Note: Descriptions are shown in the official language in which they were submitted.
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Description
METHODS FOR DIAGNOSIS AND THERAPY
OF HEMATOLOGICAL AND VIRUS-ASSOCIATED MALIGNANCIES
Technical Field
The present invention relates generally to the therapy of malignancies,
and more specifically to methods employing Her2/neu sequences for detection
and
therapy of hematological and virus-associated malignancies and lymphomas.
Background of the Invention
Hematological malignancies, such as leukemias, are neoplastic disorders
of hematopoetic stem cells. Such diseases are characterized by abnormal growth
and
maturation of hematopoetic cells and can result in a variety of symptoms,
including
bone marrow failure and organ failure. Treatment for many hematological
malignancies remains difficult and existing therapies are not universally
effective.
While treatments involving specific immunotherapy appear to have considerable
potential, such treatments have been limited by the small number of known
tumor-
associated antigens.
Her-2/neu, the product of the Her2/neu oncogene (also known as p185 or
c-erbB2; see, e.g., U.S. Patent No. 5,869,445) is a self antigen that is known
to be
overexpressed in adenocarcinomas of the breast, ovary, colon and lung. The
Her2/neu
proto-oncogene encodes a tyrosine kinase with homology to epidermal growth
factor.
Her2/neu protein is expressed during fetal development, but in adults is
detectable only
in small amounts in a limited number of normal tissues. Her2/neu has been
found to be
expressed on leukemic blasts of some patients suffering from acute lymphatic
leukemia
(ALL; EP 771,565B1) and on malignant lymphoma cells of a patient afflicted
with
aggressive diffuse lymphoma (Imamura et al., Leukemia and Lymphoma 4:4129-
422).
Her2/neu has not, ~ however, been detected on blasts from patients with acute
myelogenous leukemia (AML) or chronic myelogenous leukemia (CML), in chronic
or
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accelerated phase or in blast crisis. Thus, Her-2/neu has not appeared to be
generally
useful as a marker or therapeutic target for hematological malignancies.
Other prevalent malignancies that are often difficult to treat are virus-
associated conditions, such as malignancies associated with Epstein Barr Virus
(EBV)
infection. Such malignancies include lymphomas in immunocompromised patients
(e.g., AIDS patients and organ transplant recipients), nasopharynxcarcinoma
and breast
cancer. EBV-associated malignancies are common in immunocompromised
individuals
and are endemic in certain Asian populations. To date, there is no generally
effective
treatment for such conditions.
Accordingly, there remains a need in the art for improved methods for
detecting and treating hematological and virus-associated malignancies. The
present
invention fulfills these needs and further provides other related advantages.
Summary of the Invention
Briefly stated, the present invention provides compositions and methods
for detecting and treating hematological and virus-associated malignancies.
Within
certain aspects, the present invention provides methods for inhibiting the
development
of a hematological malignancy or virus-associated malignancy in a patient.
Such
methods may comprise administering to a patient an effective amount of a
polypeptide
comprising at least an immunogenic portion of Her2/neu, or a variant thereof
that
differs only in conservative substitutions such that the immunogenicity of the
variant is
not substantially diminished. Alternatively, a polynucleotide encoding such a
polypeptide, and antigen-presenting cell expressing such a polypeptide, or an
antibody,
or antigen-binding fragment thereof that specifically binds Her2/neu may be
administered. Hematological malignancies include AML, CML, CLL, MDS,
myelomas, Hodgkin lymphomas and non-Hodgkin lymphomas. Virus-associated
malignancies include malignancies associated with EBV, cytomegalovirus or
adenovirus, such as lymphomas and nasopharynxcarcinoma.
Within further aspects, methods for inhibiting the development of a
hematological or virus-associated malignancy in a patient comprise the steps
of: (a)
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incubating CD4+ and/or CD8+ T cells isolated from a patient with at least one
component selected from the group consisting of: (i) a polypeptide comprising
at least
an immunogenic portion of Her2/neu; (ii) a polynucleotide encoding such a
polypeptide; and (iii) an antigen-presenting cell that expresses such a
polypeptide; such
that T cells proliferate; and (b) administering to the patient an effective
amount of the
proliferated T cells. Proliferated T cells may, but need not, be cloned prior
to
administration to the patient. The patient may be afflicted with a
hematological or
virus-associated malignancy, in which case the methods provide treatment for
the
disease, or a patient considered at risk for the disease may be treated
prophylactically.
The present invention further provides methods for determining the
presence or absence of a hematological or virus-associated malignancy in a
patient,
comprising the steps of: (a) contacting a biological sample obtained from a
patient with
a binding agent that specifically binds to Her2/neu; (b) detecting in the
sample an
amount of polypeptide that binds to the binding agent; and (c) comparing the
amount of
polypeptide to a predetermined cut-off value.
Within related aspects, methods are provided for monitoring the
progression of a hematological or virus-associated malignancy in a patient,
comprising
the steps of: (a) contacting a biological sample obtained from a patient at a
first point in
time with a binding agent that specifically binds to Her2/neu; (b) detecting
in the
sample an amount of polypeptide that binds to the binding agent; (c) repeating
steps (a)
and (b) using a biological sample obtained from the patient at a subsequent
point in
time; and (d) comparing the amount of polypeptide detected in step (c) to the
amount
detected in step (b).
The present invention provides, within further aspects, methods for
determining the presence or absence of a hematological or virus-associated
malignancy
in a patient, comprising the steps of: (a) contacting a biological sample
obtained from a
patient with an oligonucleotide that hybridizes to a polynucleotide that
encodes
Her2/neu; (b) detecting in the sample an amount of a polynucleotide that
hybridizes to
the oligonucleotide; and (c) comparing the amount of polynucleotide to a
predetermined
cut-off value.
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In related aspects, methods are provided for monitoring the progression
of a hematological or virus-associated malignancy in a patient, comprising the
steps of:
(a) contacting a biological sample obtained from a patient with an
oligonucleotide that
hybridizes to a polynucleotide that encodes Her2/neu; (b) detecting in the
sample an
amount of a polynucleotide that hybridizes to the oligonucleotide; (c)
repeating steps (a)
and (b) using a biological sample obtained from the patient at a subsequent
point in
time; and (d) comparing the amount of polynucleotide detected in step (c) to
the amount
detected in step (b).
Methods are further provided for inhibiting the development of a
hematological or virus-associated malignancy in a patient, comprising: (a)
contacting
bone marrow, peripheral blood, or a fraction of bone marrow or peripheral
blood with T
cells that specifically react with Her2/neu, wherein the step of contacting is
performed
under conditions and for a time sufficient to permit the removal of Her2/neu
positive
cells to less than 10% of the number of myeloid or lymphatic cells in the bone
marrow,
peripheral blood or a fraction of bone marrow or peripheral blood; and (b)
administering
to a patient the bone marrow, peripheral blood or fraction from which Her2/neu
positive
cells have been removed.
These and other aspects of the invention will become evident upon
reference to the following detailed description and attached drawings. All
references
disclosed herein are hereby incorporated by reference in their entirety as if
each were
individually noted for incorporation.
Brief Description of the Drawings
Figures lA-1D are graphs illustrating the results of FACS staining for
Her2/. Figures 1A shows FACS staining of the Her2/neu overexpressing breast
cancer
cell line SKBR3, Figure 1B shows FAGS staining of human lymphoma cells,
Figures
1 C shows staining of human AML cells and Figure 1 D shows staining of human
CLL
cells using a control antibody (negative control; dotted line) and a Her2/neu
ECD
specific antibody (solid line). Abbreviations: MFI = mean fluorescence
intensity;
positive = % of cells staining positive for Her2/neu.
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Detailed Description of the Invention
As noted above, the present invention provides methods for detecting
and treating hematological and virus-associated malignancies. The invention is
based,
5 in part, on the discovery that Her2/neu is overexpressed in patients with
hematological
malignancies, as well as in EBV-transduced B-cells and EBV-induced lymphomas.
It
has further been found, within the context of the present invention, that
inoculating
SCID mice with EBV-infected B-cells induces lymphomas that overexpress
Her2/neu in
all animals. Vaccination with Her2/neu may be effective in preventing and/or
treating
hematological malignancies, including adult and pediatric AML, CML, ALL, CLL,
myelodysplastic syndromes (MDS), myeloproliferative syndromes (MPS), secondary
leukemia, myeloma, Hodgkin lymphoma and Non-Hodgkin lymphoma. Such
vaccination may also be used to prevent and/or treat virus-associated
malignancies, such
as conditions resulting from EBV infection. Alternatively, antibody therapy
may be
used for treatment and/or prevention of hematological and virus-associated
malignancies. Further, Her2/neu expression may be used for the diagnosis of
such
malignancies, monitoring therapy and purging bone marrow for transplantation.
HER-2/NEU POLYPEPTIDES
Certain methods provided herein employ Her-2/neu polypeptides. Such
polypeptides may comprise at least an immunogenic portion of a native Her2/neu
protein. Alternatively, a Her-2/neu polypeptide may comprise a variant of such
a
portion that differs only in conservative substitutions such that the
immunogenicity of
the variant is not substantially diminished, relative to the native
immunogenic portion.
As noted above, Her2/neu is the product of the Her2/neu oncogene, also
known as p185 or c-erbB2 (.see, e.g., U.S. Patent No. 5,869,445). "Her2/neu,"
as used
herein refers to published Her2/neu sequences, including human sequence,
alleles
thereof and homologs from other species.
An "immunogenic portion," as used herein is a portion of a protein that
is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface
antigen
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receptor. Such immunogenic portions generally comprise at least 5 amino acid
residues, more preferably at least 10, and still more preferably at least 20
amino acid
residues of Her2/neu. Certain preferred immunogenic portions include peptides
in
which an N-terminal leader sequence and/or transmembrane domain have been
deleted.
Other preferred immunogenic portions may contain a small N- and/or C-terminal
deletion (e.g., 1-30 amino acids, preferably 5-15 amino acids), relative to
the mature
protein. Preferred immunogenic portions are derived from the extracellular
domain of
Her2/neu. The sequence of a human Her2/neu protein and certain immunogenic
portions thereof are provided within, for example, U.S. Patent No. 5,726,023.
Immunogenic portions may generally be identified using well known
techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed.,
243-
247 (Raven Press, 1993) and references cited therein. Such techniques include
screening portions of Her2/neu for the ability to react with Her2/neu-specific
antibodies,
antisera and/or T-cell lines or clones. As used herein, antisera and
antibodies are
"Her2/neu-specific" if they specifically bind to Her2/neu (i. e., they react
with the
protein in an ELISA or other immunoassay, and do not react detectably with
unrelated
proteins). Such antisera and antibodies may be prepared as described herein,
and using
well known techniques. An immunogenic portion of a native Her2/neu is a
portion of
Her2/neu that reacts with such antisera and/or T-cells at a level that is not
substantially
less than the reactivity of the full length polypeptide (e.g., in an ELISA
and/or T-cell
reactivity assay). Such immunogenic portions may react within such assays at a
level
that is similar to or greater than the reactivity of a full length Her2/neu.
Such screens
may generally be performed using methods well known to those of ordinary skid
in the
art, such as those described in Harlow and Lane, Antibodies: A Laboratory
Manual,
Cold Spring Harbor Laboratory, 1988. For example, a polypeptide may be
immobilized
on a solid support and contacted with patient sera to allow binding of
antibodies within
the sera to the immobilized polypeptide. Unbound sera may then be removed and
bound antibodies detected using, for example, 'z5I-labeled Protein A.
A "variant" of an immunogenic portion is a polypeptide that differs from
a native immunogenic portion of Her2/neu in one or more substitutions,
deletions
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and/or insertions, such that the immunogenicity of the polypeptide is not
substantially
diminished. In other words, the ability of a variant to react with antigen-
specific
antisera may be enhanced or unchanged, relative to the native immunogenic
portion, or
may be diminished by less than 50%, and preferably less than 20%, relative to
the
native protein. Such variants may generally be identified by modifying an
immunogenic portion and evaluating the reactivity of the modified polypeptide
with
antigen-specific antibodies or antisera as described herein.
Variants preferably exhibit at least about 70%, more preferably at least
about 90% and most preferably at least about 95% identity to a native
immunogenic
portion. The percent identity may be determined as described above.
Preferably, a
variant contains conservative substitutions. A "conservative substitution" is
one in
which an amino acid is substituted for another amino acid that has similar
properties,
such that one skilled in the art of peptide chemistry would expect the
secondary
structure and hydropathic nature of the polypeptide to be substantially
unchanged.
Amino acid substitutions may generally be made on the basis of similarity in
polarity,
charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic
nature of the
residues. For example, negatively charged amino acids include aspartic acid
and
glutamic acid; positively charged amino acids include lysine and arginine; and
amino
acids with uncharged polar head groups having similar hydrophilicity values
include
leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine;
and
serine, threonine, phenylalanine and tyrosine. Other groups of amino acids
that may
represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, asn,
ser, thr;
(2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his;
and (5) phe, tyr, trp,
his. A variant may also, or alternatively, contain nonconservative changes. In
a
preferred embodiment, variant polypeptides differ from a native sequence by
substitution, deletion or insertion of five amino acids or fewer.
As noted above, a Her2/neu polypeptide may contain sequences in
addition to the immunogenic portion or variant thereof. Such sequences may,
but need
not, be derived from Her-2/neu. Sequences that are not derived from Her-2/neu
may,
but need not, be present at the amino and/or carboxy terminus of the
polypeptide. Such
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sequences) may be used, for example, to facilitate synthesis, purification or
solubilization. Other sequences that may be present include, but are not
limited to, a
signal (or leader) sequence at the N-terminal end of the protein which co-
translationally
or post-translationally directs transfer of the protein.
Polypeptides may be prepared using any of a variety of well known
techniques. Recombinant polypeptides encoded by Her2/neu DNA sequences as
described herein may be readily prepared from the DNA sequences using any of a
variety of expression vectors known to those of ordinary skill in the art.
Expression
may be achieved in any appropriate host cell that has been transformed or
transfected
with an expression vector containing a DNA molecule that encodes a recombinant
polypeptide. Suitable host cells include prokaryotes, yeast and higher
eukaryotic cells.
Preferably, the host cells employed are E. coli, yeast or a mammalian cell
line such as
COS or CHO. Supernatants from suitable host/vector systems which secrete
recombinant protein or polypeptide into culture media may be first
concentrated using a
commercially available filter. Following concentration, the concentrate may be
applied
to a suitable purification matrix such as an affinity matrix or an ion
exchange resin.
Finally, one or more reverse phase HPLC steps can be employed to further
purify a
recombinant polypeptide.
Portions and other variants having fewer than about 100 amino acids,
and generally fewer than about 50 amino acids, may also be generated by
synthetic
means, using techniques well known to those of ordinary skill in the art. For
example,
such polypeptides may be synthesized using any of the commercially available
solid-
phase techniques, such as the Merrifield solid-phase synthesis method, where
amino
acids are sequentially added to a growing amino acid chain. See Merrifield, J.
Am.
Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis of
polypeptides is
commercially available from suppliers such as Perkin Elmer/Applied BioSystems
Division (Foster City, CA), and may be operated according to the
manufacturer's
instructions. Crude product can be further purified by gel filtration, HPLC,
partition
chromatography or ion-exchange chromatography, using well known procedures.
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Within certain specific embodiments, a polypeptide may be a . fusion
protein. A fusion partner may, for example, assist in providing T helper
epitopes (an
immunological fusion partner), preferably T helper epitopes recognized by
humans, or
may assist in expressing the protein (an expression enhancer) at higher yields
than the
native recombinant protein. Certain preferred fusion partners are both
immunological
and expression enhancing fusion partners. Other fusion partners may be
selected so as
to increase the solubility of the protein or to enable the protein to be
targeted to desired
intracellular compartments. Still further fusion partners include affinity
tags, which
facilitate purification of the protein.
Fusion proteins may generally be prepared using standard techniques,
including chemical conjugation. Preferably, a fusion protein is expressed as a
recombinant protein, allowing the production of increased levels, relative to
a non-fused
protein, in an expression system. Briefly, DNA sequences encoding the
polypeptide
components may be assembled separately, and ligated into an appropriate
expression
vector. The 3' end of the DNA sequence encoding one polypeptide component is
ligated, with or without a peptide linker, to the 5' end of a DNA sequence
encoding the
second polypeptide component so that the reading frames of the sequences are
in phase.
This permits translation into a single fusion protein that retains the
biological activity of
both component polypeptides.
A peptide linker sequence may be employed to separate the first and
second polypeptide components by a distance sufficient to ensure that each
polypeptide
folds into its secondary and tertiary structures. Such a peptide linker
sequence is
incorporated into the fusion protein using standard techniques well known in
the art.
Suitable peptide linker sequences may be chosen based on the following
factors:
(1) their ability to adopt a flexible extended conformation; (2) their
inability to adopt a
secondary structure that could interact with functional epitopes on the first
and second
polypeptides; and (3) the lack of hydrophobic or charged residues that might
react with
the polypeptide functional epitopes. Preferred peptide linker sequences
contain Gly,
Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may
also be
used in the linker sequence. Amino acid sequences which may be usefully
employed as
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linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy
et al.,
Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Patent No. 4,935,233 and
U.S.
Patent No. 4,751,180. The linker sequence may generally be from 1 to about 50
amino
acids in length. Linker sequences are not required when the first and second
5 polypeptides have non-essential N-terminal amino acid regions that can be
used to
separate the functional domains and prevent steric interference.
The ligated DNA sequences are operably linked to suitable
transcriptional or translational regulatory elements. The regulatory elements
responsible for expression of DNA are located only 5' to the DNA sequence
encoding
10 the first polypeptides. Similarly, stop codons required to end translation
and
transcription termination signals are only present 3' to the DNA sequence
encoding the
second polypeptide.
Fusion proteins are also provided that comprise a polypeptide as
described herein together with an unrelated immunogenic protein. Preferably,
the
immunogenic protein is capable of eliciting a recall response. Examples of
such
proteins include tetanus, tuberculosis and hepatitis proteins (see, e.g.,
Stoute et al., New
Engl. J. Med. 336:86-91, 1997).
Within preferred embodiments, an immunological fusion partner is
derived from protein D, a surface protein of the gram-negative bacterium
Haemophilus
influenza B (WO 91/18926). Preferably, a protein D derivative comprises
approximately the first third of the protein (e.g., the first N-terminal 100-
110 amino
acids), and a protein D derivative may be lipidated. Within certain preferred
embodiments, the first 109 residues of a Lipoprotein D fusion partner is
included on the
N-terminus to provide the polypeptide with additional exogenous T-cell
epitopes and to
increase the expression level in E. coli (thus functioning as an expression
enhancer).
The lipid tail ensures optimal presentation of the antigen to antigen present
cells. Other
fusion partners include the non-structural protein from influenzae virus, NS 1
(hemaglutinin). Typically, the N-terminal 81 amino acids are used, although
different
fragments that include T-helper epitopes may be used.
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Iri another embodiment, the immunological fusion partner is the protein
known as LYTA, or a portion thereof (preferably a C-terminal portion). LYTA is
derived from Streptococcus pneumoniae, which synthesizes an N-acetyl-L-alanine
amidase known as amidase LYTA (encoded by the LytA gene; Gene 43:265-292,
1986). LYTA is an autolysin that specifically degrades certain bonds in the
peptidoglycan backbone. The C-terminal domain of the LYTA protein is
responsible
for the affinity to the choline or to some choline analogues such as DEAE.
This
property has been exploited for the development of E. coli C-LYTA expressing
plasmids useful for expression of fusion proteins. Purification of hybrid
proteins
containing the C-LYTA fragment at the amino terminus has been described (see
Biotechnology 10:795-798, 1992). Within a preferred embodiment, a repeat
portion of
LYTA may be incorporated into a fusion protein. A repeat portion is found in
the C-
terminal region starting at residue 178. A particularly preferred repeat
portion
incorporates residues 188-305.
In general, polypeptides (including fusion proteins) and polynucleotides
as described herein are isolated. An "isolated" polypeptide or polynucleotide
is one that
is removed from its original environment. For example, a naturally-occurring
protein is
isolated if it is separated from some or all of the coexisting materials in
the natural
system. Preferably, such polypeptides are at least about 90% pure, more
preferably at
least about 95% pure and most preferably at least about 99% pure. A
polynucleotide is
considered to be isolated if, for example, it is cloned into a vector that is
not a part of
the natural environment.
HER2/NEU POLYNUCLEOTIDES
Any polynucleotide that encodes at least a portion of a Her2/neu
polypeptide as described herein is encompassed by the present invention.
Preferred
polynucleotides comprise at least 15 consecutive nucleotides, preferably at
least 30
consecutive nucleotides and more preferably at least 45 consecutive
nucleotides, that
encode a portion of Her2/neu. Polynucleotides complementary to any such
sequences
are also encompassed by the present invention. Polynucleotides may be single-
stranded
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(coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or
synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which
contain introns and correspond to a DNA molecule in a one-to-one manner, and
mRNA
molecules, which do not contain introns. Additional coding or non-coding
sequences
may, but need not, be present within a polynucleotide of the present
invention, and a
polynucleotide may, but need not, be linked to other molecules and/or support
materials.
Polynucleotides may comprise a native sequence (i. e., an endogenous
sequence that encodes Her2/neu or a portion thereof) or may comprise a variant
of such
a sequence. Polynucleotide variants may contain one or more substitutions,
additions,
deletions and/or insertions such that the immunogenicity of the encoded
polypeptide is
not diminished, relative to native Her2/neu. The effect on the immunogenicity
of the
encoded polypeptide may generally be assessed as described herein. Variants
preferably exhibit at least about 70% identity, more preferably at least about
80%
identity and most preferably at least about 90% identity to a polynucleotide
sequence
that encodes a native Her2/neu or a portion thereof.
The percent identity for two polynucleotide or polypeptide sequences
may be readily determined by comparing sequences using computer algorithms
well
known to those of ordinary skill in the art, such as Megalign, using default
parameters.
Comparisons between two sequences are typically performed by comparing the
sequences over a comparison window to identify and compare local regions of
sequence
similarity. A "comparison window" as used herein, refers to a segment of at
least about
20 contiguous positions, usually 30 to about 75, or 40 to about 50, in which a
sequence
may be compared to a reference sequence of the same number of contiguous
positions
after the two sequences are optimally aligned. Optimal alignment of sequences
for
comparison may be conducted, for example, using the Megalign program in the
Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using
default parameters. Preferably, the percentage of sequence identity is
determined by
comparing two optimally aligned sequences over a window of comparison of at
least 20
positions, wherein the portion of the polynucleotide or polypeptide sequence
in the
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window may comprise additions or deletions (i. e., gaps) of 20 % or less,
usually S to 15
%, or 10 to 12%, relative to the reference sequence (which does not contain
additions or
deletions). The percent identity may be calculated by determining the number
of
positions at which the identical nucleic acid bases or amino acid residue
occurs in both
sequences to yield the number of matched positions, dividing the number of
matched
positions by the total number of positions in the reference sequence (i. e.,
the window
size) and multiplying the results by 100 to yield the percentage of sequence
identity.
Variants may also, or alternatively, be substantially homologous to a
native gene, or a portion or complement thereof. Such polynucleotide variants
are
capable of hybridizing under moderately stringent conditions to a naturally
occurring
DNA sequence encoding a native Her2/neu (or a complementary sequence).
Suitable
moderately stringent conditions include prewashing in a solution of 5 X SSC,
0.5%
SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-65°C, 5 X SSC,
overnight; followed
by washing twice at 65°C for 20 minutes with each of 2X, O.SX and 0.2X
SSC
containing 0.1% SDS.
It will be appreciated by those of ordinary skill in the art that, as a result
of the degeneracy of the genetic code, there are many nucleotide sequences
that encode
a polypeptide as described herein. Some of these polynucleotides bear minimal
homology to the nucleotide sequence of any native gene. Nonetheless,
polynucleotides
that vary due to differences in codon usage are specifically contemplated by
the present
invention.
Polynucleotides may be prepared using any of a variety of techniques.
94:2150-2155, 1997). For example, polynucleotides may be amplified from cDNA
prepared from cells expressing Her2/neu, such as certain breast tumor cells.
Such
polynucleotides may be amplified via polymerase chain reaction (PCR). For this
approach, sequence-specific primers may be designed based on known sequences,
and
may be purchased or synthesized.
An amplified portion may be used to isolate a full length gene from a
suitable library (e.g., a breast tumor eDNA library) using well known
techniques.
Within such techniques, a library (cDNA or genomic) is screened using one or
more
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polynucleotide probes or primers suitable for amplification. Preferably, a
library is
size-selected to include larger molecules. Random primed libraries may also be
preferred for identifying 5' and upstream regions of genes. Genomic libraries
are
preferred for obtaining introns and extending 5' sequences.
For hybridization techniques, a partial sequence may be labeled (e.g., by
nick-translation or end-labeling with 32P) using well known techniques. A
bacterial or
bacteriophage library is then screened by hybridizing filters containing
denatured
bacterial colonies (or lawns containing phage plaques) with the labeled probe
(see
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques
are
selected and expanded, and the DNA is isolated for further analysis. cDNA
clones may
be analyzed to determine the amount of additional sequence by, for example,
PCR using
a primer from the partial sequence and a primer from the vector. Restriction
maps and
partial sequences may be generated to identify one or more overlapping clones.
The
complete sequence may then be determined using standard techniques, which may
involve generating a series of deletion clones. The resulting overlapping
sequences are
then assembled into a single contiguous sequence. A full length cDNA molecule
can be
generated by ligating suitable fragments, using well known techniques.
Alternatively, there are numerous amplification techniques for obtaining
a full length coding sequence from a partial cDNA sequence. Within such
techniques,
amplification is generally performed via PCR. Any of a variety of commercially
available kits may be used to perform the amplification step. Primers may be
designed
using, for example, software well known in the art. Primers are preferably 22-
30
nucleotides in length, have a GC content of at least 50% and anneal to the
target
sequence at temperatures of about 68°C to 72°C. The amplified
region may be
sequenced as described above, and overlapping sequences assembled into a
contiguous
sequence.
One such amplification technique is inverse PCR (see Triglia et al., Nucl.
Acids Res. 16:8186, 1988), which uses restriction enzymes to generate a
fragment in the
known region of the gene. The fragment is then circularized by intramolecular
ligation
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and used as a template for PCR with divergent primers derived from the known
region.
Within an alternative approach, sequences adjacent to a partial sequence may
be
retrieved by amplification with a primer to a linker sequence and a primer
specific to a
known region. The amplified sequences are typically subjected to a second
round of
5 amplification with the same linker primer and a second primer specific to
the known
region. A variation on this procedure, which employs two primers that initiate
extension in opposite directions from the known sequence, is described in WO
96/38591. Another such technique is known as "rapid amplification of cDNA
ends" or
RACE. This technique involves the use of an internal primer and an external
primer,
10 which hybridizes to a polyA region or vector sequence, to identify
sequences that are 5'
and 3' of a known sequence. Additional techniques include capture PCR
(Lagerstrom et
al., PCR Methods Applic. 1:111-19, 1991) and walking PCR (Parker et al., Nucl.
Acids.
Res. 19:3055-60, 1991). Other methods employing amplification may also be
employed to obtain a full length cDNA sequence.
15 Polynucleotide variants may generally be prepared by any method
known in the art, including chemical synthesis by, for example, solid phase
phosphoramidite chemical synthesis. Modifications in a polynucleotide sequence
may
also be introduced using standard mutagenesis techniques, such as
oligonucleotide-
directed site-specific mutagenesis (see Adelman et al., DNA 2:183, 1983).
Alternatively, RNA molecules may be generated by in vitro or in vivo
transcription of
DNA sequences, provided that the DNA is incorporated into a vector with a
suitable
RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to
prepare an encoded polypeptide, as described herein. In addition, or
alternatively, a
portion may be administered to a patient such that the encoded polypeptide is
generated
in vivo (e.g., by transfecting antigen-presenting cells, such as dendritic
cells, with a
cDNA construct encoding a Her2/neu, and administering the transfected cells to
the
patient).
A portion of a sequence complementary to a coding sequence (i. e., an
antisense polynucleotide) may also be used as a probe or to modulate Her2/neu
expression. cDNA constructs that can be transcribed into antisense RNA may
also be
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16
introduced into cells or tissues to facilitate the production of antisense
RNA. An
antisense polynucleotide may be used, as described herein, to inhibit
expression of a
Her2/neu. Antisense technology can be used to control gene expression through
triple-
helix formation, which compromises the ability of the double helix to open
sufficiently
for the binding of polymerases, transcription factors or regulatory molecules
(see Gee et
al., In Huber and Carr, Molecular and Immunologic Approaches, Futura
Publishing Co.
(Mt. Kisco, NY; 1994)). Alternatively, an antisense molecule may be designed
to
hybridize with a control region of a gene (e.g., promoter, enhancer or
transcription
initiation site), and block transcription of the gene; or to block translation
by inhibiting
binding of a transcript to ribosomes.
A portion of a coding sequence or of a complementary sequence may
also be designed as a probe or primer to detect gene expression. Probes may be
labeled
with a variety of reporter groups, such as radionuclides and enzymes, and are
preferably
at least 10 nucleotides in length, more preferably at least 20 nucleotides in
length and
still more preferably at least 30 nucleotides in length. Primers, as noted
above, are
preferably 22-30 nucleotides in length.
Any polynucleotide may be further modified to increase stability in vivo.
Possible modifications include, but are not limited to, the addition of
flanking
sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl
rather
than phosphodiesterase linkages in the backbone; and/or the inclusion of
nontraditional
bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-,
thio- and
other modified forms of adenine, cytidine, guanine, thymine and uridine.
Her2/neu polynucleotides may be joined to a variety of other nucleotide
sequences using established recombinant DNA techniques. For example, a
polynucleotide may be cloned into any of a variety of cloning vectors,
including
plasmids, phagemids, lambda phage derivatives and cosmids. Vectors of
particular
interest include expression vectors, replication vectors, probe generation
vectors and
sequencing vectors. In general, a vector will contain an origin of replication
functional
in at least one organism, convenient restriction endonuclease sites and one or
more
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17
selectable markers. Other elements will depend upon the desired use, and will
be
apparent to those of ordinary skill in the art.
Within certain embodiments, polynucleotides may be formulated so as to
permit entry into a cell of a mammal, and expression therein. Such
formulations are
particularly useful for therapeutic purposes, as described below. Those of
ordinary skill
in the art will appreciate that there are many ways to achieve expression of a
polynucleotide in a target cell, and any suitable method may be employed. For
example, a polynucleotide may be incorporated into a viral vector such as, but
not
limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or
other pox virus
(e.g., avian pox virus). Techniques for incorporating DNA into such vectors
are well
known to those of ordinary skill in the art. A retroviral vector may
additionally transfer
or incorporate a gene for a selectable marker (to aid in the identification or
selection of
transduced cells) and/or a targeting moiety; such as a gene that encodes a
ligand for a
receptor on a specific target cell, to render the vector target specific.
Targeting may
also be accomplished using an antibody, by methods known to those of ordinary
skill in
the art.
Other formulations for therapeutic purposes include colloidal dispersion
systems, such as macromolecule complexes, nanocapsules, microspheres, beads,
and
lipid-based systems including oil-in-water emulsions, micelles, mixed
micelles, and
liposomes. A preferred colloidal system for use as a delivery vehicle in vitro
and in
vivo is a liposome (i. e., an artificial membrane vesicle). The preparation
and use of
such systems is well known in the art.
BINDING AGENTS
The present invention further employs agents, such as antibodies and
antigen-binding fragments thereof, that specifically bind to Her2/neu. As used
herein,
an antibody, or antigen-binding fragment thereof, is said to "specifically
bind" to
Her2/neu if it reacts at a detectable level (within, for example, an ELISA)
with
Her2/neu, and does not react detectably with unrelated proteins under similar
conditions. As used herein, "binding" refers to a noncovalent association
between two
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18
separate molecules such that a complex is formed. The ability to bind may be
evaluated
by, for example, determining a binding constant for the formation of the
complex. The
binding constant is the value obtained when the concentration of the complex
is divided
by the product of the component concentrations. In general, two compounds are
said to
"bind," in the context of the present invention, when the binding constant for
complex
formation exceeds about 103 L/mol. The binding constant maybe determined using
methods well known in the art.
Binding agents may be further capable of differentiating between
patients with and without a hematological malignancy. Such binding agents
generate a
signal indicating the presence of a hematological malignancy in at least about
20% of
patients with the disease, and will generate a negative signal indicating the
absence of
the disease in at least about 90% of individuals without the disease. To
determine
whether a binding agent satisfies this requirement, biological samples (e.g.,
blood, sera,
urine and/or tumor biopsies) from patients with and without a hematological
malignancy (as determined using standard clinical tests) may be assayed as
described
herein for the presence of polypeptides that bind to the binding agent. It
will be
apparent that a statistically significant number of samples with and without
the disease
should be assayed. Each binding agent should satisfy the above criteria;
however, those
of ordinary skill in the art will recognize that binding agents may be used in
combination to improve sensitivity.
Any agent that satisfies the above requirements may be a binding agent.
For example, a binding agent may be a ribosome, with or without a peptide
component,
an RNA molecule or a polypeptide. In a preferred embodiment, a binding agent
is an
antibody or an antigen-binding fragment thereof. Antibodies may be prepared by
any of
a variety of techniques known to those of ordinary skill in the art. See,
e.g., Harlow and
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In
general, antibodies can be produced by cell culture techniques, including the
generation
of monoclonal antibodies as described herein, or via transfection of antibody
genes into
suitable bacterial or mammalian cell hosts, in order to allow for the
production of
recombinant antibodies. In one technique, an immunogen comprising the
polypeptide is
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19
initially injected into any of a wide variety of mammals (e.g., mice, rats,
rabbits, sheep
or goats). In this step, the polypeptides of this invention may serve as the
immunogen
without modification. Alternatively, particularly for relatively short
polypeptides, a
superior immune response may be elicited if the polypeptide is joined to a
carrier
protein, such as bovine serum albumin or keyhole limpet hemocyanin. The
immunogen
is injected into the animal host, preferably according to a predetermined
schedule
incorporating one or more booster immunizations, and the animals are bled
periodically.
Polyclonal antibodies specific for the polypeptide may then be purified from
such
antisera by, for example, affinity chromatography using the polypeptide
coupled to a
suitable solid support.
Monoclonal antibodies specific for an antigenic polypeptide of interest
may be prepared, for example, using the technique of Kohler and Milstein, Eur.
J.
Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods
involve
the preparation of immortal cell lines capable of producing antibodies having
the
desired specificity (i.e., reactivity with the polypeptide of interest). Such
cell lines may
be produced, for example, from spleen cells obtained from an animal immunized
as
described above. The spleen cells are then immortalized by, for example,
fusion with a
myeloma cell fusion partner, preferably one that is syngeneic with the
immunized
animal. A variety of fusion techniques may be employed. For example, the
spleen cells
and myeloma cells may be combined with a nonionic detergent for a few minutes
and
then plated at low density on a selective medium that supports the growth of
hybrid
cells, but not myeloma cells. A preferred selection technique uses HAT
(hypoxanthine,
aminopterin, thymidine) selection. After a sufficient time, usually about 1 to
2 weeks,
colonies of hybrids are observed. Single colonies are selected and their
culture
supernatants tested for binding activity against the polypeptide. Hybridomas
having
high reactivity and specificity are preferred.
Monoclonal antibodies may be isolated from the supernatants of growing
hybridoma colonies. In addition, various techniques may be employed to enhance
the
yield, such as injection of the hybridoma cell line into the peritoneal cavity
of a suitable
vertebrate host, such as a mouse. Monoclonal antibodies may then be harvested
from
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the ascites fluid or the blood. Contaminants may be removed from the
antibodies by
conventional techniques, such as chromatography, gel filtration,
precipitation, and
extraction. The polypeptides of this invention may be used in the purification
process
in, for example, an affinity chromatography step.
5 Within certain embodiments, the use of antigen-binding fragments of
antibodies may be preferred. Such fragments include Fab fragments, which may
be
prepared using standard techniques. Briefly, immunoglobulins may be purified
from
rabbit serum by affinity chromatography on Protein A bead columns (Harlow and
Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and
digested
10 by papain to yield Fab and Fc fragments. The Fab and Fc fragments may be
separated
by affinity chromatography on protein A bead columns.
Monoclonal antibodies, and fragments thereof, of the present invention
may be coupled to one or more therapeutic agents, such as radionuclides,
differentiation
inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides
include 9°Y,
15 '23h i2sh »'I, '~6Re, '88Re, Z"At, and Z'2Bi. Preferred drugs include
methotrexate, and
pyrimidine and purine analogs. Preferred differentiation inducers include
phorbol esters
and butyric acid. Preferred toxins include ricin, abrin, diptheria toxin,
cholera toxin,
gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.
For
certain in vivo and ex vivo therapies, an antibody or fragment thereof is
preferably
20 coupled to a cytotoxic agent, such as a radioactive or chemotherapeutic
moiety.
A therapeutic agent may be coupled (e.g., covalently bonded) to a
suitable monoclonal antibody either directly or indirectly (e.g., via a linker
group). A
direct reaction between an agent and an antibody is possible when each
possesses a
substituent capable of reacting with the other. For example, a nucleophilic
group, such
as an amino or sulfllydryl group, on one may be capable of reacting with a
carbonyl-
containing group, such as an anhydride or an acid halide, or with an alkyl
group
containing a good leaving group (e.g., a halide) on the other.
Alternatively, it may be desirable to couple a therapeutic agent and an
antibody via a linker group. A linker group can function as a spacer to
distance an
antibody from an agent in order to avoid interference with binding
capabilities. A
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21
linker group can also serve to increase the chemical reactivity of a
substituent on an
agent or an antibody, and thus increase the coupling efficiency. An increase
in
chemical reactivity may also facilitate the use of agents, or functional
groups on agents,
which otherwise would not be possible.
It will be evident to those skilled in the art that a variety of bifunctional
or polyfunctional reagents, both homo- and hetero-functional (such as those
described
in the catalog of the Pierce Chemical Co., Rockford, IL), may be employed as
the linker
group. Coupling may be effected, for example, through amino groups, carboxyl
groups,
sulfhydryl groups or oxidized carbohydrate residues. There are numerous
references
describing such methodology, e.g., U.S. Patent No. 4,671,958, to Rodwell et
al.
Where a therapeutic agent is more potent when free from the antibody
portion of the immunoconjugates of the present invention, it may be desirable
to use a
linker group which is cleavable during or upon internalization into a cell. A
number of
different cleavable linker groups have been described. The mechanisms for the
intracellular release of an agent from these linker groups include cleavage by
reduction
of a disulfide bond (e.g., U.S. Patent No. 4,489,710,' to Spider), by
irradiation of a
photolabile bond (e.g., U.S. Patent No. 4,625,014, to Senter et al.), by
hydrolysis of
derivatized amino acid side chains (e.g., U.S. Patent No. 4,638,045, to Kohn
et al.), by
serum complement-mediated hydrolysis (e.g., U.S. Patent No. 4,671,958, to
Rodwell
et al.), and acid-catalyzed hydrolysis (e.g., U.S. Patent No. 4,569,789, to
Blattler et al.).
It may be desirable to couple more than one agent to an antibody. In one
embodiment, multiple molecules of an agent are coupled to one antibody
molecule. In
another embodiment, more than one type of agent may be coupled to one
antibody.
Regardless of the particular embodiment, immunoconjugates with more than one
agent
may be prepared in a variety of ways. For example, more than one agent may be
coupled directly to an antibody molecule, or linkers which provide multiple
sites for
attachment can be used. Alternatively, a carrier can be used.
A carrier may bear the agents in a variety of ways, including covalent
bonding either directly or via a linker group. Suitable carriers include
proteins such as
albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides and
polysaccharides
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22
such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.). A
carrier may
also bear an agent by noncovalent bonding or by encapsulation, such as within
a
liposome vesicle (e.g., U.S. Patent Nos. 4,429,008 and 4,873,088). Carriers
specific for
radionuclide agents include radiohalogenated small molecules and chelating
compounds. For example, U.S. Patent No. 4,735,792 discloses representative
radiohalogenated small molecules and their synthesis. A radionuclide chelate
may be
formed from chelating compounds that include those containing nitrogen and
sulfur
atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
For
example, U.S. Patent No. 4,673,562, to Davison et al. discloses representative
chelating
compounds and their synthesis.
A variety of routes of administration for the antibodies and
immunoconjugates may be used. Typically, administration will be intravenous,
intramuscular, subcutaneous or in the bed of a resected tumor. It will be
evident that the
precise dose of the antibody/immunoconjugate will vary depending upon the
antibody
used, the antigen density on the tumor, and the rate of clearance of the
antibody.
T CELLS
Immunotherapeutic compositions may also, or alternatively, comprise T
cells specific for Her2/neu. Such cells may generally be prepared in vitro or
ex vivo,
using standard procedures. For example, T cells may be isolated from bone
marrow,
peripheral blood or a fraction of bone marrow or peripheral blood of a
patient, using a
commercially available cell separation system, such as the CEPRATET"" system,
available from CellPro Inc., Bothell WA (see also U.S. Patent No. 5,240,856;
U.S.
Patent No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243).
Alternatively,
T cells may be derived from related or unrelated humans, non-human mammals,
cell
lines or cultures.
T cells may be stimulated with a Her2/neu polypeptide, Her2/neu
polynucleotide and/or an antigen presenting cell (APC) that expresses a
Her2/neu
polypeptide. Such stimulation is performed under conditions and for a time
sufficient
to permit the generation of T cells that are specific for the polypeptide.
Preferably, a
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23
Her2/neu polypeptide or polynucleotide is present within a delivery vehicle,
such as a
microsphere, to facilitate the generation of specific T cells.
T cells are considered to be specific for Her2/neu if the T cells kill target
cells coated with Her2/neu or expressing a gene encoding Her2/neu. T cell
specificity
may be evaluated using any of a variety of standard techniques. For example,
within a
chromium release assay or proliferation assay, a stimulation index of more
than two
fold increase in lysis and/or proliferation, compared to negative controls,
indicates T
cell specificity. Such. assays may be performed, for example, as described in
Chen et
al., Cancer Res. 54:1065-1070, 1994. Alternatively, detection of the
proliferation of
T cells may be accomplished by a variety of known techniques. For example, T
cell
proliferation can be detected by measuring an increased rate of DNA synthesis
(e.g., by
pulse-labeling cultures of T cells with tritiated thymidine and measuring the
amount of
tritiated thymidine incorporated into DNA). Contact with a Her2/neu
polypeptide ( 100
ng/ml - 100 pg/ml, preferably 200 ng/ml - 25 pg/ml) for 3 - 7 days should
result in at
least a two fold increase in proliferation of the T cells. Contact as
described above for
2-3 hours should result in activation of the T cells, as measured using
standard cytokine
assays in which a two fold increase in the level of cytokine release (e.g.,
TNF or IFN-y)
is indicative of T cell activation (see Coligan et al., Current Protocols in
Immunology,
vol. l, Wiley Interscience (Greene 1998)). T cells that have been activated in
response
to a Her2/neu polypeptide, polynucleotide or polypeptide-expressing APC may be
CD4+
and/or CD8+. Her2/neu-specific T cells may be expanded using standard
techniques.
Within preferred embodiments, the T cells are derived from a patient, or from
a related
or unrelated donor, and are administered to the patient following stimulation
and
expansion.
For therapeutic purposes, CD4+ or CD8+ T cells that proliferate in
response to a Her2/neu polypeptide, polynucleotide or APC can be expanded in
number
either in vitro or in vivo. Proliferation of such T cells in vitro may be
accomplished in a
variety of ways. For example, the T cells can be re-exposed to a Her2/neu
polypeptide
(e.g., a short peptide corresponding to an immunogenic portion of Her2/neu)
with or
without the addition of T cell growth factors, such as interleukin-2, and/or
stimulator
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24
cells that synthesize a Her2/neu polypeptide. Alternatively, one or more T
cells that
proliferate in the presence of Her2/neu can be expanded in number by cloning.
Methods for cloning cells are well known in the art, and include limiting
dilution.
Following expansion, the cells may be administered back to the patient as
described, for
example, by Chang et al., C'rit. Rev. Oncol. Hematol. 22:213, 1996.
PHARMACEUTICAL COMPOSITIONS AND VACCINES
Within certain aspects, polypeptides, polynucleotides, T cells and/or
binding agents described herein may be incorporated into pharmaceutical
compositions
or immunogenic compositions (i. e., vaccines). Pharmaceutical compositions
comprise
one or more such compounds and a physiologically acceptable carrier. Vaccines
may
comprise one or more such compounds and a non-specific immune response
enhancer.
A non-specific immune response enhancer may be any substance that enhances an
immune response to an exogenous antigen. Examples of non-specific immune
response
enhancers include adjuvants, biodegradable microspheres (e.g., polylactic
galactide) and
liposomes (into which the compound is incorporated; see e.g., Fullerton, U.S.
Patent
No. 4,235,877). Vaccine preparation is generally described in, for example,
M.F.
Powell and M.J. Newman, eds., "Vaccine Design (the subunit and adjuvant
approach),"
Plenum Press (NY, 1995). Pharmaceutical compositions and vaccines within the
scope
of the present invention may also contain other compounds, which may be
biologically
active or inactive.
A pharmaceutical composition or vaccine may contain a polynucleotide
(DNA or RNA) encoding one or more of the polypeptides as described above, such
that
the polypeptide is generated in situ. As noted above, a polynucleotide may be
present
within any of a variety of delivery systems known to those of ordinary skill
in the art,
including nucleic acid expression systems, bacteria and viral expression
systems.
Numerous gene delivery techniques are well known in the art, such as those
described
by Rolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, and
references
cited therein. Appropriate nucleic acid expression systems contain the
necessary DNA
sequences for expression in the patient (such as a suitable promoter and
terminating
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signal). Bacterial delivery systems involve the administration of a bacterium
(such as
Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the
polypeptide
on its cell surface or secretes such an epitope. In a preferred embodiment,
DNA may be
introduced using a viral expression system (e.g., vaccinia or other pox virus,
retrovirus,
5 or adenovirus), which may involve the use of a non-pathogenic (defective),
replication
competent virus. Suitable systems are disclosed, for example, in Fisher-Hoch
et al.,
Proc. Natl. Acac~ Sci. USA 86:317-321, 1989; Flexner et al., Ann. N. Y. Acacl.
Sci.
569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Patent Nos.
4,603,112,
4,769,330, and 5,017,487; WO 89/01973; U.S. Patent No. 4,777,127; GB
2,200,651;
10 EP 0,345,242; WO 91/02805; Berkner, Biotechnigues 6:616-627, 1988;
Rosenfeld et
al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA
91:215-219,
1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90:11498-11502, 1993;
Guzman et
al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir. Res. 73:1202-
1207, 1993.
Techniques for incorporating DNA into such expression systems are well known
to
15 those of ordinary skill in the art. DNA may also be "naked," as described,
for example,
in Ulmer et al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science
259:1691-1692, 1993. The uptake of naked DNA may be increased by coating the
DNA onto biodegradable beads, which are efficiently transported into the
cells.
While any suitable carrier known to those of ordinary skill in the art may
20 be employed in the pharmaceutical compositions of this invention, the type
of carrier
will vary depending on the mode of administration. Compositions of the present
invention may be formulated for any appropriate manner of administration,
including
for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal,
subcutaneous
or intramuscular administration. For parenteral administration, such as
subcutaneous
25 injection, the carrier preferably comprises water, saline, alcohol, a fat,
a wax or a buffer.
For oral administration, any of the above carriers or a solid carrier, such as
mannitol,
lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose,
glucose,
sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres
(e.g., polylactate polyglycolate) may also be employed as carriers for the
pharmaceutical compositions of this invention. Suitable biodegradable
microspheres
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26
are disclosed, for example, in U.S. Patent Nos.4,897,268; 5,075,109;
5,928,647;
5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252.
Such compositions may also comprise buffers (e.g., neutral buffered
saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose,
sucrose or
dextrans), mannitol, proteins, polypeptides or amino acids such as glycine,
antioxidants,
chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum
hydroxide)
and/or preservatives. Alternatively, compositions of the present invention may
be
formulated as a lyophilizate. Compounds may also be encapsulated within
liposomes
using well known technology.
Any of a variety of non-specific immune response enhancers may be
employed in the vaccines of this invention. For example, an adjuvant may be
included.
Most adjuvants contain a substance designed to protect the antigen from rapid
catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of
immune
responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis
derived
proteins. Suitable adjuvants are commercially available as, for example,
Freund's
Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI);
Merck
Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham);
aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate;
salts of
calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated
sugars;
canonically or anionically derivatized polysaccharides; polyphosphazenes;
biodegradable microspheres; monophosphoryl lipid A and quit A. Cytokines, such
as
GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
Within the vaccines provided herein, the adjuvant composition is
preferably designed to induce an immune response predominantly of the Thl
type.
High levels of Thl-type cytokines (e.g., IFN-y, IL-2 and IL-12) tend to favor
the
induction of cell mediated immune responses to an administered antigen. In
contrast,
high levels of Th2-type cytokines (e.g., IL-4, IL-5, IL-6, IL-10 and TNF-(3)
tend to
favor the induction of humoral immune responses. Following application of a
vaccine
as provided herein, a patient will support an immune response that includes
Thl- and
Th2-type responses. Within a preferred embodiment, in which a response is
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27
predominantly Thl-type, the level of Thl-type cytokines will increase to a
greater
extent than the level of Th2-type cytokines. The levels of these cytokines may
be
readily assessed using standard assays. For a review of the families of
cytokines, see
Mosmann and Coffman, Ann. Rev. Immunol. 7:145-173, 1989.
Preferred adjuvants for use in eliciting a predominantly Thl-type
response include, for example, a combination of monophosphoryl lipid A,
preferably 3-
de-O-acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt.
MPL adjuvants are available from Ribi ImmunoChem Research Inc. (Hamilton, MT;
see US Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094). Also
preferred is
AS-2 (SmithKline Beecham). CpG-containing oligonucleotides (in which the CpG
dinucleotide is unmethylated) also induce a predominantly Thl response. Such
oligonucleotides are well known and are described, for example, in WO
96/02555.
Another preferred adjuvant is a saponin, preferably QS21, which may be used
alone or
in combination with other adjuvants. For example, an enhanced system involves
the
combination of a monophosphoryl lipid A and saponin derivative, such as the
combination of QS21 and 3D-MPL as described in WO 94/00153, or a less
reactogenic
composition where the QS21 is quenched with cholesterol, as described in WO
96/33739. Other preferred formulations comprises an oil-in-water emulsion and
tocopherol. A particularly potent adjuvant formulation involving QS21, 3D-MPL
and
tocopherol in an oil-in-water emulsion is described in WO 95/17210. Any
vaccine
provided herein may be prepared using well known methods that result in a
combination of antigen, immune response enhancer and a suitable carrier or
excipient.
The compositions described herein may be administered as part of a
sustained release formulation (i.e., a formulation such as a capsule or sponge
that effects
a slow release of compound following administration). Such formulations may
generally be prepared using well known technology and administered by, for
example,
oral, rectal or subcutaneous implantation, or by implantation at the desired
target site.
Sustained-release formulations may contain a polypeptide, polynucleotide or
antibody
dispersed in a carrier matrix and/or contained within a reservoir surrounded
by a rate
controlling membrane. Carriers for use within such formulations are
biocompatible,
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and may also be biodegradable; preferably the formulation provides a
relatively
constant level of active component release. The amount of active compound
contained
within a sustained release formulation depends upon the site of implantation,
the rate
and expected duration of release and the nature of the condition to be treated
or
prevented.
Any of a variety of delivery vehicles may be employed within
pharmaceutical compositions and vaccines to facilitate production of an
antigen-specific
immune response that targets tumor cells. Delivery vehicles include antigen
presenting
cells (APCs), such as dendritic cells, macrophages, B cells, monocytes and
other cells
that may be engineered to be efficient APCs. Such cells may, but need not, be
genetically modified to increase the capacity for presenting the antigen, to
improve
activation and/or maintenance of the T cell response, to have anti-tumor
effects per se
and/or to be immunologically compatible with the receiver (i. e., matched HLA
haplotype). APCs may generally be isolated from any of a variety of biological
fluids
and organs, including tumor and peritumoral tissues, and may be autologous,
allogeneic, syngeneic or xenogeneic cells.
Certain preferred embodiments of the present invention use dendritic
cells or progenitors thereof as antigen-presenting cells. Dendritic cells are
highly potent
APCs (Banchereau and Steinman, Nature 392:245-251, 1998) and have been shown
to
be effective as a physiological adjuvant for eliciting prophylactic or
therapeutic
antitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529, 1999).
In
general, dendritic cells may be identified based on their typical shape
(stellate in situ,
with marked cytoplasmic processes (dendrites) visible in vitro) and based on
the lack of
differentiation markers of B cells (CD 19 and CD20), T cells (CD3), monocytes
(CD 14)
and natural killer cells (CD56), as determined using standard assays.
Dendritic cells
may, of course, be engineered to express specific cell-surface receptors or
ligands that
are not commonly found on dendritic cells in vivo or ex vivo, and such
modified
dendritic cells are contemplated by the present invention. As an alternative
to dendritic
cells, secreted vesicles antigen-loaded dendritic cells (called exosomes) may
be used
within a vaccine (see Zitvogel et al., Nature Med. 4:594-600, 1998).
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Dendritic cells and progenitors may be obtained from peripheral blood,
bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltrating cells,
lymph
nodes, spleen, skin, umbilical cord blood or any other suitable tissue or
fluid. For
example, dendritic cells may be differentiated ex vivo by adding a combination
of
cytokines such as GM-CSF, IL-4, IL-13 and/or TNFoc to cultures of monocytes
harvested from peripheral blood. Alternatively, CD34 positive cells harvested
from
peripheral blood, umbilical cord blood or bone marrow may be differentiated
into
dendritic cells by adding to the culture medium combinations of GM-CSF, IL-3,
TNFa,
CD40 ligand, LPS, flt3 ligand and/or other compounds) that induce maturation
and
proliferation of dendritic cells. Dendritic cells may alternatively be
generated from
leukemic and lymphoma cells.
Dendritic cells are conveniently categorized as "immature" and "mature"
cells, which allows a simple way to discriminate between two well
characterized
phenotypes. However, this nomenclature should not be construed to exclude all
possible intermediate stages of differentiation. Immature dendritic cells are
characterized as APC with a high capacity for antigen uptake and processing,
which
correlates with the high expression of Fcy receptor, mannose receptor and DEC-
205
marker. The mature phenotype is typically characterized by a lower expression
of these
markers, but a high expression of cell surface molecules responsible for T
cell
activation such as class I and class II MHC, adhesion molecules (e.g., CD54
and CD11)
and costimulatory molecules (e.g., CD40, CD80 and CD86).
APCs may generally be transfected with a polynucleotide encoding a
Her2/neu polypeptide such that the polypeptide, or an immunogenic portion
thereof, is
expressed on the cell surface. Such transfection may take place ex vivo, and a
composition or vaccine comprising such transfected cells may then be used for
therapeutic purposes, as described herein. Alternatively, a gene delivery
vehicle that
targets a dendritic or other antigen presenting cell may be administered to a
patient,
resulting in transfection that occurs in vivo. In vivo and ex vivo
transfection of dendritic
cells, for example, may generally be performed using any methods known in the
art,
such as those described in WO 97/24447, or the gene gun approach described by
Mahvi
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et al., Immunology and cell Biology 75:456-460, 1997. Antigen loading of
dendritic
cells may be achieved by incubating dendritic cells or progenitor cells with
the
Her2/neu polypeptide, DNA (naked or within a plasmid vector) or RNA; or with
antigen-expressing recombinant bacterium or viruses (e.g., vaccinia, fowlpox,
5 adenovirus or lentivirus vectors). Prior to loading, the polypeptide may be
covalently
conjugated to an immunological partner that provides T cell help (e.g., a
carrier
molecule). Alternatively, a dendritic cell may be pulsed with a non-conjugated
immunological partner, separately or in the presence of the polypeptide.
1O THERAPEUTIC METHODS
In further aspects of the present invention, the compositions described
herein may be used for immunotherapy of hematological malignancies including
adult
and pediatric AML, CML, ALL, CLL, myelodysplastic syndromes (MDS),
myeloproliferative syndromes (MPS), secondary leukemia, multiple myeloma,
Hodgkin
15 lymphoma and Non-Hodgkin lymphomas. Such compositions may further be used
for
immunotherapy of virus-associated malignancies (i.e., malignancies in which
viral
infection is detectable). Viruses that may be associated with malignancies
include, but
are not limited to, EBV, adenovirus and cytomegalovirus. EBV-associated
malignancies include, for example, lymphomas and nasopharynxcarcinoma. Certain
20 EBV-associated malignancies are post-transplant lymphomas (e.g., following
transplant
of an organ such as liver, heart, kidney or bone marrow) and lymphomas in
immunocompromised patients (such as AIDS patients). In addition, compositions
described herein may be used for therapy of diseases associated with an
autoimmune
response against hematopoetic precursor cells, such as leukopenia and
pancytopenia
25 (e.g., severe aplastic anemia). In particular, such therapies may
effectively treat or
prevent such diseases caused by immunity to Her2/neu (i.e., the presence of
antibodies
to Her2/neu that induce apoptosis in hematopoetic precursor cells).
Immunotherapy may be performed using any of a variety of techniques,
in which compounds or cells provided herein function to remove Her2/neu-
expressing
30 cells from a patient. Such removal may take place as a result of enhancing
or inducing
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an immune response in a patient specific for Her2/neu or a cell expressing
Her2/neu.
Alternatively, Her2/neu-expressing cells may be removed ex vivo (e.g., by
treatment of
autologous bone marrow, peripheral blood or a fraction of bone marrow or
peripheral
blood). Fractions of bone marrow or peripheral blood may be obtained using any
standard technique in the art.
Within such methods, pharmaceutical compositions and vaccines are
typically administered to a patient. As used herein, a "patient" refers to any
warm-
blooded animal, preferably a human. A patient may or may not be afflicted with
a
hematological malignancy or virus-associated malignancy. Accordingly, the
above
pharmaceutical compositions and vaccines may be used to prevent the
development of a
malignancy or to treat a patient afflicted with a malignancy. A hematological
malignancy or virus-associated malignancy may be diagnosed using criteria
generally
accepted in the art. Pharmaceutical compositions and vaccines may be
administered
either prior to or following surgical removal of primary tumors and/or
treatment such as
administration of radiotherapy or conventional chemotherapeutic drugs, or bone
marrow
transplantation (autologous, allogeneic or syngeneic).
Within certain embodiments, immunotherapy may be active
immunotherapy, in which treatment relies on the in vivo stimulation of the
endogenous
host immune system to react against tumors with the administration of immune
response-modifying agents (such as polypeptides and polynucleotides as
provided
herein).
Within other embodiments, immunotherapy may be passive
immunotherapy, in which treatment involves the delivery of agents with
established
tumor-immune reactivity (such as effector cells or antibodies) that can
directly or
indirectly mediate antitumor effects and does not necessarily depend on an
intact host
immune system. Examples of effector cells include T cells as discussed above,
T
lymphocytes (such as CD8+ cytotoxic T lymphocytes and CD4+ T-helper tumor-
infiltrating lymphocytes), killer cells (such as Natural Killer cells and
lymphokine-
activated killer cells), B cells and antigen-presenting cells (such as
dendritic cells and
macrophages) expressing a polypeptide provided herein. T cell receptors and
antibody
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32
receptors specific for the polypeptides recited herein may be cloned,
expressed and
transferred into other vectors or effector cells for adoptive immunotherapy.
The
polypeptides provided herein may also be used to generate antibodies or anti-
idiotypic
antibodies (as described above and in U.S. Patent No. 4,918,164) for passive
immunotherapy.
Effector cells may generally be obtained in sufficient quantities for
adoptive immunotherapy by growth in vitro, as described herein. Culture
conditions for
expanding single antigen-specific effector cells to several billion in number
with
retention of antigen recognition in vivo are well known in the art. Such in
vitro culture
conditions typically use intermittent stimulation with antigen, often in the
presence of
cytokines (such as IL-2) and non-dividing feeder cells. As noted above,
immunoreactive polypeptides as provided herein may be used to rapidly expand
antigen-specific T cell cultures in order to generate a sufficient number of
cells for
immunotherapy. In particular, antigen-presenting cells, such as dendritic,
macrophage
or B cells, may be pulsed with immunoreactive polypeptides or transfected with
one or
more polynucleotides using standard techniques well known in the art. For
example,
antigen-presenting cells can be transfected with a polynucleotide having a
promoter
appropriate for increasing expression in a recombinant virus or other
expression system.
Cultured effector cells for use in therapy must be able to grow and distribute
widely,
and to survive long term in vivo. Studies have shown that cultured effector
cells can be
induced to grow in vivo and to survive long term in substantial numbers by~
repeated
stimulation with antigen supplemented with IL-2 (see, for example, Cheever et
al.,
Immunological Reviews 157:177, 1997).
Alternatively, a vector expressing a polypeptide recited herein may be
introduced into antigen presenting cells taken from a patient and clonally
propagated ex
vivo for transplant back into the same patient. Transfected cells may be
reintroduced
into the patient using any means known in the art, preferably in sterile form
by
intravenous, intracavitary, intraperitoneal or intratumor administration.
The compositions provided herein may be used alone or in combination
with conventional therapeutic regimens such as surgery, irradiation,
chemotherapy
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and/or bone marrow transplantation (autologous, syngeneic, allogeneic or
unrelated).
As discussed in greater detail below, binding agents and T cells as provided
herein may
be used for purging of autologous stem cells. Such purging may be beneficial
prior to,
for example, bone marrow transplantation or transfusion of blood or components
thereof. Binding agents, T cells, antigen presenting cells (APC) and
compositions
provided herein may further be used for expanding and stimulating (or priming)
autologous, allogeneic, syngeneic or unrelated Her2/neu-specific T-cells in
vitro and/or
in vivo. Such Her2/neu-specific T cells may be used, for example, within donor
lymphocyte infusions.
Routes and frequency of administration of the therapeutic compositions
described herein, as well as dosage, will vary from individual to individual,
and may be
readily established using standard techniques. In general, the pharmaceutical
compositions and vaccines may be administered by injection (e.g.,
intracutaneous,
intramuscular, intravenous or subcutaneous), intranasally (e.g., by
aspiration) or orally.
Preferably, between 1 and 10 doses may be administered over a 52 week period.
Preferably, 6 doses are administered, at intervals of 1 month, and booster
vaccinations
may be given periodically thereafter. Alternate protocols may be appropriate
for
individual patients. A suitable dose is an amount of a compound that, when
administered as described above, is capable of promoting an anti-tumor immune
response, and is at least 10-50% above the basal (i.e., untreated) level. Such
response
can be monitored by measuring the anti-tumor antibodies in a patient or by
vaccine-
dependent generation of cytolytic effector cells capable of killing the
patient's tumor
cells in vitro. Such vaccines should also be capable of causing an immune
response that
leads to an improved clinical outcome (e.g., more frequent remissions,
complete or
partial or longer disease-free survival) in vaccinated patients as compared to
non-
vaccinated patients. In general, for pharmaceutical compositions and vaccines
comprising one or more polypeptides, the amount of each polypeptide present in
a dose
ranges from about 100 ~g to 5 mg per kg of host. Suitable dose sizes will vary
with the
size of the patient, but will typically range from about 0.1 mL to about 5 mL.
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In general, an appropriate dosage and treatment regimen provides the
active compounds) in an amount sufficient to provide therapeutic and/or
prophylactic
benefit. Such a response can be monitored by establishing an improved clinical
outcome (e.g., more frequent remissions, complete or partial, or longer
disease-free
survival) in treated patients as compared to non-treated patients. Increases
in
preexisting immune responses to Her2/neu generally correlate with an improved
clinical
outcome. Such immune responses may generally be evaluated using standard
proliferation, cytotoxicity or cytokine assays, which may be performed using
samples
obtained from a patient before and after treatment.
Within further aspects, methods for inhibiting the development of a
malignant disease associated with Her2/neu expression involve the
administration of
autologous T cells that have been activated in response to a Her2/neu
polypeptide or
Her2/neu-expressing APC, as described above. Such T cells may be CD4+ and/or
CD8+, and may be proliferated as described above. The T cells may be
administered to
the individual in an amount effective to inhibit the development of a
malignant disease.
Typically, about 1 x 109 to 1 x 101 T cells/MZ are administered intravenously,
intracavitary or in the bed of a resected tumor. It will be evident to those
skilled in the
art that the number of cells and the frequency of administration will be
dependent upon
the response of the patient.
Within certain embodiments, T cells may be stimulated prior to an
autologous bone marrow transplantation. Such stimulation may take place in
vivo or in
vitro. For in vitro stimulation, bone marrow and/or peripheral blood (or a
fraction of
bone marrow or peripheral blood) obtained from a patient may be contacted with
a
Her2/neu polypeptide, a polynucleotide encoding a Her2/neu polypeptide and/or
an
APC that expresses a Her2/neu polypeptide under conditions and for a time
sufficient to
permit the stimulation of T cells as described above. Bone marrow, peripheral
blood
stem cells and/or Her2/neu-specific T cells may then be administered to a
patient using
standard techniques.
Within related embodiments, T cells of a related or unrelated donor may
be stimulated prior to a syngeneic or allogeneic (related or unrelated) bone
marrow
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transplantation. Such stimulation may take place in vivo or in vitro. For in
vitro
stimulation, bone marrow and/or peripheral blood (or a fraction of bone marrow
or
peripheral blood) obtained from a related or unrelated donor may be contacted
with a
Her2/neu polypeptide, Her2/neu polynucleotide and/or APC that expresses a
Her2/neu
5 polypeptide under conditions and for a time sufficient to permit the
stimulation of T
cells as described above. Bone marrow, peripheral blood stem cells and/or
Her2/neu-
specific T cells may then be administered to a patient using standard
techniques.
Within other embodiments, Her2/neu-specific T cells as described herein
may be used to remove cells expressing Her2/neu from autologous bone marrow,
10 peripheral blood or a fraction of bone marrow or peripheral blood (e.g.,
CD34+ enriched
peripheral blood (PB) prior to administration to a patient). Such methods may
be
performed by contacting bone marrow or PB with such T cells under conditions
and for
a time sufficient to permit the reduction of Her2/neu expressing cells to less
than 10%,
preferably less than 5% and more preferably less than 1%, of the total number
of
15 myeloid or lymphatic cells in the bone marrow or peripheral blood. The
extent to which
such cells have been removed may be readily determined by standard methods
such as,
for example, qualitative and quantitative PCR analysis, morphology,
immunohistochemistry and FACS analysis. Bone marrow or PB (or a fraction
thereof)
may then be administered to a patient using standard techniques.
DIAGNOSTIC METHODS
In general, a hematological or virus-associated malignancy may be
detected in a patient based on the presence of Her2/neu protein and/or
polynucleotide in
a biological sample (such as blood, sera, urine and/or tumor biopsies)
obtained from the
patient. In other words, Her2/neu may be used as a marker to indicate the
presence or
absence of such a malignancy. The binding agents provided herein generally
permit
detection of the level of antigen that binds to the agent in the biological
sample.
Polynucleotide primers and probes may be used to detect the level of mRNA
encoding
Her2/neu, which is also indicative of the presence or absence of a
hematological or
virus-associated malignancy. In general, Her2/neu sequence should be present
at a level
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that is at least three fold higher in a sample obtained from a patient
afflicted with a
hematological or virus-associated malignancy than in the sample obtained from
an
individual not so afflicted.
There are a variety of assay formats known to those of ordinary skill in
the art for using a binding agent to detect polypeptide markers in a sample.
See, e.g.,
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory,
1988. In general, the presence or absence of a hematological malignancy in a
patient
may be determined by (a) contacting a biological sample obtained from a
patient with a
binding agent; (b) detecting in the sample a level of polypeptide that binds
to the
binding agent; and (c) comparing the level of polypeptide with a predetermined
cut-off
value.
In a preferred embodiment, the assay involves the use of binding agent
immobilized on a solid support to bind to and remove the polypeptide from the
remainder of the sample. The bound polypeptide may then be detected using a
detection reagent that,contains a reporter group and specifically binds to the
binding
agent/polypeptide complex. Such detection, reagents may comprise, for example,
a
binding agent that specifically binds to the polypeptide or an antibody or
other agent
that specifically binds to the binding agent, such as an anti-immunoglobulin,
protein G,
protein A or a lectin. Alternatively, a competitive assay may be utilized, in
which a
polypeptide is labeled with a reporter group and allowed to bind to the
immobilized
binding agent after incubation of the binding agent with the sample. The
extent to
which components of the sample inhibit the binding of the labeled polypeptide
to the
binding agent is indicative of the reactivity of the sample with the
immobilized binding
agent. Suitable polypeptides for use within such assays include full length
Her2/neu
and portions thereof to which the binding agent binds, as described above.
The solid support may be any material known to those of ordinary skill
in the art to which the Her2/neu polypeptide may be attached. For example, the
solid
support may be a test well in a microtiter plate or a nitrocellulose or other
suitable
membrane. Alternatively, the support may be a bead or disc, such as glass,
fiberglass,
latex or a plastic material such as polystyrene or polyvinylchloride. The
support may
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also be a magnetic particle or a fiber optic sensor, such as those disclosed,
for example,
in U.S. Patent No. 5,359,681. The binding agent may be immobilized on the
solid
support using a variety of techniques known to those of skill in the art,
which are amply
described in the patent and scientific literature. In the context of the
present invention,
the term "immobilization" refers to both noncovalent association, such as
adsorption,
and covalent attachment (which may be a direct linkage between the agent and
functional groups on the support or may be a linkage by way of a cross-linking
agent).
Immobilization by adsorption to a well in a microtiter plate or to a membrane
is
preferred. In such cases, adsorption may be achieved by contacting the binding
agent,
in a suitable buffer, with the solid support for a suitable amount of time.
The contact
time varies with temperature, but is typically between about 1 hour and about
1 day. In
general, contacting a well of a plastic microtiter plate (such as polystyrene
or
polyvinylchloride) with an amount of binding agent ranging from about 10 ng to
about
10 fig, and preferably about 100 ng to about 1 pg, is sufficient to immobilize
an
adequate amount of binding agent.
Covalent attachment of binding agent to a solid support may generally be
achieved by first reacting the support with a bifunctional reagent that will
react with
both the support and a functional group, such as a hydroxyl or amino group, on
the
binding agent. For example, the binding agent may be covalently attached to
supports
having an appropriate polymer coating using benzoquinone or by condensation of
an
aldehyde group on the support with an amine and an active hydrogen on the
binding
partner (.see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at
A12-A13).
In certain embodiments, the assay is a two-antibody sandwich assay.
This assay may be performed by first contacting an antibody that has been
immobilized
on a solid support, commonly the well of a microtiter plate, with the sample,
such that
polypeptides within the sample are allowed to bind to the immobilized
antibody.
Unbound sample is then removed from the immobilized polypeptide-antibody
complexes and a detection reagent (preferably a second antibody capable of
binding to a
different site on the polypeptide) containing a reporter group is added. The
amount of
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detection reagent that remains bound to the solid support is then determined
using a
method appropriate for the specific reporter group.
More specifically, once the antibody is immobilized on the support as
described above, the remaining protein binding sites on the support are
typically
blocked. Any suitable blocking agent known to those of ordinary skill in the
art, such
as bovine serum albumin or Tween 20TM (Sigma Chemical Co., St. Louis, MO). The
immobilized antibody is then incubated with the sample, and polypeptide is
allowed to
bind to the antibody. The sample may be diluted with a suitable diluent, such
as
phosphate-buffered saline (PBS) prior to incubation. In general, an
appropriate contact
time (i.e., incubation time) is a period of time that is sufficient to detect
the presence of
polypeptide within a sample obtained from an individual with a hematological
malignancy. Preferably, the contact time is sufficient to achieve a level of
binding that
is at least about 95% of that achieved at equilibrium between bound and
unbound
polypeptide. Those of ordinary skill in the art will recognize that the time
necessary to
achieve equilibrium may be readily determined by assaying the level of binding
that
occurs over a period of time. At room temperature, an incubation time of about
30
minutes is generally sufficient.
Unbound sample may then be removed by washing the solid support
with an appropriate buffer, such as PBS containing 0.1% Tween 20TM. The second
antibody, which contains a reporter group, may then be added to the solid
support.
Preferred reporter groups include those groups recited above.
The detection reagent is then incubated with the immobilized antibody-
polypeptide complex for an amount of time sufficient to detect the bound
polypeptide.
An appropriate amount of time may generally be determined by assaying the
level of
binding that occurs over a period of time. Unbound detection reagent is then
removed
and bound detection reagent is detected using the reporter group. The method
employed for detecting the reporter group depends upon the nature of the
reporter
group. For radioactive groups, scintillation counting or autoradiographic
methods are
generally appropriate. Spectroscopic methods may be used to detect dyes,
luminescent
groups and fluorescent groups. Biotin may be detected using avidin, coupled to
a
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39
different reporter group (commonly a radioactive or fluorescent group or an
enzyme).
Enzyme reporter groups may generally be detected by the addition of substrate
(generally for a specific period of time), followed by spectroscopic or other
analysis of
the reaction products.
To determine the presence or absence of a hematological malignancy, the
signal detected from the reporter group that remains bound to the solid
support is
generally compared to a signal that corresponds to a predetermined cut-off
value. In
one preferred embodiment, the cut-off value for the detection of a
hematological or
virus-associated malignancy is the average mean signal obtained when the
immobilized
antibody is incubated with samples from patients without the malignancy. In
general, a
sample generating a signal that is three standard deviations above the
predetermined
cut-off value is considered positive for the malignancy. In an alternate
preferred
embodiment, the cut-off value is determined using a Receiver Operator Curve,
according to the method of Sackett et al., Clinical Epidemiology: A Basic
Science for
Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this
embodiment,
the cut-off value may be determined from a plot of pairs of true positive
rates (i.e.,
sensitivity) and false positive rates (100%-specificity) that correspond to
each possible
cut-off value for the diagnostic test result. The cut-off value on the plot
that is the
closest to the upper left-hand corner (i.e., the value that encloses the
largest area) is the
most accurate cut-off value, and a sample generating a signal that is higher
than the cut-
off value determined by this method may be considered positive. Alternatively;
the cut
off value may be shifted to the left along the plot, to minimize the false
positive rate, or
to the right, to minimize the false negative rate. In general, a sample
generating a signal
that is higher than the cut-off value determined by this method is considered
positive for
a malignancy.
In a related embodiment, the assay is performed in a flow-through or
strip test format, wherein the binding agent is immobilized on a membrane,
such as
nitrocellulose. In the flow-through test, polypeptides within the sample bind
to the
immobilized binding agent as the sample passes through the membrane. A second,
labeled binding agent then binds to the binding agent-polypeptide complex as a
solution
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containing the second binding agent flows through the membrane. The detection
of
bound second binding agent may then be performed as described above. In the
strip test
format, one end of the membrane to which binding agent is bound is immersed in
a
solution containing the sample. The sample migrates along the membrane through
a
5 region containing second binding agent and to the area of immobilized
binding agent.
Concentration of second binding agent at the area of immobilized antibody
indicates the
presence of a hematological or virus-associated malignancy. Typically, the
concentration of second binding agent at that site generates a pattern, such
as a line, that
can be read visually. The absence of such a pattern indicates a negative
result. In
10 general, the amount of binding agent immobilized on the membrane is
selected to
generate a visually discernible pattern when the biological sample contains a
level of
polypeptide that would be sufficient to generate a positive signal in the two-
antibody
sandwich assay, in the format discussed above. Preferred binding agents for
use in such
assays are antibodies and antigen-binding fragments thereof. Preferably, the
amount of
15 antibody immobilized on the membrane ranges from about 25 ng to about 1 pg,
and
more preferably from about 50 ng to about 500 ng. Such tests can typically be
performed with a very small amount of biological sample.
Of course, numerous other assay protocols exist that are suitable for use
with the Her2/neu sequences or binding agents of the present invention. The
above
20 descriptions are intended to be exemplary only. For example, it will be
apparent to
those of ordinary skill in the art that the above protocols may be readily
modified to use
Her2/neu polypeptides to detect antibodies that bind to such polypeptides in a
biological
sample. The detection of Her2/neu-specific antibodies may correlate with the
presence
of a hematological or virus-associated malignancy.
25 A malignancy may also, or alternatively, be detected based on the
presence of T cells that specifically react with Her2/neu in a biological
sample. Within
certain methods, a biological sample comprising CD4+ and/or CD8+ T cells
isolated
from a patient is incubated with a Her2/neu polypeptide, a polynucleotide
encoding
such a polypeptide and/or an APC that expresses such a polypeptide, and the
presence
30 or absence of specific activation of the T cells is detected. Suitable
biological samples
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include, but are not limited to, isolated T cells. For example, T cells may be
isolated
from a patient by routine techniques (such as by Ficoll/Hypaque density
gradient
centrifugation of peripheral blood lymphocytes). T cells may be incubated in
vitro for
2-9 days (typically 4 days) at 37°C with Mtb-81 or Mtb-67.2 polypeptide
(e.g., 5 - 25
~g/ml). It may be desirable to incubate another aliquot of a T cell sample in
the
absence of Her2/neu polypeptide to serve as a control. For CD4+ T cells,
activation is
preferably detected by evaluating proliferation of the T cells. For CD8+ T
cells,
activation is preferably detected by evaluating cytolytic activity. A level of
proliferation that is at least two fold greater and/or a level of cytolytic
activity that is at
least 20% greater than in disease-free patients indicates the presence of a
hematological
or virus-associated malignancy in the patient.
As noted above, a hematological or virus-associated malignancy may
also, or alternatively, be detected based on the level of mRNA encoding
Her2/neu in a
biological sample. For example, at least two oligonucleotide primers may be
employed
in a polymerase chain reaction (PCR) based assay to amplify a portion of
Her2/neu
cDNA derived from a biological sample, wherein at least one of the
oligonucleotide
primers is specific for (i.e., hybridizes to) a polynucleotide encoding the
Her2/neu
protein. The amplified cDNA is then separated and detected using techniques
well
known in the art, such as gel electrophoresis. Similarly, oligonucleotide
probes that
specifically hybridize to a polynucleotide encoding Her2/neu may be used in a
hybridization assay to detect the presence of polynucleotide encoding Her2/neu
in a
biological sample.
To permit hybridization under assay conditions, oligonucleotide primers
and probes should comprise an oligonucleotide sequence that has at least about
60%,
preferably at least about 75% and more preferably at least about 90%, identity
to a
portion of a polynucleotide encoding Her2/neu that is at least 10 nucleotides,
and
preferably at least 20 nucleotides, in length. Preferably, oligonucleotide
primers and/or
probes hybridize to a polynucleotide encoding a polypeptide described herein
under
moderately stringent conditions, as defined above. Oligonucleotide primers
and/or
probes which may be usefully employed in the diagnostic methods described
herein
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preferably are at least 10-40 nucleotides in length. Techniques for both PCR
based
assays and hybridization assays are well known in the art (see, for example,
Mullis et
al., Cold Spring Harbor Symp. Quant. Biol., 51:263, 1987; Erlich ed., PCR
Technology,
Stockton Press, NY, 1989).
One preferred assay employs RT-PCR, in which PCR is applied in
conjunction with reverse transcription. Typically, RNA is extracted from a
biological
sample such as a biopsy tissue and is reverse transcribed to produce cDNA
molecules.
PCR amplification using at least one specific primer generates a cDNA
molecule, which
may be separated and visualized using, for example, gel electrophoresis.
Amplification
may be performed on biological samples taken from a test patient and from an
individual who is not afflicted with a hematological or virus-associated
malignancy.
The amplification reaction may be performed on several dilutions of cDNA
spanning
two orders of magnitude. A two-fold or greater increase in expression in
several
dilutions of the test patient sample as compared to the same dilutions of the
sample
from a normal individual is typically considered positive.
In another embodiment, Her2/neu may be used as a marker for
monitoring the progression or therapy of a hematological or virus-associated
malignancy. In this embodiment, assays as described above for the diagnosis of
a
hematological or virus-associated malignancy may be performed over time, and
the
change in the level of reactive polypeptide(s) evaluated. For example, the
assays may
be performed every 24-72 hours for a period of 6 months to 1 year, and
thereafter
performed as needed. In general, a malignancy is progressing in those patients
in whom
the level of polypeptide detected by the binding agent increases over time. In
contrast,
the malignancy is not progressing when the level of reactive polypeptide
either remains
constant or decreases with time.
Certain in vivo diagnostic assays may be performed directly on a tumor.
One such assay involves contacting tumor cells with a binding agent. The bound
binding agent may then be detected directly or indirectly via a reporter
group. Such
binding agents may also be used in histological applications. Alternatively,
polynucleotide probes may be used within such applications.
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As noted above, to improve sensitivity, multiple markers may be assayed
within a given sample. It will be apparent that binding agents specific for
different
proteins provided herein may be combined within a single assay. Further,
multiple
primers or probes may be used concurrently. The selection of markers may be
based on
routine experiments to determine combinations that results in optimal
sensitivity.
Further diagnostic applications include the detection of extramedullary
disease (e.g., cerebral infiltration of blasts in leukemias). Within such
methods, a
binding agent may be coupled to a tracer substance, and the diagnosis is
performed in
vivo using well known techniques. Coupled binding agent may be administered as
described above, and extramedullary disease may be detected based on assaying
the
presence of tracer substance. Alternatively, a tracer substance may be
associated with a
T cell specific for Her2/neu, permitting detection of extramedullary disease
based on
assays to detect the location of the tracer substance.
1 S DIAGNOSTIC KITS
The present invention further provides kits for use within any of the
above diagnostic methods. Such kits typically comprise two or more components
necessary for performing a diagnostic assay. Components may be compounds,
reagents, containers and/or equipment. For example, one container within a kit
may
contain a monoclonal antibody or fragment thereof that specifically binds to
Her2/neu.
Such antibodies or fragments may be provided attached to a support material,
as
described above. One or more additional containers may enclose elements, such
as
reagents or buffers, to be used in the assay. Such kits may also, or
alternatively, contain
a detection reagent as described above that contains a reporter group suitable
for direct
or indirect detection of antibody binding.
Alternatively, a kit may be designed to detect the level of mRNA
encoding Her2/neu in a biological sample. Such kits generally comprise at
least one
oligonucleotide probe or primer, as described above, that hybridizes to a
polynucleotide
encoding Her2/neu. Such an oligonucleotide may be used, for example, within a
PCR
or hybridization assay. Additional components that may be present within such
kits
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include a second oligonucleotide and/or a diagnostic reagent or container to
facilitate
the detection of a polynucleotide encoding Her2/neu.
The following Examples are offered by way of illustration and not by
way of limitation.
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EXAMPLES
Example 1
Her2/neu Expression in Leukemia Patients
5 This Example illustrates the use of real time PCR analysis to identify
Her2/neu as a marker for hematological malignancies.
A leukemia cDNA panel was generated from mononuclear cells isolated
from bone marrow aspirates and peripheral blood samples using Lymphoprep
(Nycomed, Oslo, Norway). RNA was extracted according to standard protocols. To
10 assess the purity of mRNA, all RNA samples were analyzed on denaturing
formamide
agarose gels and control amplification of beta-actin cDNA was performed. 2 p.g
of
RNA was used for reverse transcription according to~ standard protocols.
This panel was analyzed using real time PCR to compare the relative
level of Her2/neu mRNA in bone marrow and peripheral blood of leukemia
patients
15 (AML and CML) with normal peripheral blood and bone marrow. The real time
PCR
analysis showed overexpression of Her2/neu mRNA in 33% of leukemia patients,
relative to normal peripheral blood and bone marrow. Results are presented in
Table I.
Table I
20 Real Time PCR Results
Her2neu
Tissue Type Replicate 1 Replicate 2 Mean
Human Cell Line 1056.500 1051.900 1054.200
Human Cell Line 88.225 22.914 55.570
Human Cell Line 4466.600 2070.300 3268.450
CML 300.300 245.740 273.020
CML 42.688 123.070 82.879
AML 10.948 12.681 11.815
AML 84.732 105.420 95.076
AML 78.452 214.420 146.436
AML 93.151 25.111 59.131
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AML 72.619 103.450 88.035
AML 3 3.164 12.790 22.977
AML 652.650 221.860 435.255
AML 7530.000 7065.400 7297.700
AML 307.210 1739.300 1023.255
AML 319.710 462.850 391.280
AML 77.453 118.680 98.067
AML 99.729 49.020 74.375
AML 145.360 81.941 113.651
Bone Marrow Normal145.740 118.990 132.365
Bone Marrow Normal107.110 267.250 187.180
Bone Marrow Normal384.270 382.110 383.190
Whole Blood 1 0 0 0
Whole Blood 2 0 0 0
Whole Blood 3 0 0 0
Whole Blood 4 0 0 0
Whole Blood 5 0 0 0
Whole Blood 6 0 0 0
Whole Blood 7 0 0 0
Whole Blood 8 0 0 0
Whole Blood 9 0 0 0
Whole Blood 10 0 0 0
Whole Blood 11 0 0 0
Whole Blood 12 0 0 0
Whole Blood 13 0 0 0
Whole Blood 14 0 0 0
Whole Blood 15 0 0 0
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Example 2
Her2/neu Immune Responses in Leukemia Patients
This Example illustrates the detection of preexisting antibody and T cell
responses in leukemia patients, and the examination of Her2/neu expression in
leukemic
cells using FACS analysis.
For Her2/neu staining, cells were stained in buffer consisting of 10%
FCS (fetal calf serum) in HBSS (Hank's balanced salt solution) containing 0.1%
NaN3.
Cells were incubated for 20 minutes on ice with 10~g/ml biotin-conjugated
Herceptin
(Genentech) or control as the primary step, followed by PE-streptavidin (1:000
dilution,
Fisher) for detection. Cells were then washed twice between staining steps
with
staining buffer and resuspended in PBS prior to analysis. Cells were analyzed
by flow
cytometry using a Becton Dickinson FACSCalibur instrument using standard
techniques. Ten thousand events were collected for analysis.
Results of FACS staining for Her2/neu are presented in Figures lA-1D.
Figures 1A shows FACS staining of the Her2/neu overexpressing breast cancer
cell line
SKBR3. Also shown are staining of human lymphoma cells (Figure 1B), human AML
cells (Figure 1 C) and human CLL cells (Figure 1 D) using a control antibody
(negative
control) and a Her2/neu ECD specific antibody. FACS analyzes showed Her2/neu
overexpression in the positive control SKBR3, as well as the clinical samples
of a
lymphoma, AML and CLL patient.
The human leukemia cell line K562 (American Type Culture Collection)
was also examined for Her2/neu expression using FACS analysis. FACS staining
using
a Her2/neu specific antibody showed a heterogeneous positive staining of this
cell line.
Antibody responses to Her2/neu were determined by Western blot
analysis using recombinant Her2/neu protein (extracellular domain, ECD).
Antibodies
used were WT C-19 and WT 180 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Western blot analysis was performed according to established protocols. As the
primary antibody, sera from patients with acute myeloid leukemia as well as
sera form
healthy normal individuals were used, in a 1:500 dilution. A donkey-polyclonal
Antihuman-IgG peroxidase-conjugated second antibody (Jackson ImmunoResearch
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Lab, Inc.) was used in a 1:10,000 dilution. The blots were then developed by
using a
chemiluminescent reaction (Amersham ECL) after which they were exposed to
Kodak
X-OMATTM AR film (Eastman Kodak Company, NY). The film was developed and
examined.
SCID mice were inoculated with EBV-infected B-cells (see Lacerda et
al., J. Exp. Med. 183:1214-1228, 1996). Lymphomas were found to be induced in
all
animals (a good model for lymphomas in immunocompromised organisms). The
lymphomas overexpressed Her2/neu. Taken together, these data indicate that EBV-
associated malignancies overexpressing Her2/neu may be treated by Her2/neu-
based
immunotherapy, as described herein.
From the foregoing, it will be evident that although specific
embodiments of the invention have been described herein for the purpose of
illustrating
the invention, various modifications may be made without deviating from the
spirit and
scope of the invention. Accordingly, the present invention is not limited
except as by
1 S the appended claims.
