form after
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deglycosylation. In addition, these antisera do not detect the mutant
protein, suggesting that the detected epitope requires intact threonines,
which are the putative glycosylation sites. Bgl and Bg2 were tested as
non-purified 4- or 8-week-bleed antisera at dilutions of 1/600 and 1/300
respectively.
[0018] Figure 6 illustrates that AF-Bgl specifically detects B/bog in human
malignant gliomas. The antibody AF-Bgl was affinity-purified from rabbit
antiserum and compared with a pan-BEHAB/brevican antibody on
membrane-enriched samples from human malignant gliomas and age
matched controls. AF-Bg1 specifically detected B/bog by Western blotting,
and exclusively detected this form in gliomas. AF-Bgl did not cross-react
with other forms of BEHAB/brevican or other protein bands. Bgl
specifically detects B/bA, in human malignant gliomas.
[0019] Figure 7 illustrates that AF-Bgl distinguishes between malignant
and benign low-grade tumors. Total homogenates from a benign
epileptogenic grade II oligodendroglioma (1), a malignant grade 11
oligodendroglioma (2), and non-neural scar tissue from a brain tumor
sample (3) were processed for SDS-PAGE and probed with pan-
BEHAB/brevican (Pan-B/b) and AF-Bgl antibodies. AF-Bgl provides an
unequivocal diagnosis of the tissues (1) and (2) as benign and malignant
gliomas, respectively, while the combined use of pan-BEHAB/brevican
helps avoiding false negatives for AF-Bgl when comparing tissues (1) and
(3).
[0020] Figure 8 illustrates that AF-Bgl specifically detects glioma cells by
immunohistochemistry. A surgical sample of a GBM was fresh frozen,
cryostat sectioned and post-fixed briefly with 4% parafomaldehyde followed
by acetone. The section was incubated with AF=Bg1 (1:100) overnight and
further processed for DAB immunohistochemistry. AF-Bgl specifically
detected glioma cells within this tumor and showed no reactivity with
normal surrounding brain tissue. AF-Bgl represent a novel reagent which
specifically detects glioma cells by immunohistochemistry.
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DETAILED DESCRIPTION OF THE INVENTION
[0021] As used herein, each of the following terms has the meaning
associated with it in this section.
[0022] The articles "a" and "an" are used herein to refer to one or to more
than one (i.e. to at least one) of the grammatical object of the article. By
way of example, "an element" means one element or more than one
element.
[0023] The term "glycosylation-variant BEHAB antibody" refers to an
antibody that binds to a glycosylation-variant BEHAB isoform such as
under- or un-glycosylated BEHAB but does not bind to normal glycosylated
BEHAB, i.e., BEHAB expressed in normal adult brain cells.
[0024] The term "antibody," as used herein, refers to an immunoglobulin
molecule that is able to specifically bind to a specific epitope on an
antigen.
Antibodies can be intact immunoglobulins derived from natural sources or
from recombinant sources and can be immunoreactive portions of intact
immunoglobulins. Antibodies are typically tetramers of immunoglobulin
molecules. The antibodies in the present invention may exist in a variety of
forms including, for example, polyclonal antibodies, monoclonal antibodies,
Fv, Fab and F(ab)2, as well as single chain antibodies, chimeric antibodies,
humanized antibodies and human antibodies.
[0025] By the term "synthetic antibody" or "recombinant antibody" as
used herein, is meant an antibody that is generated using recombinant
DNA technology, such as, for example, an antibody expressed by a
bacteriophage as described herein. The term should also be construed to
mean an antibody that has been generated by the synthesis of a DNA
molecule encoding the antibody and which DNA molecule expresses an
antibody protein, or an amino acid sequence specifying the antibody,
wherein the DNA or amino acid sequence has been obtained using
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synthetic DNA or amino acid sequence technology which is available and
well known in the art.
[0026] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the population are
identical except for possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies are highly specific, being
directed against a single antigenic site. Furthermore, in contrast to
polyclonal antibody preparations which include different antibodies directed
against different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that they may
be synthesized uncontaminated by other antibodies. The modifier
"monoclonal" indicates the character of the antibody as being obtained
from a substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any particular
method. For example, the monoclonal antibodies to be used in accordance
with the present invention may be made by the hybridoma (murine or
human) method first described by Kohler et al., Nature, 256:495 (1975), or
may be made by recombinant DNA methods (see, e.g., U.S. Pat.
No.4,816,567). The "monoclonal antibodies" may also be isolated from
phage antibody libraries using the techniques described in Clackson et al.,
Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597
(1991), for example.
[0027] "Humanized" forms of non-human (e.g. murine) antibodies are
specific chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab)2 or other antigen-binding
subsequences of antibodies) that contain sequences derived from non-
human immunoglobulin and human immunoglobulin sequences. For the
most part, humanized antibodies are human immunoglobulins (recipient
antibody) in which residues from the complementarity determining regions
(CDRs) of the recipient antibody are replaced by residues from the CDRs
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of a non-human species (donor antibody) such as mouse, rat or rabbit
having the desired specificity, affinity and capacity. In some instances, Fv
framework region (FR) residues of the human immunoglobulin are replaced
by corresponding non-human FR residues. Furthermore, the humanized
antibody may comprise residues which are found neither in the recipient
antibody nor in the imported CDR or FR sequences. These modifications
are made to further refine and optimize antibody performance. In general,
the humanized antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all or
substantially all of the FR residues are those of a human immunoglobulin
consensus sequence. The humanized antibody optimally also will comprise
at least a portion of an immunoglobulin constant region (Fc), typically that
of a human immunoglobulin.
[0028] By the term "applicator" as the term is used herein, is meant any
device including, but not limited to, a hypodermic syringe, a pipette, and the
like, for administering the mutant BEHAB nucleic acid, protein, and/or
glycosylation-variant BEHAB antibodies and the antisense BEHAB nucleic
acid of the invention to a mammal.
[0029] "BEHAB," "full-length BEHAB," or "endogenous BEHAB" as the
terms are used synonymously herein, refers to the Brain-Enriched
Hyaluronan Binding molecule, otherwise known as brevican. Full-length
BEHAB has a molecular weight of greater than about 150 kDa in rats and
mice. Human BEHAB has a molecular weight greater than about 160 kDa,
but less than 163 kDa and is exemplified by the amino acid sequence set
forth in SEQ ID NO:1 and the nucleotide sequence set forth in SEQ ID
NO:4.
[0030] "Biological sample," as that term is used herein, means a sample
obtained from or in a mammal that can be used to assess the level of
glycosylation-variant BEHAB antibody binding. Such a sample includes,
but is not limited to, a central nervous system (CNS) sample such as a
neural tissue sample, a brain sample or a cerebrospinal fluid sample.
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[0031] By "complementary to a portion or all of the nucleic acid encoding
BEHAB" is meant a nucleic acid sequence which does not encode a
BEHAB protein. Rather, the sequence which is being expressed in the
cells is identical to the non-coding strand of the nucleic acid encoding a
BEHAB protein and thus, does not encode BEHAB protein.
[0032] A "fragment" of BEHAB may be at least 6 amino acids in length.
In some embodiments, the fragment of a BEHAB proteiri is at least 10
amino acids. In some embodiments, the fragment of a BEHAB protein is
about 15 amino acids. In some embodiments, the fragment of a BEHAB
protein is about 20 amino acids. In some embodiments, the fragment of a
BEHAB protein is about 40 amino acids. In some embodiments, the
fragment of a BEHAB protein is about 60 amino acids. In some
embodiments, the fragment of a BEHAB protein is about 80 amino acids.
In other embodiments, the fragment of a BEHAB protein is about 100
amino acids, even more preferably, at least about 200, yet more preferably,
at least about 300, even more preferably, at least about 400, yet more
preferably, at least about 500, even more preferably, about 600, and more
preferably, even more preferably, at least about 700, yet more preferably,
at least about 800, even more preferably, about 850, and more preferably,
at least about 884 amino acids in length.
[0033] Percent sequence identity for polypeptides is typically measured
using sequence analysis software. Protein analysis software matches
sequences using measuresof similarity assigned to various substitutions,
deletions and other modifications, including conservative amino acid
substitutions. For instance, GCG contains programs such as "Gap" and
"Bestfit" which can be used with default parameters, as specified with the
programs, to determine sequence homology or sequence identity between
closely related polypeptides, such as homologous polypeptides from
different species of organisms or between a wild type protein and a mutein
thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be
compared using FASTA using default or recommended parameters, see
GCG Version 6.1. (University of Wisconsin WI) FASTA (e.g., FASTA2 and
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FASTA3) provides alignments and percent sequence identity of the regions
of the best overlap between the query and search sequences (Pearson,
Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.
132:185-219 (2000)). Another preferred algorithm when comparing a
sequence of the invention to a database containing a large number of
sequences from different organisms is the computer program BLAST,
especially blastp or tblastn, using default parameters, as supplied with the
programs. See, e.g., Altschul et al., J. Mol. Biol. 215:403-410 (1990);
Altschul et al., Nucleic Acids Res. 25:3389-402 (1997).
[0034] The term "immunoconjugate" refers to the operative association of
the antibody with another effective agent and is not intended to refer solely
to any type of operative association, and is particularly not limited to
chemical "conjugation".
[0035] As used herein, a "glycosylation-variant BEHAB isoform" and
"glycosylation-variant BEHAB" means a BEHAB protein having fewer 0-
linked sugars compared to normal glycosylated BEHAB as expressed in
normal adult brain tissue, or a portion thereof. Such a protein may be
underglycosylated or unglycosylated as compared to the fully glycosylated
protein.
[0036] "Underglycosylated BEHAB isoform" and "underglycosylated
BEHAB" are used herein to refer to a BEHAB protein having the primary
amino acid sequence of a full-length BEHAB protein, or a fragment thereof,
but having less 0-glycosylation content than full-length BEHAB protein, but
still having at least one 0-linked sugar or carbohydrate associated with the
protein.
[0037] "Unglycosylated BEHAB isoform" and "unglycosylated BEHAB"
are used herein to refer to a BEHAB protein having the primary amino acid
sequence of a full-length BEHAB protein, or fragment thereof, but having
no 0-linked sugars or carbohydrates associated with the protein.
Unglycosylated BEHAB is used interchangeably with B/bQg to refer to the
isoform of BEHAB exclusively expressed by malignant glioma cells.
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[0038] As used herein, an "instructional material" includes a publication,
a recording, a diagram, or any other medium of expression which can be
used to communicate the usefulness of the composition of the invention for
its designated use. The instructional material of the kit of the invention
may, for example, be affixed to a container which contains the composition
or be shipped together with a container which contains the composition.
Alternatively, the instructional material may be shipped separately from the
container with the intention that the instructional material and the
composition be used cooperatively by the recipient.
[0039] A "malignant high grade glioma" is used herein to refer to a grade
III or grade IV glioma using the histologic grading system for grading
gliomas and the level of differentiation in glioma cells and tissues.
According to the same grading system, a "benign low grade glioma" is used
herein to refer to a grade I or grade II glioma. According to the grading
system, Grade I gliomas are well-differentiated (low grade), Grade II
gliomas are moderately differentiated (intermediate grade), Grade III
gliomas are poorly differentiated (high grade) and Grade IV gliomas are
undifferentiated (high grade). The criteria for grading gliomas are those
according to the American Joint Committee on Cancer. AJCC Cancer
Staging Manual. 6th ed. New York, NY: Springer, 2002.
[0040] "Naturally-occurring" or "normal" as applied to an object refers to
the fact that the object can be found in nature. For example, a polypeptide
or polynucleotide sequence that is present in an organism (including
viruses) that can be isolated from a source in nature and which has not
been intentionally modified by man is naturally-occurring.
[0041] In the context of the present invention, the following abbreviations
for the commonly occurring nucleic acid bases are used. "A" refers to
adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to
thymidine, and "U" refers to uridine.
[0042] Unless otherwise specified, a "nucleotide sequence encoding an
amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino acid
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sequence. Nucleotide sequences that encode proteins and RNA may
include introns.
[0043] For purposes herein, "stringent conditions" are defined for solution
phase hybridization as aqueous hybridization (i.e., free of formamide) in 6X
SSC (where 20X SSC contains 3.0 M NaCI and 0.3 M sodium citrate), 1%
SDS at 65oC for at least 8 hours, followed by one or more washes in 0.2X
SSC, 0.1 % SDS at 65 C.
[0044] By describing two polynucleotides as "operably linked" is meant
that a single-stranded or double-stranded nucleic acid moiety comprises
the two polynucleotides arranged within the nucleic acid moiety in such a
manner that at least one of the two polynucleotides is able to exert a
physiological effect by which it is characterized upon the other. By way of
example, a promoter operably linked to the coding region of a gene is able
to promote transcription of the coding region.
[0045] The direction of 5' to 3' addition of nucleotides to nascent RNA
transcripts is referred to as the transcription direction. The DNA strand
having the same sequence as an mRNA is referred to as the "coding
strand"; sequences on the DNA strand which are located 5' to a reference
point on the DNA are referred to as "upstream sequences"; sequences on
the DNA strand which are 3' to a reference point on the DNA are referred to
as "downstream sequences."
[0046] "Treating" a primary malignant glioma is used herein to refer to a
situation where the severity of a symptom of a primary glioma, including the
volume of the tumor or the frequency with which any symptom or sign of
the tumor is experienced by a patient, or both, is reduced for at least some
period of time. The term also is used herein to refer to a situation where
the rate of tumor progression is reduced or survival 1ime is increased.
[0047] "Primer" refers to a polynucleotide that is capable of specifically
hybridizing to a designated polynucleotide template and providing a point of
initiation for synthesis of a complementary polynucleotide. Such synthesis
occurs when the polynucleotide primer is placed under conditions in which
synthesis is induced, i.e., in the presence of nucleotides, a complementary
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polynucleotide template, and an agent for polymerization such as DNA
polymerase. A primer is typically single-stranded, but may be double-
stranded. Primers are typically deoxyribonucleic acids, but a wide variety
of synthetic and naturally occurring primers are useful for many
applications. A primer is complementary to the template to which it is
designed to hybridize to serve as a site for the initiation of synthesis, but
need not reflect the exact sequence of the template. In such a case,
specific hybridization of the primer to the template depends on the
stringency of the hybridization conditions. Primers can be labeled with, e.g.,
chromogenic, radioactive, or fluorescent moieties and used as detectable
moieties.
[0048] A host cell that comprises a recombinant polynucleotide is
referred to as a "recombinant host cell." A gene which is expressed in a
recombinant host cell wherein the gene comprises a recombinant
polynucleotide, produces a "recombinant polypeptide."
[0049] A "recombinant polypeptide" is one which is produced upon
expression of a recombinant polynucleotide.
[0050] "Polypeptide" refers to a polymer composed of amino acid
residues, related naturally occurring structural variants and synthetic non-
naturally occurring analogs thereof linked via peptide bonds. Synthetic
polypeptides can be synthesized, for example, using an automated
polypeptide synthesizer.
[0051] Conventional notation is used herein to portray polypeptide
sequences: the left-hand end of a polypeptide sequence is the amino-
terminus; the right-hand end of a polypeptide sequence is the carboxyl-
terminus.
[0052] An antibody that "specifically binds" glycosylation-variant BEHAB
refers to an antibody that binds an epitope of a glycosylation-variant
BEHAB, such as under- or un-glycosylated BEHAB, but that does not
substantially bind unrelated proteins in a sample, and does not bind
glycosylated BEHAB.
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[0053] A"therapeutic treatment is a treatment administered to a subject
who exhibits signs of pathology for the purpose of diminishing or
eliminating those signs.
[0054] A therapeutically effective amount" of a compound is that amount
of compound which is sufficient to provide a beneficial effect to the subject
to which the compound is administered.
[0055] A"vector" is a composition of matter which comprises an isolated
nucleic acid and which can be used to deliver the isolated nucleic acid to
the interior of a cell. Numerous vectors are known in the art including, but
not limited to, linear polynucleotides, polynucleotides associated with ionic
or amphiphilic compounds, plasmids, and viruses. Thus, the term vector"
includes an autonomously replicating plasmid or a virus. The term should
also be construed to include non-plasmid and non-viral compounds which
facilitate transfer of nucleic acid into cells, such as, for example,
polylysin
compounds, liposomes, and the like. Examples of viral vectors include, but
are not limited to, adenoviral vectors, adeno-associated virus vectors,
retroviral vectors, and the like.
[0056] "Expression vector" refers to a vector comprising a recombinant
polynucleotide comprising expression control sequences operatively linked
to a nucleotide sequence to be expressed. An expression vector
comprises sufficient cis-acting elements for expression; other elements for
expression can be supplied by the host cell or in an in vitro expression
system. Expression vectors include all those known in the art, such as
cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that
incorporate the recombinant polynucleotide.
[0057] The current invention pertains to antibodies capable of specifically
binding glycosylation-variant BEHAB proteins, such as under- or
unglycosylated BEHAB, while not binding glycosylated BEHAB. Preferably,
the antibody specifically binds unglycosylated BEHAB also referred to
herein as B/bog. Unglycosylated BEHAB is found only on the cell surface
of malignant gliomas. Thus, the antibodies of the current invention are
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capable of distinguishing between normal and malignant glioma brain
tissue.
10058] The antibodies of the present invention specifically bind
mammalian glycosylation-variant BEHAB. In various embodiments, the
antibodies specifically bind human glycosylation-variant BEHAB. In some
embodiments, the antibodies bind glycosylation-variant BEHAB from more
than one mammalian species.
[0059] The present invention is not limited to the antibodies enumerated
herein, but rather also includes glycosylation-variant BEHAB antibodies
identified and generated in the future. Additional antibodies specific for
glycosylation-variant BEHAB, including underglycosylated and
unglycosylated BEHAB, can be generated using the methods described
and exemplified herein.
(0060] In some embodiments, the glycosylation-variant BEHAB lacks
glycosylation at one or more 0-linked glycosylation sites. In some
embodiments, the glycosylation-variant BEHAB is substantially lacking 0-
linked glycosylation. In other embodiments, the glycosylation-variant
BEHAB is completely lacking in 0-linked glycosylation.
[0061] In some embodiments, antibodies are raised against a BEHAB
polypeptide that lacks 0-linked glycosylation at one or more sites that are
glycosylated when the polypeptide is expressed in a normal
(untransformed) adult brain cell. In some embodiments, the BEHAB
immunogen contains no O-linked glycosylation. The immunogen can be a
full length BEHAB polypeptide or an immunogenic portion as long as the
portion contains at least one unglycosylated 0-linked glycosylation site that
is glycosylated in BEHAB expressed in a normal adult brain cell.
[0062] In some embodiments, a BEHAB polypeptide containing 0-linked
glycosylation sites not glycosylated in the immunogen can be produced by
expressing a nucleic acid encoding said BEHAB polypeptide in an
organism that does not glycosylate the proteins it produces, such as E. coli.
The polypeptide isolated, from a non-glycosylating prokaryotic species can
then be used to generate antibodies, as is described herein.
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[0063] In preferred embodiments, BEHAB polypeptide immunogens
comp(se one or more clusters of 0-linked glycosylation sites. It is well-
known how to identify potential 0-linked glycosylation sites. 0-linked
saccharides usually are attached via a glycosidic bond on a threonine or
serine residue, and in some cases, on hydroxylysine or hydroxyproline. In
preferred embodiments, an 0-linked glycosylation site is located at the N or
C terminal of the immunogen thereby increasing exposure of the residue to
the antibody-producing cells. The use of such immunogens allows for the
generation of glycosylation-variant BEHAB antibodies specific for
unglycosylated 0-linked glycosylation sites on glycosylation-variant BEHAB
and portions thereof.
[0064] In some embodiments said BEHAB immunogen is a BEHAB
peptide of any length that comprises the sequence from the threonine at
position 540 to the threonine at position 545 of SEQ ID NO: 1 wherein one
or more of the threonines are unglycosylated. In other embodiments, said
BEHAB immunogen is a BEHAB peptide of at least 6 amino acids in length.
In preferred embodiments, said BEHAB immunogen is a BEHAB peptide of
at least 10 amino acids in length. In other embodiments, said BEHAB
immunogen is a BEHAB peptide of at least 15, 20, 25 or 30 amino acids in
length. In some embodiments, two of the threonines are unglycosylated.
In a preferred embodiment, all three of the threonines (at positions 540,
542 and 545) are unglycosylated. As demonstrated herein, the BEHAB
polypeptide immunogen can be Bgl peptide (SEQ ID NO: 2) or Bg2
peptide (SEQ ID NO: 3) lacking glycosylation on one or more of the
threonine residues. Thus, the skilled artisan, when armed with the present
disclosure and the methods disclosed herein, would readily be able to
identify other potential glycosylation sites in a BEHAB primary amino acid
sequence, generate peptides for immunizing an animal comprising these
potential glycosylation sites, and generate antibodies that specifically bind
glycosylation-variant BEHAB. Such antibodies are useful in therapeutic
treatments, including, but not limited to immunizing a mammal against the
formation of gliomas, treating gliomas, detecting a glioma in a mammal
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either in vivo or in vitro, and other methods and uses disclosed elsewhere
herein.
[0065] The invention should not be construed as being limited solely to
the antibodies disclosed herein or to any particular immunogenic portion of
the proteins of the invention. According to the invention, the BEHAB
polypeptide can be from any mammal. Where the BEHAB polypeptide is
human BEHAB, the amino acid sequence can be any BEHAB amino acid
sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence
in SEQ ID NO:1 containing one or more 0-linked glycosylation sites.
[0066] The current invention should also be construed to include anti-
glycosylation variant BEHAB antibodies produced against a BEHAB
polypeptide encoded by a nucleic acid that hybridizes, under stringent
conditions, to the nucleic acid sequence of SEQ ID N0:4.
[0067] One skilled in the art would also appreciate, based upon the
disclosure provided herein, that the antibodies can be used to
immunoprecipitate and/or immune-affinity purify their cognate antigen using
methods well-known in the art.
[0068] The invention encompasses polyclonal, monoclonal, synthetic
antibodies, and the like. The antibodies can be of any isotype, i.e., IgM,
IgG, IgE, IgA or IgD or any sub-isotype thereof.One skilled in the art would
understand, based upon the disclosure provided herein, that the crucial
feature of the antibody of the invention is that the antibody binds
specifically with glycosylation-variant BEHAB, including under- and un-
glycosylated BEHAB, and does not bind to fully glycosylated BEHAB. That
is, the antibody of the invention recognizes glycosylation-variant BEHAB,
including under- and un-glycosylated BEHAB, or a fragment thereof (e.g.,
an immunogenic portion, glycosylation-variant or antigenic determinant
thereof), but does not recognize the corresponding glycosylated
polypeptide on Western blots, in immunostaining of cells, and
immunoprecipitates BEHAB, including glycosylation-variant BEHAB, using
standard methods well-known in the art.
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[0069] In some embodiments, the antibodies of the present invention are
glycosylation-variant BEHAB antagonists. Such antagonists or neutralizing
antibodies, reduce one or more biological activity of glycosylation-variant
BEHAB expressed exclusively by malignant glioma cells. In one
embodiment, the biological activity is associated with glioma invasion or
spreading. Such antagonist antibodies may reduce the ability of
glycosylation-variant BEHAB to promote glioma invasion. In some
embodiments, said antagonist antibodies slow the rate of glioma tumor
progression and/or growth. In some embodiments, said antagonist
antibodies stop glioma tumor progression and/or growth. In other
embodiments, said antagonist antibodies lead to glioma regression and/or
size reduction. In a highly preferred embodiment, the antagonist antibodies
cause the glioma tumor not to increase in weight or volume or to decrease
in weight or volume.
[0070] The present invention also provides an anti-glycosylation variant
BEHAB antibody that is modified or derivatized to improve one or more of
its properties.
[0071] In some embodiments, a glycosylation-variant BEHAB antibody
can be derivatized with a chemical group, such as polyethylene glycol
(PEG), in order to facilitate conjugation to additional moieties. Other
linking
groups, such as peptide spacers, can be enzymatically cleaved after
antibody delivery, and can be utilized to temporarily attach a moiety such
as an immunotoxin. Additional linkers include acid sensitive spacers and
peptide linkers that include a cleavage site for peptidases and/or
proteinases which may be present at a disease site (for e.g., a tumors).
Peptide linkers that include a cleavage site for urokinase, pro-urokinase,
plasmin, plasminogen, TGFR, staphylokinase, Thrombin, Factor IXa, Factor
Xa or a metalloproteinase (MMP), such as an interstitial collagenase, a
gelatinase or a stromelysin, are described by U.S. Pat. No. 5,877,289,
incorporated herein by reference.
[0072] In another embodiments, glycosylation-variant BEHAB antibodies
may also be derivatized to introduce functional groups permitting the
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attachment of the therapeutic agent(s) through an, optionally, biologically
releasable bond. Antibodies may be derivatized to introduce hydrazide,
hydrazine, primary amine or secondary amine side chains as a means of
facilitating the conjugation of therapeutic agents through a Schiffs base
linkage, a hydrazone or acyl hydrazone bond or a hydrazide linker (see
U.S. Pat. Nos. 5,474,765 and 5,762,918, each specifically incorporated
herein by reference).
[0073] In various embodiments, the antibody or an antigen-binding
portion of the antibody is linked to an imaging agent or detectable label
known in the art useful for in vitro or in vivo imaging. Useful detection
agents with which an antibody or antigen-binding portion of the invention
may be derivatized include fluorescent compounds, including fiuorescein,
fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-
naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the
like. An antibody can also be labeled with enzymes that are useful for
detection, such as horseradish peroxidase, (3-galactosidase, luciferase,
alkaline phosphatase, glucose oxidase, and the like. When an antibody is
labeled with a detectable enzyme, it is detected by adding additional
reagents that the enzyme uses to produce a reaction product that can be
discerned. For example, when the agent horseradish peroxidase is
present, the addition of hydrogen peroxide and diaminobenzidine leads to a
detectable, colored reaction product. An antibody can also be labeled with
biotin, and detected through indirect measurement of avidin or streptavidin
binding. An antibody can also be labeled with a predetermined polypeptide
epitope recognized by a secondary reporter (e.g., leucine zipper pair
sequences, binding sites for secondary antibodies, metal binding domains,
epitope tags). In some embodiments, labels are attached by spacer arms
of various lengths to reduce potential steric hindrance.
[00741 In other embodiments, the invention provides immunoconjugates
comprising an anti-glycosylation-variant BEHAB antibody of the invention.
In various embodiments, an antibody of the invention can be joined to
another antibody (e.g., a bispecific antibody or a diabody), a detection
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agent, a label, a cytotoxic agent, a pharmaceutical agent, and/or a protein
or peptide that can mediate association of the antibody or antibody portion
with another molecule (such as a streptavidin core region or a polyhistidine
tag).
[0075] The preparation of immunoconjugates and immunotoxins is
generally well known in the art (see, e.g., U.S. Pat. No. 4,340,535,
incorporated herein by reference). Each of the following patents and patent
applications relating to immunotoxin generation, purification and use are
incorporated herein by reference: U.S. application Ser. No. 07/846,349;
08/295,868 (U.S. Pat. No. 6,004,554); 08/205,330 (U.S. Pat. No.
5,855,866); 08/350,212 (U.S. Pat. No. 5,965,132); 08/456,495 (U.S. Pat.
No. 5,776,427); 08/457,487 (U.S. Pat. No. 5,863,538); 08/457,229 and
08/457,031 (U.S. Pat. No. 5,660,827) and 08/457,869 (U.S. Pat. No.
6,051,230).
[0076] In various embodiments, the antibody or an antigen-binding
portion of the antibody is linked to a therapeutic agent. In some
embodiments, the anti-glycosylation-variant BEHAB antibody, either in
conjugated or unconjugated (naked) form is administered with one or more
additional therapeutic agent. Such additional therapeutic agents can be co-
formulated or co-administered with or administered separately from the
anti-glycosylation variant BEHAB antibody of the invention. The
therapeutic agent can be any agent suitable for treating malignant glioma.
[0077] In embodiments where agents are used in combination with an
anti-glycosylation-variant BEHAB antibody in a non-targeted form, the
agent, particularly therapeutic agents, will generally be used according to
their standard use in the art. In other embodiments, glycosylation-variant
BEHAB antibodies may be used in combined compositions in which the
therapeutic agent is in the form of a prodrug. In such embodiments, the
activating component that is capable of converting the prodrug to the
functional form of the drug may be operatively associated with the
glycosylation-variant BEHAB antibodies of the present invention.
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[0078] In some embodiments the glycosylation-variant BEHAB antibody
therapeutic agent is operatively attached to an anti-angiogenic agent.
Exemplary anti-angiogenic agents include, but are not limited to,
angiostatin, endostatin, any one of the angiopoietins, vasculostatin,
canstatin and maspin.
[0079] In some embodiments the glycosylation-variant BEHAB antibody
therapeutic agent of the present invention may be linked to an anti-tubulin
agent. Such anti-tubulin agents refer to any agent, drug, prodrug or
combination thereof that inhibits cell mitosis by directly or indirectly
inhibiting tubulin activities, such as tubulin polymerization or
depolymerization, necessary for cell mitosis. Exemplary anti-tubulin drugs
include, but are not limited to, colchicine, taxanes (such as taxol), vinca
alkaloids (such as vinbiastine, vincristine and vindescine) and
combretastatins. Exemplary combretastatins are combretastatin A, B
and/or D, including A-1, A-2, A-3, A-4, A-S, A-6, B-1, B-2, B-3, B-4, D-1 and
D-2 and prodrug forms thereof.
[0080] In some embodiments the glycosylation-variant BEHAB antibody
therapeutic agent is operatively attached to cytotoxic, cytostatic or other
anti-cellular proliferation 'agents which have the ability to kill or suppress
the
growth or cell division of endothelial cells. Exemplary chemotherapeutic
agents include, but are not limited to, steroids, cytokines, anti-metabolites
(such as cytosine arabinoside, fluorouracil, methotrexate or aminopterin),
anthracyclines, mitomycin C, vinca alkaloids, antibiotics, demecolcine,
etoposide, mithramycin and anti-tumor alkylating agents (such as
chlorambucil or melphalan). Examples of anti-cellular agents include, but
are not limited to, DNA synthesis inhibitors such as daunorubicin,
doxorubicin, and adriamycin. In addition, the use of anti-cellular and
cytotoxic agents may be used to generate glycosylation-variant BEHAB
antibody immunotoxins while the use of coagulation factors may be used to
generate glycosylation-variant BEHAB antibody coaguligands. In some
embodiments, the use of two or more therapeutic agents may be
contemplated. Examples of such combinations include, but are not limited
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to, radiotherapeutic agents, anti-angiogenic agents, apoptosis-inducing
agents, anti-tubulin drugs, anti-cellular and cytotoxic agents and
coagulation factors. The attachment of other agents such as
neocarzinostatin, macromycin, trenimon and a-amanitin has been
described (see U.S. Pat. Nos. 5,660,827; 5,855,866; and 5,965,132; each
incorporated herein.)
[0081] In embodiments wherein a therapeutic composition is intended to
have a toxic effect on targeted cells, preferred agents for conjugation to
glycosylation-variant BEHAB antibodies include, but are not limited to,
epipodophyllotoxins, bacterial endotoxin, ribosome inactivating proteins
(such as saporin or gelonin; a-sarcin), aspergillin, restrictocin,
ribonucleases (such as placental ribonuclease), diphtheria toxin,
pseudomonas exotoxin, and daunomycin.
[0082] The following patents and patent applications are specifically
incorporated herein by reference for the purposes of further supplementing
the present teachings regarding tumor targeting and treatment with
immunotoxins anti-cellular and cytotoxic agents: U.S. application Ser. No.
07/846,349; 08/295,868 (U.S. Pat. No. 6,004,554); 08/205,330 (U.S. Pat.
No. 5,855,866); 08/350,212 (U.S. Pat. No. 5,965,132); 08/456,495 (U.S.
Pat. No. 5,776,427); 08/457,487 (U.S. Pat. No. 5,863,538); 08/457,229 and
08/457,031 (U.S. Pat. No. 5,660,827) and 08/457,869 (U.S. Pat. No.
6,051,230).
[0083] According to the invention, anti-convulsive agents, anti-
inflammatory agents or other agents that prevent or reduce swelling in the
CNS tissue, including the brain and spinal chord, diuretics, antibiotics or
other anti-infective agents may be administered in conjunction with an anti-
glycosylation variant antibody of the invention.
[0084] In other embodiments, an antibody of the invention is crosslinked
to one or more antibodies (of the same specificity or of different
specificity,
e.g., to create bispecific antibodies). Suitable crosslinkers include those
that are heterobifunctional, having two distinctly reactive groups separated
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by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide
ester) or homobifunctional (e.g., disuccinimidyl suberate).
[0085] Agents may be conjugated to antibodies of the current invention
via 0-linked and N-linked carbohydrate moieties. Accordingly, antibodies
may be modified to recreate or create additional glycosylation sites for
conjugate addition. The addition of such carbohydrate attachment point
may be achieved by by engineering appropriate amino acid sequences (i.e.
Asn-X-Ser, Asn-X-Thr, Ser or Thr) into the primary sequence amino acid
sequence without disrupting antibody activity.
[0086] The current invention also provides compositions, pharmaceutical
compositions, therapeutic kits and medicinal cocktails comprising a
biologically effective amount of at least one glycosylation-variant BEHAB
antibody, or an antigen-binding fragment or immunoconjugate of such a
glycosylation-variant BEHAB antibody, and a biologically effective amount
of at least a second biological agent, component or system. Such a
second biological agent, component or system may comprise components
for modification of the antibody and/or for attaching other agents to the
antibody. Certain preferred second biological agents, components or
systems include prodrugs or components for making and using prodrugs,
including components for making the prodrug itself and components for
adapting the antibodies of the inverition to function in such prodrug
embodiments.
[0087] The present invention encompasses various kits which comprise
an antibody that specifically binds glycosylation-variant BEHAB, i.e., an
antibody of the invention. In some embodiments, such kits may include
one or more of: an applicator, and instructional materials that describe use
of the antibody to perform the methods of the invention. Although model
kits are described below, the contents of other useful kits will be apparent
to the skilled artisan in light of the present disclosure. Each of these kits
is
contemplated within the present invention.
[0088] The present invention comprises a kit for detecting a
glycosylation-variant BEHAB isoform. The kit comprises an antibody to a
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glycosylation-variant BEHAB isoform. Such antibodies are disclosed are
set forth elsewhere herein. The kit further comprises an instructional
material comprising information on how to use the antibody for the
detection of a glycosylation-variant BEHAB isoform, including instructions
to accomplish the methods set forth elsewhere herein.
[0089] The present invention further comprises a kit for diagnosing a
malignant glioma in a mammal. The kit comprises an antibody that
specifically binds a glycosylation-variant BEHAB isoform, an applicator and
a instructional method for the use of the kit. Uses of an applicator and
methods for the diagnosis of a malignant glioma are disclosed elsewhere
herein.
[0090] The present invention further comprises a kit for diagnosing a
malignant glioma in a mammal. The kit comprises an antibody that
specifically binds a glycosylation-variant BEHAB isoform, an applicator and
a instructional method for the use of the kit. Uses of an applicator and
methods for the diagnosis of a malignant glioma are disclosed elsewhere
herein.
[0091] The invention also includes a kit for treating a malignant glioma.
The kit includes a composition comprising an antibody that specifically
binds a glycosylation-variant BEHAB isoform, or a fragment thereof, a
pharmaceutically acceptable carrier, and an applicator. Methods for using
an antibody and applicator are set forth elsewhere
herein. The instructional material comprises the methods disclosed herein
for the treatment of a malignant glioma.
[0092] The present invention encompasses-glycosylation-variant BEHAB
antibodies produced according to the methods taught herein other than
those exemplified herein. Such antibodies also are useful as, among other
things, therapeutics, diagnostic tools for primary malignant gliomas,
research tools for elucidating the interaction of the neural extracellular
matrix with cancer causing mutations, dysfunctions, and the like.
[0093] The invention encompasses antibodies that bind to about,
substantially, essentially or at the same epitope as a glycosylation-variant
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BEHAB antibody raised against proteins or peptides comprising any one of
the amino acid sequences, or portions thereof, in SEQ ID NOs:1-3. Such
antibodies may be identified by comparison to a reference antibody. The
identification of competing and cross-competing antibodies can be readily
accomplished using any one of variety of immunological screening assays
in which antibody competition can be assessed. All such assays are
routine in the art and are further described herein in detail. U.S. Pat. No.
5,660,827, issued Aug. 26, 1997, is specifically incorporated herein by
reference for purposes including even further supplementing the present
teaching concerning how to make antibodies that bind to the same or
substantially the same epitope as a given antibody.
[0094] For example, where the test antibodies to be examined are
obtained from different source animals, or are even of a different isotype, a
simple competition assay may be employed in which the control and test
antibodies are admixed (or pre-adsorbed) and applied to a glycosylation-
variant BEHAB antigen composition. By glycosylation-variant BEHAB
antigen composition is meant any composition that contains a BEHAB-
binding antigen unique to glycosylation-variant BEHAB as described
herein. Thus, protocols based upon ELISAs and Western blotting are
suitable for use in such competition studies.
[0095] In certain embodiments, one would or pre-mix the reference or
control antibodies with varying amounts of the test antibodies (e.g., 1:10 or
1:100) for a period of time prior to applying to an antigen composition. In
other embodiments, the control and varying amounts of test antibodies can
simply be admixed during exposure to the antigen composition. By using
species or isotype secondary antibodies one will be able to detect only the
bound control antibodies, the binding of which will be reduced by the
presence of a test antibody that recognizes substantially the same epitope.
Antibodies that cross-compete with a reference antibody can be identified
by conducting the competition experiment in two directions, i.e.,
determining if the test antibody competes for binding with the reference
antibody and vice versa.
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[0096] In conducting an antibody competition study between a control
antibody and any test antibody (irrespective of species or isotype), one may
first label the control with a detectable label, such as, e.g., biotin or an
enzymatic (or even radioactive) label to enable subsequent identification. In
these cases, one would pre-mix or incubate the labeled control antibodies
with the test antibodies to be examined at various ratios (e.g., 1:10 or
1:100) and (optionally after a suitable period of time) then assay the
reactivity of the labeled control antibodies and compare this with a control
value in which no potentially competing test antibody was included in the
incubation.
[0097] The assay may again be any one of a range of immunological
assays based upon antibody hybridization, wherein control antibodies
would be detected by means of detecting their label, e.g., using streptavidin
in the case of biotinylated antibodies or a chromogenic substrate in
connection with an enzymatic label (such as 3,3'5,5'-tetramethylbenzid-ine
(TMB) substrate with peroxidase enzyme) or by detecting of a radioactive
label. An antibody which binds to the same epitope as the control
antibodies will be able to effectively compete for binding and thus will
significantly reduce control antibody binding, as evidenced by a reduction in
bound label.
[0098] The reactivity of the (labeled) control antibodies in the absence of
a completely irrelevant antibody would be the control high value. The
control low value would be obtained by incubating a mixture of labeled and
unlabelled antibodies wherein direct competition would reduce the binding
of the labeled antibodies. For example, a significant reduction in labeled
antibody reactivity in the presence of a test antibody is indicative of a test
antibody which recognizes the same epitope (i.e., cross-reacts with the
labeled antibody).
[0099] The antibodies of the present invention are designed to recognize
stretches of amino acids in BEHAB/brevican that are normally modified by
0-linked sugars but not in glycosylation-variant BEHAB, for example, as
expressed by high grade malignant gliomas. Antibodies generated to
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recognize such epitopes are able to detect only glycosylation-variant
BEHAB and are unable to detect other isoforms of the protein - possibly
due to negative steric interactions between the antibody and the
carbohydrates present on the immunogen. In some embodiments,
glycosylation-variant BEHAB antibodies are generated against
"glycosylation hotspots," i.e., 10-15 amino acid long stretches comprising 3
or more putative glycosylation sites. According to the methods of the
invention, glycosylation sites are predicted from the primary amino acid
structure of BEHAB/brevican, for example, using 0-glycosylation prediction
software (e.g. Net-O-Glyc). Predicted 0-glycosylation sites are then
independently confirmed. For example, in the present invention, the region
from amino acid 537 to 550 of SEQ ID NO:1 (N-GPPTETLPTPRERN-C),
was identified as containing three putative 0-glycosylation sites (threonines
noted in bold). Threonine mutagenesis and BEHAB deglycosylation
experiments (described elsewhere herein) confired that the threonine
residues are glycosylated in BEHAB. Immunogenic peptides comprising N-
GPPTETLPTPRE-C (AA 537-548) and N-TETLPTPRERN-C (AA 540-550),
named Bgl (SEQ ID NO:2) and Bg2 (SEQ ID NO:3), respectively can be
used as immunogens to generate the antibodies of the present invention
according to the techniques describe herein.
[0100] The antibodies or antigen-binding portions of the present invention
can be prepared according to several methods known in the art. For
example, phage display techniques can be used to provide libraries
containing a repertoire of antibodies that specifically bind glycosylation-
variant BEHAB or the peptides Bgl and Bg2 comprising one or more
unglycosylated 0-glycosylation sites. These libraries can then be screened
to identify and isolate antibodies with optimal levels of specificity for
glycosylation-variant BEHAB.
[0101] For example, glycosylation-variant BEHAB antibodies of the
present invention can be isolated by screening a recombinant combinatorial
antibody library. The library can be from any mammal, including humans.
In some embodiments, the library is a scFv phage display library,
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generated using VL and VH cDNAs prepared from mRNA isolated from B
cells. In some embodiments, the B cells are human B cells. Methods for
preparing and screening such libraries are known in the art. Kits for
generating phage display libraries are commercially available (e.g., the
Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01;
and the Stratagene SurfZAPTM phage display kit, catalog no. 240612).
There also are other methods and reagents that can be used in generating
and screening antibody display libraries (see, e.g., U.S. Pat. No. 5,223,409:
PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791, WO
92/15679, WO 93/01288, WO 92/01047, and WO 92/09690; Fuchs et al.,
Bio/Technology 9:1370-1372 (1991); Hay, et al., Hum. Antibod.
Hybridomas, 3:81-85 (1992); Huse, et al., Science, 246:1725-1281 (1989);
McCafferty et al., Nature, 348:552-554 (1990); Griffiths, et al., EMBO J.,
12:725-734 (1993); Hawkins, et al., J. Mol. Biol., 226:889-896 (1992);
Clackson, et al., Nature 352:624-628 (1991); Gram, et al., Proc. Natl. Acad.
Sci. USA, 89:3576-3580 (1992); Garrad, et al., Bio/Technology, 9:1373-
1377 (1991); Hoogenboom, et al.; Nuc. Acid Res., 19:4133-4137 (1991);
Barbas, et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991); and
Griffiths, et al., EMBO J., 13:3245-3260 (1994); which are all incorporated
herein by reference).
[0102] Another method for preparing a library of antibodies for use in
phage display techniques comprises the steps of immunizing a non-human
animal comprising human immunoglobulin loci with glycosylation-variant
BEHAB or an antigenic portion thereof as described above (such as Bgl or
Bg2) to create an immune response, extracting antibody-producing cells
from the immunized animal, isolating RNA encoding heavy and light chains
of antibodies of the invention from the extracted cells, reverse transcribing
the RNA to produce cDNA, amplifying the cDNA using primers and
inserting the cDNA into a phage display vector such that antibodies are
expressed on the phage. For production of such repertoires, it is
unnecessary to immortalize the B cells from the immunized animal.
Rather, the primary B cells can be used directly as a source of DNA. The
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mixture of cDNAs obtained from B cells, e.g., derived from spleens, is used
to prepare an expression library, for example, a phage display library
transfected into E. coli. Ultimately, clones from the library are identified
that
produce binding affinities of a desired magnitude for the antigen and the
DNA encoding the product responsible for such binding is recovered and
manipulated for standard recombinant expression. Phage display libraries
may also be constructed using previously manipulated nucleotide
sequences and screened in a similar fashion. In general, the cDNAs
encoding heavy and light chains are independently supplied or linked to
form Fv analogs for production in the phage library. The phage library is
then screened for the antibodies with the highest affinities for glycosylation-
variant BEHAB and the genetic material is recovered from the appropriate
clone. Further rounds of screening can increase affinity of the original
antibody isolated.
[0103] In one embodiment, to isolate and produce glycosylation-variant
BEHAB antibodies with the desired characteristics, a glycosylation-variant
BEHAB antibody as described herein is first used to select human heavy
and light chain sequences having similar binding activity toward
glycosylation-variant BEHAB, using the epitope imprinting methods
described in PCT Publication No. WO 93/06213, incorporated herein by
reference. The antibody libraries used in this method are preferably scFv
libraries prepared and screened as described in PCT Publication No. WO
92/01047, McCafferty, et al., Nature, 348:552-554 (1990); and Griffiths, et
al., EMBO J. 12:725-734 (1993), all incorporated herein by reference. The
scFv antibody libraries can be screened using glycosylation-variant BEHAB
as the antigen. The phage library is screened for the antibodies with the
highest affinities for glycosylation-variant BEHAB and the genetic material
recovered from the appropriate clone. Further rounds of screening can
increase affinity of the original antibody isolated.
[0104] Once initial human VL and VH domains are selected, "mix and
match" experiments can then be performed, in which different pairs of the
initially selected VL and VH segments are screened for glycosylation-variant
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BEHAB binding to select preferred VLNH pair combinations. These mix
and match experiments can also be performed after the VH and VL
segments have been randomly mutated for optimized binding as described
below. Additionally, to further improve the quality of the antibody, the VL
and VH segments of the preferred VLNH pair(s) can be randomly mutated,
preferably within the CDR3 region of VH and/or VL, in a process analogous
to the in vivo somatic mutation process responsible for affinity maturation of
antibodies during a natural immune response. This in vitro affinity
maturation can be accomplished, for example, by amplifying VH and VL
domains using PCR primers complimentary to the VH CDR3 or VL CDR3,
respectively, which primers have been "spiked" with a random mixture of
the four nucleotide bases at certain positions such that the resultant PCR
products encode VH and VL segments into which random mutations have
been introduced into the VH and/or VL CDR3 regions. These randomly
mutated VH and VL segments can be re-screened for binding to
glycosylation-variant BEHAB, and sequences that exhibit high affinity and a
low off rate for glycosylation-variant BEHAB can be selected.
[0105] Following screening and isolation of an anti- BEHAB antibody of
the invention from a recombinant immunoglobulin display library, nucleic
acids encoding the selected antibody can be recovered from the display
package (e.g., from the phage genome) and subcloned into other
expression vectors by standard recombinant DNA techniques. If desired,
the nucleic acid can further be manipulated to create other antibody forms
of the invention, as described below. To express a recombinant human
antibody isolated by screening of a combinatorial library, the DNA encoding
the antibody is cloned into a recombinant expression vector and introduced
into a host cell, as described below.
[0106] In another embodiment, glycosylation-variant BEHAB antibodies
can be produced by immunizing a non-human animal with glycosylation-
variant BEHAB or an antigenic portion thereof (such as Bgl or Bg2)
comprising one or more 0-linked glycosylation sites that are glycosylated in
BEHAB expressed in normal adult, brain tissue but non glycosylated in the
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immunogen. For example, the non-human animal can be a rabbit, rat,
mouse, goat, chicken, pig, primate or other non-human mammal. In
preferred embodiments, the non-human animal is a mouse or rabbit.
[0107] Methods of production of polyclonal antibodies are known to those
of skill in the art. A non-human animal is immunized with the protein using
a standard adjuvant, such as Freund's adjuvant, and a standard
immunization protocol. The animal's immune response to the immunogen
preparation is monitored by taking test bleeds and determining the titer of
reactivity to the beta subunits. When appropriately high titers of antibody to
the immunogen are obtained, blood is collected from the animal and
antisera are prepared. Further fractionation of the antisera to enrich for
antibodies reactive to the protein can be done if desired (see, Harlow &
Lane, supra). In the case of antibodies directed against a peptide coupled
to a carrier protein, it is desirable to purify the antisera further using
immunoaffinity chromatography on carrier protein-Sepharose.
Alternatively, peptide-Sepharose may be used to purify the antisera.
[0108] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Briefly, spleen cells from an animal
immunized with a desired antigen are immortalized, commonly by fusion
with a myeloma cell (see, Kohler& Milstein, Eur. J. Immunol. 6:511-519
(1976)). Alternative methods of immortalization include transformation with
Epstein Barr Virus, oncogenes, or retroviruses, or other methods well
known in the art. Colonies arising from single immortalized cells are
screened for production of antibodies of the desired specificity and affinity
for the antigen, and yield of the monoclonal antibodies produced by such
cells may be enhanced by various techniques, including injection into the
peritoneal cavity of a vertebrate host. Alternatively, one may isolate DNA
sequences which encode a monoclonal antibody or a binding fragment
thereof by screening a DNA library from human B cells according to the
general protocol outlined by Huse, et al., Science 246:1275-1281 (1989).
[0109] Monoclonal antibodies and polyclonal sera are collected and
titered against the immunogen protein in an immunoassay, for example, a
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solid phase immunoassay with the immunogen immobilized on a solid
support. Typically, polyclonal antisera with a titer of 104 or greater are
selected and tested for their cross reactivity against non-equine IgE
proteins using a competitive binding immunoassay. Specific polyclonal
antisera and monoclonal antibodies will usually bind with a Kd of at least
about 0.1 mM, more usually at least about 1 pM, preferably at least about
0.1 pM or better, and most preferably, 0.01 pM or better.
[0110] In a preferred embodiment, the immunized animal is a non-human
animal that expresses human immunoglobulin genes wherein splenic B
cells are fused to a myeloma cell line from the same species as the non-
human animal. In a more preferred embodiment, the immunized animal is
a XENOMOUSE mouse and the myeloma cell line is a non-secretory
mouse myeloma.
[0111] Thus, in one embodiment, the invention provides methods for
producing a cell line that produces a glycosylation-variant BEHAB antibody
by (a) immunizing a non-human transgenic animal described herein with
glycosylation-variant BEHAB or an antigenic portion thereof that contains
one or more 0-linked glycosylation sites that are not glycosylated (such as
Bgl or Bg2); (b) allowing the transgenic animal to mount an immune
response to said glycosylation-variant BEHAB or an antigenic portion
thereof; (c) isolating antibody-producing cells from transgenic animal; (d)
immortalizing the antibody-producing cells; (e) creating individual
monoclonal populations of the immortalized antibody-producing cells; and
(f) screening the immortalized antibody-producing cells to identify a
glycosylation-variant BEHAB antibody.
[0112] In another aspect, the invention provides a cell line that produces
a human glycosylation-variant BEHAB antibody. In some embodiments the
cell line is immortalized. In some embodiments the cell line is a hybridoma
cell line. In some embodiments, the hybridomas are mouse hybridomas,
as described above. In other embodiments, the hybridomas are produced
in a non-human, non-mouse species such as rats, sheep, pigs, goats,
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cattle or horses. In another embodiment, the hybridomas are human
hybridomas.
[0113] In another embodiment, a transgenic animal is immunized with
glycosylation-variant BEHAB or an antigenic portion thereof as described
above, primary cells, e.g., spleen or peripheral blood B cells, are isolated
from an immunized transgenic animal and individual cells producing
antibodies specific for the desired antigen are identified. Polyadenylated
mRNA from each individual cell is isolated and reverse transcription
polymerase chain reaction (RT-PCR) is performed using sense primers that
anneal to variable domain sequences, e.g., degenerate primers that
recognize most or all of the FR1 regions of human heavy and light chain
variable domain genes and anti-sense primers that anneal to constant or
joining region sequences. cDNAs of the heavy and light chain variable
domains are then cloned and expressed in any suitable host cell, e.g., a
myeloma cell, as chimeric antibodies with respective immunoglobulin
constant regions, such as the heavy chain and K or A constant domains.
See Babcook, J.S. et al., Proc. Natl. Acad. Sci. USA 93:7843-48, 1996,
incorporated herein by reference. Glycosylation-variant BEHAB antibodies
may then be identified and isolated as described herein.
[0114] After immunization of an animal with a glycosylation-variant
BEHAB antigen, anti-glycosylation-variant BEHAB antibodies can also be
obtained from serum obtained from the animal by bleeding or sacrificing the
animal. The serum may be used as it is obtained from the animal, an
immunoglobulin fraction may be obtained from the serum, or the anti-
glycosylation-variant BEHAB antibodies may be purified from the serum.
[0115] In other embodiments, human B cells are immunized in vitro in the
presence of glycosylation-variant BEHAB antigens and then immortalized
by fusion to a myeloma cell. The resulting hybrids are then screened by,
for e.g., ELISA-based assays, for secretion of glycosylation-variant
BEHAB-specific monoclonal antibodies. Examples of antibodies generated
in such a fashion include those specific for human mesothelin and
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granulocyte-macrophage colony-stimulating factor (GM-CSF) (Li et. al.,
2006, PNAS, 103: 3557-3562).
[0116] To express the glycosylation-variant BEHAB antibodies of the
present invention, DNA fragments encoding VH and VL regions can first be
obtained using any of the methods described above. Various mutations,
deletions, and/or additions can also be introduced into the DNA sequences
using standard methods known to those of skill in the art. For example,
mutagenesis can be carried out using standard methods, such as PCR-
mediated mutagenesis, in which the mutated nucleotides are incorporated
into the PCR primers such that the PCR product contains the desired
mutations or site-directed mutagenesis. One type of substitution, for
example, that may be made is to change one or more cysteines in the
antibody, which may be chemically reactive, to another residue, such as,
without limitation, alanine or serine. For example, there can be a
substitution of a non-canonical cysteine. The substitution can be made in a
CDR or framework region of a variable domain or in the constant domain of
an antibody. In some embodiments, the cysteine is canonical.
[0117] The antibodies may also be mutated in the variable domains of
the heavy and/or light chains, e.g., to alter a binding property of the
antibody. For example, a mutation may be made in one or more of the
CDR regions to increase or decrease the KD of the antibody for
glycosylation-variant BEHAB, to increase or decrease koff, or to alter the
binding specificity of the antibody. Techniques in site-directed mutagenesis
are well-known in the art. See, e.g., Sambrook, et al. and Ausubel, et al.,
supra, which is incorporated herein by reference.
[0118] A mutation may also be made in a framework region or constant
domain to increase the half-life of a glycosylation-variant BEHAB antibody.
See, e.g., PCT Publication No. WO 00/09560, incorporated herein by
reference. A mutation in a framework region or constant domain can also
be made to alter the immunogenicity of the antibody, to provide a site for
covalent or non-covalent binding to another molecule, or to alter such
properties as complement fixation, FcR binding and antibody-dependent
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cell-mediated cytotoxicity (ADCC). According to the invention, a single
antibody may have mutations in any one or more of the CDRs or
framework regions of the variable domain or in the constant domain.
[0119] In a process known as "germlining", certain amino acids in the VH
and VL sequences can be mutated to match those found naturally in
germline VH and VL sequences. In particular, the amino acid sequences of
the framework regions in the VH and VL sequences can be mutated to
match the germline sequences to reduce the risk of immunogenicity when
the antibody is administered. Germline DNA sequences for human VH and
VL genes are known in the art (see e.g., the "Vbase" human germline
sequence database; see also Kabat, E. A., et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health
and Human Services, NIH Publication No. 91-3242; TomliNSOn, et al.
(1992) J. Mol. Biol. 227:776-798; and Cox, et al. Eur. J. Immunol. 24:827-
836 (1994); the contents of each of which are expressly incorporated
herein by reference).
[0120] The removal of potential proteolytic sites in the antibody may also
be contemplated. Such sites may occur in a CDR or framework region of a
variable domain or in the constant domain of an antibody. Substitution of
cysteine residues and removal of proteolytic sites may decrease the risk of
heterogeneity in the antibody product and thus increase its homogeneity.
Another type of amino acid substitution is to eliminate asparagine-glycine
pairs, which form potential deamidation sites, by altering one or both of the
residues. In another example, the C-terminal lysine of the heavy chain of a
glycosylation-variant BEHAB antibody of the invention can be cleaved.
[0121] Once DNA fragments encoding the VH and VL segments of the
present invention are obtained, these DNA fragments can be further
manipulated by standard recombinant DNA techniques, for example to
convert the variable region genes to full-length antibody chain genes, to
Fab fragment genes, or to a scFv gene. In these manipulations, a VL- or
VH-encoding DNA fragment is operatively linked to another DNA fragment
encoding another protein, such as an antibody constant region or a flexible
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linker. The term "operatively linked", as used in this context, is intended to
mean that the two DNA fragments are joined such that the amino acid
sequences encoded by the two DNA fragments remain in-frame.
[0122] The isolated DNA encoding the VH region can be converted to a
full-length heavy chain gene by operatively linking the VH-encoding DNA to
another DNA molecule encoding heavy chain constant regions (CH1, CH2
and CH3). The sequences of human heavy chain constant region genes
are known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Ed., U.S. Department of Health
and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1, IgG2,
IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an
IgG1 or IgG2 constant region. The IgG1 constant region sequence can be
any of the various alleles or allotypes known to occur among different
individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypes
represent naturally occurring amino acid substitution in the IgG1 constant
regions. For a Fab fragment heavy chain gene, the VH-encoding DNA can
be operatively linked to another DNA molecule encoding only the heavy
chain CH1 constant region. The CH1 heavy chain constant region may be
derived from any of the heavy chain genes.
[0123] The isolated DNA encoding the VL region can be converted to a
full-length light chain gene (as well as a Fab light chain gene) by
operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of human
light chain constant region genes are known in the art (see e.g., Kabat, E.
A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Ed.,
U.S. Department of Health and Human Services, NIH Publication No. 91-
3242) and DNA fragments encompassing these regions can be obtained by
standard PCR amplification. The light chain constant region can be a
kappa or lambda constant region. The kappa constant region may be any
of the various alleles known to occur among different individuals, such as
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Inv(1), Inv(2), and Inv(3). The lambda constant region may be derived from
any of the three lambda genes.
[0124] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible linker, e.g.,
encoding the amino acid sequence (GIy4-Ser)3, such that the VH and VL
sequences can be expressed as a contiguous single-chain protein, with the
VL and VH regions joined by the flexible linker (see e.g., Bird et al. Science
242:423-426 (1988); Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-
5883 (1988); McCafferty, et al., Nature, 348:552-554 (1990)). The single
chain antibody may be monovalent, if only a single VH and VL are used,
bivalent, if two VH and VL are used, or polyvalent, if more than two VH and
VL are used. Bispecific or polyvalent antibodies may be generated that
bind specifically to glycosylation-variant BEHAB and to another molecule.
[0125] In another embodiment, a fusion antibody or immunoadhesin may
be made that comprises all or a portion of a glycosylation-variant BEHAB
antibody of the invention linked to another polypeptide. In another
embodiment, only the variable domains of the glycosylation-variant BEHAB
antibody are linked to the polypeptide. In another embodiment, the VH
domain of a glycosylation-variant BEHAB antibody is linked to a first
polypeptide, while the VL domain of a glycosylation-variant BEHAB
antibody is linked to a second polypeptide that associates with the first
polypeptide in a manner such that the VH and VL domains can interact with
one another to form an antigen binding site. In another preferred
embodiment, the VH domain is separated from the VL domain by a linker
such that the VH and VL domains can interact with one another. The VH-
linker-VL antibody is then linked to the polypeptide of interest. In addition,
fusion antibodies can be created in which two (or more) single-chain
antibodies are linked to one another. This is useful if one wants to create a
divalent or polyvalent antibody on a single polypeptide chain, or if one
wants to create a bispecific antibody.
[0126] In other embodiments, other modified antibodies may be prepared
using glycosylation-variant BEHAB antibody encoding nucleic acid
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molecules. For instance, "Kappa bodies" (III, et al., Protein Eng. 10: 949-
57 (1997)), "Minibodies' (Martin, et al., EMBO J., 13: 5303-9 (1994)),
"Diabodies" (Holliger, et al., Proc. Nati. Acad. Sci. USA, 90: 6444-6448
(1993)), or "Janusins" (Traunecker, et al., EMBO J., 10:3655-3659 (1991)
and Traunecker, et al., Int. J. Cancer,'(Suppl.) 7:51-52 (1992)) may be
prepared using standard molecular biological techniques following the
teachings of the specification.
[0127] Bispecific antibodies or antigen-binding fragments can be
produced by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol.,
79:315-321 (1990), Kostelny, et al., J. Immunol., 148:1547-1553 (1992). In
addition, bispecific antibodies may be formed as "diabodies" or "Janusins."
In some embodiments, the bispecific antibody binds to two different
epitopes of glycosylation-variant BEHAB. In some embodiments, the
modified antibodies described above are prepared using one or more of the
variable domains or CDR regions from a human glycosylation-variant
BEHAB antibody provided herein.
[0128] To express the antibodies and antigen-binding portions of the
invention, DNAs encoding partial or full-length light and heavy chains,
obtained as described above, are inserted into expression vectors such
that the genes are operatively linked to transcriptional and translational
control sequences. In this context, the term "operatively linked" is intended
to mean that an antibody gene is ligated into a vector such that
transcriptional and translational control sequences within the vector serve
their intended function of regulating the transcription and translation of the
antibody gene. The expression vector and expression control sequences
are chosen to be compatible with the expression host cell used. Expression
vectors include plasmids, retroviruses, adenoviruses, adeno-associated
viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco
mosaic virus, cosmids, YACs, EBV derived episomes, and the like. In
some embodiments, the antibody light chain gene and the antibody heavy
chain gene can be inserted into separate vectors. In some embodiments,
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both genes are inserted into the same expression vector. The antibody
genes are inserted into the expression vector by standard methods (e.g.,
ligation of complementary restriction sites on the antibody gene fragment
and vector, or blunt end ligation if no restriction sites are present).
[0129] A convenient vector is one that encodes a functionally complete
human CH or CL immunoglobulin sequence, with appropriate restriction
sites engineered so that any VH or VL sequence can easily be inserted and
expressed, as described above. In such vectors, splicing usually occurs
between the splice donor site in the inserted J region and the splice
acceptor site preceding the human C domain, and also at the splice regions
that occur within the human CH exons. Polyadenylation and transcription
termination occur at native chromosomal sites downstream of the coding
regions. The recombinant expression vector also can encode a signal
peptide that facilitates secretion of the antibody chain from a host cell. The
antibody chain gene may be cloned into the vector such that the signal
peptide is linked in-frame to the amino terminus of the immunoglobulin
chain. The signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a non-
immunoglobulin protein).
[0130] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that control
the expression of the antibody chain genes in a host cell. It will be
appreciated by those skilled in the art that the design of the expression
vector, including the selection of regulatory sequences may depend on
such factors as the choice of the host cell to be transformed, the level of
expression of protein desired, and so forth. Preferred regulatory
sequences for mammalian host cell expression include viral elements that
direct high levels of protein expression in mammalian cells, such as
promoters and/or enhancers derived from retroviral LTRs, cytomegalovirus
(CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40)
(such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus
major late promoter (AdMLP)), polyoma and strong mammalian promoters
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such as native immunoglobulin and actin promoters. For further description
of viral regulatory elements, and sequences thereof, see e.g., U.S. Pat. No.
5,168,062, U.S. Pat. No. 4,510,245 and U.S. Pat. No. 4,968,615. Methods
for expressing antibodies in plants, including a description of promoters and
vectors, as well as transformation of plants is known in the art. See, e.g.,
U.S. Pat. No. 6,517,529, incorporated herein by reference. Methods of
expressing polypeptides in bacterial cells or fungal cells, e.g., yeast cells,
are also well known in the art.
[0131] In addition to the antibody chain genes and regulatory sequences,
the recombinant expression vectors of the invention may carry additional
sequences, such as sequences that regulate replication of the vector in
host cells (e.g., origins of replication) and selectable marker genes. The
selectable marker gene facilitates selection of host cells into which the
vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665
and 5,179,017, incorporated herein by reference). For example, typically
the selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector has been
introduced. Preferred selectable marker genes include the dihydrofolate
reductase (DHFR) gene (for use in dhfr-host cells with methotrexate
selection/amplification), the neomycin phosphotransferase gene (for G418
selection), and the glutamate synthetase gene.
[0132] Nucleic acid molecules encoding glycosylation-variant BEHAB
antibodies and vectors comprising these nucleic acid molecules can be
used for transfection of a suitable mammalian, plant, bacterial or fungal
host cell, including yeast. Transformation can be by any known method for
introducing polynucleotides into a host cell. Methods for introduction of
heterologous polynucleotides into mammalian cells are well known in the
art and include dextran-mediated transfection, calcium phosphate
precipitation, polybrene-mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in liposomes, and
direct microinjection of the DNA into nuclei. In addition, nucleic acid
molecules may be introduced into mammalian cells by viral vectors.
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Methods of transforming cells are well known in the art. See, e.g., U.S. Pat.
Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, incorporated herein
by reference). Methods of transforming plant cells are well known in the
art, including, e.g., Agrobacterium-mediated transformation, biolistic
transformation, direct injection, electroporation and viral transformation.
Methods of transforming bacterial and yeast cells are also well known in
the art.
[0133] Mammalian cell lines available as hosts for expression are well
known in the art and include many immortalized cell lines available from the
American Type Culture Collection (ATCC). These include, for example,
Chinese hamster ovary (CHO) cells, NSO cells, SP2 cells, HEK-293T cells,
NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green
monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g.,
Hep G2), A549 cells, and a number of other cell lines. Cell lines of
particular preference are selected through determining which cell lines
have high expression levels. Other cell lines that may be used are insect
cell lines, such as Sf9 or Sf21 cells. When recombinant expression vectors
encoding antibody genes are introduced into mammalian host cells, the
antibodies are produced by culturing the host cells for a period of time
sufficient to allow for expression of the antibody in the host cells or, more
preferably, secretion of the antibody into the culture medium in which the
host cells are grown. Antibodies can be recovered from the culture
medium using standard protein purification methods. Plant host cells
include, e.g., Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, and
so forth. Bacterial host cells include E. coli and Streptomyces species.
Yeast host cells include Schizosaccharomyces pombe, Saccharomyces
cerevisiae and Pichia pastoris.
[0134] Standard recombinant DNA methodologies used to obtain
antibody heavy and light chain genes, incorporate these genes into
recombinant expression vectors and introduce the vectors into host cells
are described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning:
A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),
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Ausubel, F. M., et al. (eds.) Current Protocols in Molecular Biology, Greene
Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397, the
disclosures of which are incorporated herein by reference. Further,
expression of antibodies of the invention from production cell lines can be
enhanced using a number of known techniques. For example, the
glutamine synthetase (the GS system) and DHFR gene expression
systems are common approaches for enhancing expression under certain
conditions. High expressing cell clones can be identified using
conventional techniques, such as limited dilution cloning and Microdrop
technology. The GS system is discussed in European Patent Nos. 0 216
846, 0 256 055, 0 323 997 and 0 338 841.
[0135] It is likely that antibodies expressed by different cell lines or in
transgenic animals will have different glycosylation from each other.
However, all antibodies encoded by the nucleic acid molecules provided
herein, or comprising the amino acid sequences provided herein are part of
the present invention, regardless of the glycosylation of the antibodies.
[0136] Glycosylation-variant BEHAB antibodies of the invention also can
be produced transgenically through the generation of a mammal or plant
that is transgenic for the immunoglobulin heavy and light chain sequences
of interest and production of the antibody in a recoverable form therefrom.
In connection with the transgenic production in mammals, glycosylation-
variant BEHAB antibodies can be produced in, and recovered from, the
milk of goats, cows, or other mammals. See, e.g., U.S. Pat. Nos.
5,827,690, 5,756,687, 5,750,172, and 5,741,957, incorporated herein by
reference. Methods for producing antibodies in plants are described, e.g.,
in U.S. Pat. Nos. 6,046,037 and 5,959,177, incorporated herein by
reference.
[0137] In some embodiments, non-human transgenic animals or plants
are produced by introducing one or more nucleic acid molecules encoding
a glycosylation-variant BEHAB antibody, or antigen binding portion thereof,
of the invention into the animal or plant by standard transgenic techniques.
See Hogan and U.S. Pat. No. 6,417,429, supra. The transgenic cells used
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for making the transgenic animal can be embryonic stem cells or somatic
cells or a fertilized egg. The transgenic non-human organisms can be
chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. See,
e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual,
2nd ed., Cold Spring Harbor Press (1999); Jackson, et al., Mouse Genetics
and Transgenics: A Practical Approach, Oxford University Press (2000);
and Pinkert, Transgenic Animal Technology: A Laboratory Handbook,
Academic Press (1999), all incorporated herein by reference. In some
embodiments, the transgenic non-human animals have a targeted
disruption and replacement by a targeting construct that encodes a heavy
chain and/or a light chain of interest. The glycosylation-variant BEHAB
antibodies may be made in any transgenic animal. In a preferred
embodiment, the non-human animals are mice, rats, sheep, pigs, goats,
cattle or horses. The non-human transgenic animal expresses said
encoded polypeptides in blood, milk, urine, saliva, tears, mucus and other
bodily fluids.
[0138] The antibodies of the invent-ion may be humanized using any
technology known in the art. A number of techniques for humanizing
antibodies are well known in the art. For example, Wright et al. (1992,
Critical Rev. Immunol. 12: 125-168), and in the references cited therein,
and in Gu et al. (1997, Thrombosis and Hematocyst 77: 755-759). The
present invention also includes the use of humanized antibodies
specifically reactive with epitopes of glycosylation-variant BEHAB including
under- and un-glycosylated BEHAB. Such antibodies are capable of
specifically binding glycosylation-variant BEHAB, or a fragment thereof. In
some embodiments, the humanized antibodies of the invention have a
human framework and have one or more complementarity determining
regions (CDRs) from a non-human antibody, for example a mouse
antibody, specifically reactive with glycosylation-variant BEHAB, or a
fragment thereof. The humanized antibodies to glycosylation-variant
BEHAB are useful in the detection and/or differential diagnosis of malignant
primary gliomas such as anaplastic astrocytomas, well-differentiated
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astrocytomas, glioblastomas, ependymomas, oligodendrogliomas,
ganglioneuromas, mixed gliomas, brain stem gliomas, optic nerve gliomas
and pilocystic astrocytomas.
[0139] Expression of the recombinant DNA segments can be under the
control of expression control sequences that are eukaryotic promoter
systems in vectors capable of transforming or transfecting eukaryotic host
cells or the expression control sequences that'are prokaryotic promoter
systems in vectors capable of transforming or transfecting prokaryotic host
cells. Once the vector has been incorporated into the appropriate host, the
host is maintained under conditions suitable for high level expression of the
introduced nucleotide sequences and as desired the collection and
purification of the humanized light chains, heavy chains, light/heavy chain
dimers or intact antibodies, binding fragments or other immunoglobulin
forms may follow (Beychok, Cells of Immunoglobulin Synthesis, Academic
Press, New York, (1979), which is incorporated herein by reference).
[0140] Human constant region DNA sequences from a variety of human
cells can be isolated in accordance with well known procedures.
Preferably, the human constant region DNA sequences are isolated from
immortalized B-cells as described in WO 87/02671, which is herein
incorporated by reference. DNA sequences encoding CDRs useful in
producing the antibodies of the present invention may be similarly derived
from DNA encoding monoclonal antibodies capable of binding to
glycosylation-variant BEHAB, including under- and un-glycosylated
BEHAB. Suitable cells for constant region and framework DNA sequences
and host cells in which the antibodies are expressed and secreted, can be
obtained from a number of sources, for example, American Type Culture
Coilection, Manassas, VA.
[0141] In addition to the humanized antibodies discussed above, other
modifications to native antibody sequences can be readily designed and
manufactured utilizing various recombinant DNA techniques well known to
those skilled in the art. Moreover, a variety of different human framework
regions may be used singly or in combination as a basis for humanizing
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antibodies directed to BEHAB, including glycosylation-variant BEHAB. In
general, modifications of genes may be readily accomplished using a
variety of well-known techniques, such as site-directed mutagenesis
(Gillman and Smith, Gene,8 :81-97 (1979); Roberts et al., 1987, Nature,
328: 731-734).
[0142] The present invention encompasses methods for the diagnosis of
primary gliomas, such as malignant gliomas. Examples of such gliomas
include malignant anaplastic astrocytomas, well-differentiated
astrocytomas, glioblastomas, ependymomas, oligodendrogliomas,
ganglioneuromas, mixed gliomas, brain stem gliomas, optic nerve gliomas
and pilocystic astrocytomas. Expression of glycosylation-variant BEHAB is
specifically and exclusively expressed by malignant gliomas, is not present
in other neurological pathologies and is not present in benign tumors (see
WO 2005/069852, incorporated herein by reference). The present
invention includes methods of detecting the expression of glycosylation-
variant BEHAB in a mammal with glycosylation-variant BEHAB-specific
antibodies, methods of diagnosing a primary malignant glioma and a
method of differentially diagnosing a benign glioma from a malignant
glioma. In all instances recited herein, the most preferred mammal is a
human.
10143] The invention includes methods of diagnosing a malignant glioma
in a mammal. In some embodiments, the method comprises obtaining a
biological sample.from a mammal and detecting the presence of
glycosylation-variant BEHAB in that sample by detecting glycosylation-
variant BEHAB-specific antibody binding - a specific diagnostic marker of a
malignant glioma. In some embodiments, the malignant glioma is detected
by in vivo imaging.
[0144] The invention also encompasses a method of differentially
diagnosing a malignant glioma in a mammal, including a human, in vivo or
in vitro. That is, the present invention includes a method of differentially
diagnosing a giioma either in a mammal or in a biological sample from a
mammal. The method allows for the differential diagnosis between a
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malignant or high-grade glioma and a benign or low grade glioma. The
method comprises detecting glycosylation-variant BEHAB-specific antibody
binding in a mammal suspected of having a glioma. The binding of
glycosylation-variant BEHAB-specific antibodies is an indication that the
glioma is malignant.
[0145] The invention further includes a method of diagnosing malignant
glioma progression in a mammal. The method comprises obtaining a
biological sample from a mammal and detecting glycosylation-variant
BEHAB-specific antibody binding in that sample. The presence of
glycosylation-variant BEHAB-specific antibody binding in the sample can
then be compared to samples obtained earlier or later from the same
mammal, including a human, in order to determine the expression of
glycosylation-variant BEHAB in earlier or later samples. A lesser
detectable level or no detectable level of glycosylation-variant BEHAB-
specific antibody binding in the sample indicates that the mammal is in
regression or the anti-tumor treatment administered to the animal is
effective. A higher detectable level of glycosylation-variant BEHAB-specific
antibody binding indicates that the tumor is progressing and that other
courses of therapy should be used. This is because, as disclosed
elsewhere herein, a detectable level of binding of glycosylation-variant
BEHAB-specific antibodies in a mammal is specific for a malignant or high-
grade glioma.
[0146] The invention includes a method of assessing the effectiveness of
a treatment for a malignant glioma in a mammal. The method comprises
monitoring the treatment of a malignant glioma by assessing the level of
glycosylation-variant BEHAB-specific antibody binding, before, during and
after a specified course of treatment of a malignant glioma. An increase or
lack of change in antibody binding is an indication that the glioma is still
present and that the treatment is not effective while a reduction in antibody
binding is an indication that a treatment is successful.
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[0147] The course of therapy to be assessed can include, but is not
limited to, surgery, chemotherapy, radiation therapy, and/or the multiple
modes of therapy for a glioma disclosed herein.
[01481 Detecting glycosylation-variant BEHAB-specific antibody binding
in a biological sample can be accomplished using any of the methods
disclosed herein or known in the art, ELISA, immunoblotting techniques,
Western blot analysis, protein detection techniques, SDS-PAGE
electrophoresis, and other techniques well known in the art. As an
example, a biological sample can be from the central nervous system
(CNS).
[0149] For the in vivo detection of glycosylation-variant BEHAB-specific
antibody binding, the skilled artisan can employ a labeled antibody. Such
antibodies can be generated using techniques described elsewhere herein
and then conjugated to a tag or other molecule capable of detection
through any one of a number of methods. Methods of labeling antibodies
are well known in the art and can be accomplished using techniques in
protein chemistry, described elsewhere herein. As an example, an
antibody that binds glycosylation-variant BEHAB can be labeled with or
conjugated to a radioactive isotope and the binding of the isotope tagged
antibody can be detected on a film sensitive to radioactivity, such as X-ray
film. The antibody can also be bound to a tag visible to magnetic
resonance imaging technology. Further, the present invention includes a
method in which an antibody is conjugated to fluorescent molecule, such as
green fluorescent protein, an enzyme, a radioactive isotope, or gadolinium,
and the binding of the antibody to a glycosylation-variant BEHAB isoform is
detected through an imaging system capable of visualizing a tag. Uses of
biophotonic imaging systems for the in vivo detection of fluorescent tags
are well known in the art and such systems are available commercially
(Xenogen, Alameda, CA).
[0150] The invention further provides methods for treating a malignant
glioma by administering an anti-glycosylation variant BEHAB antibody of
the invention, or an antigen-binding portion thereof, to a mammalian
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subject in need thereof, including a human subject. The antibody of the
invention may be administered alone or in combination with another anti-
glycosylation variant BEHAB antibody, one or more antibodies directed to a
different target and/or one or more additional therapeutic agents. The
antibody of the invention may be administered as a "naked" antibody, i.e., a
neutralizing antibody that reduces one or more biological activity of
unglycosylated (B/bo9) BEHAB expressed in malignant melanoma cells.
Alternatively, the antibody of the invention may be administered as an
immuno-conjugate or fusion protein.
[0151] Pharmaceutical compositions, whether for diagnosis or therapy,
comprising an anti-glycosylation variant BEHAB antibody of the invention
may be administered by any means known in the art. Those of skill in
imaging and/or treating tumors of the CNS will be familiar with such routes
of administration. In particular, the compositions of the invention may be
administered by methods designed to allow for the delivery of molecules
inside the blood brain barrier. Administration may be, for example, by
Convection-Enhanced Delivery'(CED), a means of delivering therapeutic
agents into the brain via positive-pressure infusion. With CED, one or more
catheters connected to an infusion pump are implanted into the brain and
2p the agent is then pumped directly into the target tissues: The target
tissues
then dilate in response to the pressure field allowing permeation of the
agent.
[0152] Anti-glycosylation variant BEHAB antibodies of the current
invention may also be administered by GLIADEL wafers. GLIADEL
wafers are biodegradable polymer systems which deliver drug agents when
implanted in the brain. Such wafers have been designed to deliver agents
directly into the surgical cavity created when a brain tumor is resected.
Upon exposure to the aqueous environment of the resection cavity, the
anhydride bonds in the wafer are hydrolyzed, releasing the agent which
then diffuses into the surrounding brain tissue.
[0153] Colloidal drug delivery systems are also useful for the delivery of
anti-giycosylation variant BEHAB antibodies to the brain. Colloidal drug
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delivery systems such as cationic liposomes, micellar solutions, vesicle and
liquid crystal dispersions and nanocapsule dispersions typically consist of
small particles of 10-400 nm in diameter. Such systems are suitable for
imaging and/or treating tpmors of the CNS as their drug loading and
release, shelf-life and toxicity properties have been optimized. In addition,
colloidal drug delivery systems have been shown to hydrogen bond with
the surrounding aqueous medium effectively protecting the encapsulated
drug against hydrolysis and enzymatic degradation (Kaparissides et. al., J.
Nanotech. Online, March 25, 2006). Such colloids may be comprised of,
for example, poly(ethylene glycol)-600-hydroxystearate (PEG-HS) fatty
polymers.
[0154] Direct interstitial infusion can be used for the delivery of anti-
glycosylation variant BEHAB antibodies over both small and large
dimensions of the CNS and brain tissue. Application of interstitial infusion
over a broad distance scale allows for the administration agents to a variety
of sites in the brain. Examples of direct catheter infusion pumps and
systems include, but are not limited to, Ommaya reservoirs, Infusaid pumps
((nfusaid Corp., Norwood, MA), MiniMed PIMS pumps (MiniMed, Sylmor,
CA) and the Medtronic SynchroMed system (Medtronic, Minneapolis, MN).
[0155] The antibodies and antigen-binding portions of the present
invention can also be incorporated into pharmaceutical compositions
suitable for administration to a subject. Such a pharmaceutical composition
comprises an anti-glycosylation variant BEHAB antibody or antigen-binding
portion of the invention and a pharmaceutically acceptable carrier. As used
herein, "pharmaceutically acceptable carrier" means any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic
and absorption delaying agents, and the like that are physiologically
compatible. Some examples of pharmaceutically acceptable carriers are
water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and
the like, as well as combinations thereof. In many cases, it will be
preferable to include isotonic agents, for example, sugars, polyalcohols
such as mannitol, sorbitol, or sodium chloride in the composition.
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Additional examples of pharmaceutically acceptable substances are
wetting agents or minor amounts of auxiliary substances such as wetting or
emulsifying agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the antibody.
[0156] The compositions of this invention may be in a variety of forms, for
example, liquid, semi-solid and solid dosage forms, such as liquid solutions
(e.g., injectable and infusible solutions), dispersions or suspensions,
tablets, pills, powders and liposomes. The preferred form depends on the
intended mode of administration and therapeutic application. Modes of
administration include, but are not limited to, parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular), intravenous infusion or
injection and intramuscular or subcutaneous injection. Compositions may
take such forms as suspensions, solutions, or emulsions in oily or aqueous
vehicles, and may contain formulatory agents such as suspending,
stabilizing and/or dispersing agents. Alternatively, the active ingredient
may be in powder form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
[0157] Therapeutic compositions typically must be sterile and stable
under the conditions of manufacture and storage. The composition can be
2q formulated as a solution, microemulsion, dispersion, liposome, or other
ordered structure suitable to high drug concentration. Sterile injectable
solutions can be prepared by incorporating the anti-glycosylation variant
BEHAB antibody in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required, followed by
filtered sterilization.
[0158] The compositions of the invention may include a "therapeutically
effective amount" or a "prophylactically effective amount" of an antibody or
antigen-binding portion of the invention. A "therapeutically effective
amount" refers to an amount effective, at dosages and for periods of time
necessary, to achieve the desired therapeutic result. [0159] A
therapeutically effective amount of the antibody or antigen-binding portion
may vary according to factors such as the disease state, age, sex, and
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weight of the individual, and the ability of the antibody or antibody portion
to
elicit a desired response in the individual. A therapeutically effective
amount is also one in which any toxic or detrimental effects of the antibody
or antigen-binding portion are outweighed by the therapeutically beneficial
effects. A "prophylactically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve the desired
prophylactic result. Typically, since a prophylactic dose is used in subjects
prior to or at an earlier stage of disease, the prophylactically effective
amount may be less than the therapeutically effective amount.
[0160] The present invention further includes a method of identifying that
disrupts the ability of glycosylation-variant BEHAB to promote glioma
invasion. The method comprises contacting a cell with a test compound
and comparing the level glycosylation-variant BEHAB-specific antibody
binding in the cell so contacted with said compound and comparing the
level of glycosylation-variant BEHAB-specific antibody binding in an
otherwise identical cell not contacted with the compound. If the level of
glycosylation-variant BEHAB-specific antibody binding is higher or lower in
the cell contacted with the test compound compared to the level
glycosylation-variant BEHAB-specific antibody binding in the otherwise
identical cell not contacted with the test compound, this is an indication
that
the test compound affects glycosylation-variant BEHAB mediated glioma
invasion.
[0161] Similarly, the present invention includes a method of identifying a
compound that disrupts the ability of glycosylation-variant BEHAB to
promote glioma invasion in a cell. The method comprises contacting a cell
with a test compound and comparing the level of glycosylation-variant
BEHAB-specific antibody binding in the cell contacted with the compound
with the level of glycosylation-variant BEHAB-specific antibody binding in
an otherwise identical cell, which is not contacted with the compound. If
the level of glycosylation-variant BEHAB-specific antibody binding is lower
in the cell contacted with the compound compared to the level in the cell
that was not contacted with the compound, then that is an indication that
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the test compound reduces glycosylation-variant BEHAB mediated glioma
invasion in a cell.
[0162] The skilled artisan will further appreciate that the present invention
is not limited to a method of identifying a useful compound in a cell or an
animal. That is, the present invention includes methods of identifying a
useful compound in a cell-free system. A cell-free system, as used herein,
refers to an in vitro assay wherein the components necessary for a reaction
to take place are present, but are not associated with a cell. Such
components can include cellular enzymes, transcription factors, proteins,
antibodies, nucleic acids, and the like, provided that they are substantially
free from a cell. Glycosylation-variant BEHAB-specific antibody binding
assays can be performed free of a cell or animal, including the use of
immunoprecipitation assays and the like. Thus, the present invention
includes a method of identifying a useful compound for treating a glioma in
a cell-free system.
EXPERIMENTAL EXAMPLES
[0163] The invention is now described with reference to the following
Examples. These Examples are provided for the purpose of illustration
only and the invention should in no way be construed as being limited to
these Examples, but rather should be construed to encompass any and all
variations which become evident as a result of the teaching provided
herein.
[0164] The materials and methods used in the experiments presented in
these Examples are now described.
Example 1
Production of BEHAB Antibodies
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[0165] The generation of antibodies specific to glycosylation-variant
BEHAB was accomplished as follows.
[0166] We analyzed human BEHAB/brevican using the 0-glycosylation
prediction software, Net-O-Glyc (Julenius et. al, 2005, Glycobiology,
15:153-164) to identify potential 0-linked glycosylation sites. We selected
a region from amino acid 537-550 that contained three potential 0-
glycosylation sites (threonines) in close proximity for further analysis.
[0167] To confirm that the predicted glycosylation sites are normally
glycosylated, site-directed mutagenesis was employed to eliminate three
potential glycosylation sites found within the region from amino acids 537-
550 (N-GPPTETLPTPRERN-C). Threonines were mutated to alanines (N-
GPPAEALPARERN-C) thereby eliminating the glycosylation sites at these
positions (Figure 4). Human glioma cell line, U87 (American Type Culture
Collection, Manassas, VA), was then transiently transfected with a
construct encoding normal BEHAB (B/b), i.e. fully-glycosylated, or a
construct encoding alanine-mutated BEHAB (B/bm) and media analyzed by
Western blot analysis. B/bm, was observed to run at a slightly lower
apparent molecular mass than B/b thus indicating that the amino acid
stretch corresponding to aa537-550 of BEHAB is glycosylated in normal,
secreted BEHAB/brevican.
[0168] To confirm these results, B/b and B/b,,, were both deglycosylated
with sialidase and O-glycanase. In a Western blot, both protein bands
shifted downward to the same apparent molecular weight further confirming
the previously observed difference in apparent molecular weight can be
attributed solely to glycosylation (Figure 4). These data indicate that the
region of human BEHAB from aa537-550 is glycosylated in normal
secreted BEHAB.
[0169] We designed two immunogenic peptides from the same region:
N-GPPTETLPTPRE-C (AA 537-548) and N-TETLPTPRERN-C (AA 540-
550), designated Bgl and Bg2 respectively, and synthesized the peptides
using standard peptide synthesis techniques. Two rabbits were immunized
with each immunogen and antisera collected by bleeding.
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Example 2
Determination of Anti-Bgl (AF-Bgl) and Anti-Bg2 (AF-Bg2) BEHAB
Antibody Specificity
[0170] The specificity of antisera containing anti-Bgl antibodies (AF-
BG1) from immunized rabbits to bind to glycosylation-variant
BEHAB/brevican was determined. Briefly, U87MG cells (American Type
Culture Collection, Manassas, VA) were transfected with either normal
BEHAB/brevican cDNA or cDNA encoding mutated BEHAB (see Example
1). Culture media (CM) and cell membranes (cmb) were harvested and
processed by SDS-PAGE and Western blot. CM was additionally
deglycosylated with sialidase and 0-glycanase (CM deglyc).
[0171] We have shown previously that U87 cells secrete normal
glycosylated BEHAB into the media while the underglycosylated glioma-
specific isoform is exclusively found in cell lysates.
[0172] Antiserum (11600 dilution) from 4-week-bleeds of rabbits
immunized with Bgl specifically detected the B/bog isoform in cell lysates
(Figure 5). The secreted glycosylated form of B/b in the cell media,
conversely, was not detected by this antibody, AF-Bg1, under normal
conditions but could be detected after deglycosylation with 0-glycanase
and sialidase. Thus, rabbits immunized with Bgl produced antisera that
specifically detects B/bog or enzymatically deglycosylated BEHAB/brevican
in a cell culture system. Further, serum containing AF-Bgl antibodies did
not recognize B/bm (Figure 5). AF-Bgl antiserum was subsequently affinity
purified utilizing the immunogenic peptide bound to sepharose.
[0173] The ability of antisera (1/300 dilution) containing anti-Bg2 (AF-
Bg2) antibodies from immunized rabbits to bind to glycosylation-variant
BEHAB/brevican was determined. 4-week-bleed antiserum from animals
immunized with Bg2 initially showed no immunoreactivity. After a second
immunization, 8-week-bleed antiserum containing anti-Bg2 antibodies from
rabbits specifically detected B/bog. Thus rabbits immunized with Bg2
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produced antisera that specifically detects B/bog or enzymatically
deglycosylated BEHAB/brevican in a cell culture system. In addition,
serum containing AF-Bg2 antibodies did not recognize B/bm (Figure 5). AF-
Bg2 antiserum was subsequently affinity purified to improve its affinity
utilizing the immunogenic peptide bound to sepharose. Failure of either
antiserum to detect the mutated protein indicates that the epitope bound by
the antibodies requires intact threonines.
Example 3
Detection of Gliomas With AF-Bgl
[0174] Affinity purified AF-Bgl was subsequently utilized to assay normal
versus glioma tissue by Western blot analysis. We prepared membrane-
enriched samples from human malignant gliomas and age matched
controls and processed the samples as disclosed above using SDS-PAGE
and Western blot.
[0175] It was observed that AF-Bg1 specifically detects B/bog exclusively
in the glioma samples and shows virtually no non-specific staining in either
normal tissue or gliomas (Figure 6).
[0176] The ability of AF-Bgl to specifically bind to malignant grade Il
oligodendrogliomas also was determined. Total homogenates from benign
epileptogenic grade Il oligodendroglioma, a malignant grade ll
oligodendroglioma and non-neural scar tissue from a brain tumor sample
were processed for SDS-PAGE and probed with a pan-BEHAB/brevican
antibody and AF-Bgl. It was observed that AF-Bgl specifically recognizes
malignant oligodendrogliomas but does not recognize benign tumors
(Figure 7). The combined use of pan BEHAB/brevican with a B/bog specific
antibody reduces the cause of false negatives when comparing the two
benign tissues.
[0177] The ability of AF-Bgl to effectively recognize native B/bo9 was
also ascertained. The ability of AF-Bgl to immunoprecipitate protein from
a solublized glioma homogenate was assayed and it was found that AF-
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Bg1 effectively and specifically precipitates B/bo9. In addition, preliminary
studies were conducted on fresh frozen surgical samples from GBMs and
normal tissue. Samples were cyrostat sectioned and postfixed briefly with
4% paraformaldehyde followed by acetone. The section was incubated
with AF-Bgl (1:100) overnight and further processed for DAB
immunohistochemistry. AF-Bgl specifically detected glioma cells within the
tumor and cellular profiles, showed no extracellular matrix reactivity and
showed no reactivity with normal brain tissue (Figure 8).
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