Note: Descriptions are shown in the official language in which they were submitted.
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MUTATIONS IN ERBB2 ASSOCIATED WITH CANCEROUS PHENOTYPES
Field of the Invention
The present invention relates to cancer-specific mutants of the ErbB2/Her2
gene (neu)
and uses thereof in the detection of abnormal cells and cancer. Moreover, the
invention
describes methods for the diagnosis of cancer, the detection of cancerous
cells in subjects
and the development of therapeutic agents for the treatment of cancer.
Introduction
Cancer can develop in any tissue of any organ at any age. Most cancers
detected at an
early stage are potentially curable; thus, the ability to screen patients for
early signs of
cancer, and thus allowing for early intervention, is highly desirable (See,
for instance, the
Merck Manual of Diagnosis and Therapy (1992)16th ed., Merck & Co).
Moreover, different cancers respond differently to therapy designed to treat
them. Since
all cancers arise from mutations in genes involved in cell proliferation,
differentiation and
death, anticancer therapy has been designed to target the products of these
genes to
potentiate or (usually) inhibit their activity. Modulators of tumourigenic
gene products
must be specific if they are to be therapeutically useful, so it is important
to understand
which gene product must be targeted in a particular cancer in order to
administer the
correct therapy.
Cancerous cells display unregulated growth, lack of differentiation, and
ability to invade
local tissues and metastasise. Thus cancer cells are unlike normal cells, and
are
potentially identifiable by not only their phenotypic traits, but also by
their biochemical
and molecular biological characteristics. Such characteristics are in turn
dictated by
changes in cancerous cells which occur at the genetic level in a subset of
cellular genes
known as oncogenes, which directly or indirectly control cell growth and
differentiation.
The ErbB2, Her2 and neu gene products were originally identified as separate
oncogenes
and subsequently shown to be identical. ErbB2/Her2/neu (hereafter: ErbB2) is a
protein
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tyrosine kinase closely related to epidermal growth factor (EGFR; also known
as
ErbB1/Herl), Her3/ErbB3 and Her4/ErbB4.
Initial observations of ErbB2 involvement in cancer indicated that
overexpression of
ErbB2 was involved in many human cancers and is associated with a poor
prognosis. For
example, Semba et al. Proc. Nat. Acad. Sci. 82: 6497-6501 (1985) observed
about 30-
fold amplification of ErbB2 in a human adenocarcinoma of the salivary gland;
Fukushige
et al., Molec. Cell. Biol. 6: 955-958 (1986) observed amplification and
elevated
expression of the ErbB2 gene in a gastric cancer cell line; Di Fiore et al.
Science 237:
178-182 (1987) demonstrated that overexpression alone can convert the gene for
a nonnal
growth factor receptor, namely, ErbB2, into an oncogene; Van de Vijver et al.
New Eng.
J. Med. 319: 1239-1245 (1988) found a correlation between overexpression of
NEU
protein and ductal carcinoma; and Slamon et al. Science 244: 707-712 (1989)
described
the role of HER2/NEU in breast and ovarian cancer, which together account for
one-third
of all cancers in women and approximately one-quarter of cancer-related deaths
in
females.
Overexpression of ErbB2 confers moreover Taxol resistance in breast cancers.
Yu et al.
Molec. Cell 2: 581-591 (1998) found that overexpression of ErbB2 inhibits
Taxol-induced
apoptosis. Taxol activates CDC2 kinase in MDA-MB-435 breast cancer cells,
leading to
cell cycle arrest at the G2/M phase and, subsequently, apoptosis. It appears
that ErbB2
can confer resistance to taxol-induced apoptosis by directly phosphorylating
CDC2.
The ErbB2 gene is amplified and ErbB2 is overexpressed in 25 to 30% of breast
cancers,
increasing the aggressiveness of the tumour. Slamon et al., New Eng. J. Med.
344: 783-
792 (2001), found that herceptin, a monoclonal antibody specific for the ErbB2
gene
product, increased the clinical benefit of first-line chemotherapy in
metastatic breast
cancer that overexpresses ErbB2.
More recent work confirms that overexpression of ErbB2 is correlated with
cancer. For
instance, Bhattacharya et al., (2003) BBRC 307:267-273 confirm that
"overexpression of
ErbB2 is frequent in breast cancer and has been linked to a poor prognosis".
The same is
true in lung cancer, where for example Hisch and Langer, (2004) Seminars in
Oncology
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31, Suppl 1:75-82 confirm that ErbB2 is overexpressed in 16% to 57% of
patients with
NSCLC (non-small cell lung cancer).
Recently, mutations in EGFR have been identified which show correlation
between the
effectiveness of anti-EGFR anticancer drugs and clinical outcome, which had
previously
been elusive (Paez et al., (2004) Science 304:1497-1500; Lynch et al., (2004)
New Engl.
J. Med. 350:2129-2139; reviewed in Stratton and Futreal, (2004) Nature 430:30.
From these studies, it appears that most cancers which respond to the EGFR
inhibitor
gefitinib have mutations in EGFR, which was not previously assumed to be
required for
tumorigenesis. The mutations observed, all in the catalytic kinase domain of
EGFR, were
point substitutions (G719C, L858R, L861Q) or deletions (de1E746-A750, de1L747-
T751insS, de1L747-P753insS). These mutations were observed in 14 out of 15
patients
responding to gefitinib treatment, compared to 0 out of 7 in non-responding
patients.
A known mechanism for the conversion of proto-oncogenes to oncogenes is the
appearance of single mutations in the DNA sequence, known as point mutations,
which
result in a change in the amino acid sequence of the encoded polypeptide. For
example,
ras oncogenes are not present in normal cells, but their proto-oncogene
counterparts are
present in all cells. The wild-type Ras proteins are small GTP-binding
proteins that are
involved in signal transduction. However, many ras oncogenes from viruses and
human
tumours have a point mutation in codon number 12: the codon GGC that normally
encodes a glycine is changed to GTC, which encodes a valine. Multiple
mutations have
been documented at this codon, including at least 5 different substitutions
which are
activating. This single amino acid change prevents the GTPase activity of the
Ras protein,
and renders Ras constitutively activated, since it remains GTP-bound. The
amino acids at
positions 13 and 61 are also frequently changed in ras oncogenes from human
tumours.
Mutations have previously not been shown to be associated with the activity of
ErbB2 in
lung cancer.
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Summary of the Invention
Mutations in ErbB2 genes and gene products are described herein. The mutations
described are identified in human tumours of natural origin. These mutations
are
associated with cancerous phenotypes and can be used as a basis for the
diagnosis of
cancer, cancerous cells or a predisposition to cancer in human subjects, and
for the
prediction of the efficacy of anti-ErbB2 therapy in cancer patients. Unlike
the mutations
described in EGFR, the mutations according to the invention comprise insertion
mutations as well as point mutations (substitutions).
In a first aspect of the invention, therefore, there is provided a naturally
occurring cancer-
associated mutant of a human ErbB2 polypeptide, comprising one or more
mutations.
Mutant ErbB2 is found to be associated with a number of tumours, including
glioma,
gastric tumours, and especially NSCLC adenocarcinomas. Preferably, the mutant
polypeptide which is associated with NSCLC and isolatable from patients
presenting with
NSCLC. The mutation is advantageously in the kinase domain of ErbB2.
Surprisingly, it has been found that the responsiveness of cancers to anti-
ErbB2 therapy is
dependent on the presence of a mutated ErbB2 gene in the patient. It is not
sufficient, as
has been postulated in the prior art, for the expression of ErbB2 to be
overexpressed. It is
believed that tumours which express mutated ErbB2 are ErbB2 dependent, and
that these
tumours are the tumours which respond to therapy which targets the activity of
ErbB2. In
contrast, tumours in which ErbB2 is overexpressed in a wild-type form do not
seem to
respond to anti-ErbB2 therapy.
Thus, the identification, for the first time, of mutants of ErbB2 in
association with disease
and in correlation with a therapeutic approach allows the diagnosis of tumours
as
responsive to anti-ErbB2 therapy or otherwise, and the more successful
selection of
therapy for tumours.
Mutations according to the invention may be insertions, deletions or
substitutions of
amino acids. Preferably, however, the mutation is an insertion, which
duplicates a
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particular string of amino acids, or a point substitution. Point substitutions
may comprise
substitution of one or more, for example 2 adjacent, amino acids, or 3, 4, 5
or 6 adjacent
amino acids.
5 Preferably, the insertion occurs at position 774 or 779 and is
advantageously selected
from the group consisting of ins774(AYVM) and ins779(VGS).
Preferably, the amino acid substitution occurs at any one of positions 755,
914 and 776.
Advantageously, the amino acid substitution is selected from the group
consisting of
L755P, E914K and G776S.
Moreover, there is provided a fragment of an ErbB2 polypeptide according to
the
invention, wherein said fragment comprises the mutation as described above.
In a second aspect, there is provided a nucleic acid encoding a polypeptide
according to
the first aspect of the invention, or a nucleic acid complementary thereto. In
particular,
the invention provides the complement of a nucleic acid selected from the
group
consisting of:
a nucleic acid encoding an ErbB2 polypeptide according to the first aspect of
the
invention; a nucleic acid encoding an ErbB2 polypeptide according to the first
aspect of
the invention, wherein the nucleic acid comprises one or more point mutations;
a nucleic
acid encoding an ErbB2 polypeptide according to the first aspect of the
invention,
wherein the nucleic acid comprises one or more insertions; a nucleic acid
encoding an
ErbB2 polypeptide according to the first aspect of the invention which
comprises one or
more point mutations, wherein the point mutation occurs at one or more of
positions
2263, 2704 and 2326 of ErbB2; a nucleic acid encoding an ErbB2 polypeptide
according
to the first aspect of the invention, which comprises one or more point
mutations, wherein
the point mutation is HetTT2263/4CC, HetG2740A or HetG2326A; a nucleic acid
encoding an ErbB2 polypeptide according to the first aspect of the invention
which
comprises one or more insertions, wherein the insertion occurs at one or more
of positions
2322 or 2335 of ErbB2; and a nucleic acid encoding an ErbB2 polypeptide
according to
the first aspect of the invention, which comprises one or more insertions,
wherein the
insertion is Het2322dupl2nt or Het2335ins9nt.
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In a further embodiment, the invention provides nucleic acid which hybridises
specifically to a nucleic acid selected as described above. Such a nucleic
acid can, for
example, be a primer which directs specific amplification of a nucleic acid as
described
above.
According to a third aspect, there is provided a ligand which binds
selectively to a
polypeptide according to the first aspect of the invention. Preferably the
ligand is an
immunoglobulin, for example an antibody or an antigen-binding fragment
thereof.
The mutations identified herein are somatic mutations, that is they are not
transmitted
through the germ line. Accordingly, in a fourth aspect, there is provided a
method for the
detection of oncogenic mutations, comprising the steps of:
(a) isolating a sample of naturally-occurring cellular material from a human
subject;
(b) examining nucleic acid material from at least part of one or more ErbB2
genes in said cellular material; and
(c) determining whether such nucleic acid material comprises one or more
mutations in a sequence encoding an ErbB2 polypeptide.
Preferably, the method comprises the steps of:
(a) isolating a first sample of cellular material from a naturally-occurring
tissue of
a subject which is suspected to be cancerous, and a second sample of cellular
material
from a non-cancerous tissue of the same subject;
(b) examining nucleic acid material from at least part of one or more ErbB2
genes in both said samples of cellular material; and
(c) determining whether such nucleic acid material comprises one or more
mutations in a sequence encoding an ErbB2 polypeptide; and said mutation being
present
in the naturally-occurring cellular material from the suspected cancerous
tissue but not
present in the cellular material from the non-cancerous tissue.
Advantageously, the mutation is as described above.
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In a fifth aspect, there is provided a method for the detection of oncogenic
mutations,
comprising the steps of
(a) obtaining a sample of cellular material from a subject;
(b) screening said sample with a ligand according to the invention; and
(c) detecting one or more mutant ErbB2 polypeptides in said sample.
Advantageously, the mutant ErbB2 polypeptide is a polypeptide according to the
first
aspect of the invention.
Automated methods, apparata and assays for detection of mutants according to
the
invention are also provided.
Four separate insertion mutations have been identified in clinical samples to
date, as set
forth in Table 1 below. This indicates an overall prevalence of at least 4.2%
(5/120) in
unselected primary NSCLC. The frequency in adenocarcinoma subtype of NSCLC is
at
least 5/51 (9.8%).
To emphasise the relevance of these findings, the frequency of ErbB2 mutations
is more
than twice that recently reported for EGFR mutations in an unselected series
of NSCLC
by Paez et al. (Science, (2004) 304(5676):1497-500).
These findings have immediate therapeutic and diagnostic implications. An
ErbB2
directed therapeutic (trastuzumab/Herceptin(b) has been approved for treatment
of
metastatic breast cancer and is under evaluation for use in NSCLC. The
identification
NSCLC patients with ErbB2 mutations may provide a very significant tool for
patient
stratification in more rational trial designs and diagnostic targeting of
those patients with
what may be the most responsive tumours. Other monoclonal antibodies which
target
ErbB2 are in development, such as Omnitarg (Pertuzumab), which impedes ErbB2
dimerisation. Moreover a selective ErbB2 small molecule inhibitor has been
reported
(Biochem Biophys Res Commun. 2003 Jul 25;307(2):267-73; US Patent Application
Publication 2003/0171386) that may be of interest in patients with ErbB2
mutant
tumours. Likewise inhibitors with equipotency for both EGFR and ErbB2 may be
of
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interest (Cancer Res. 2001 Oct 1;61(19):7196-203 and Bioorg Med Chem Lett.
2003 Feb
24;13(4):637-40).
Accordingly, the invention provides a method for determining whether a patient
is
expected to be responsive to anti-ErbB2 therapy, comprising the steps of:
(a) isolating a sample of naturally-occurring cellular material from a human
subj ect;
(b) examining nucleic acid material from at least part of one or more ErbB2
genes in said cellular material; and
(c) determining whether such nucleic acid material comprises one or more
mutations in a sequence encoding an ErbB2 polypeptide.
The method may also be practised at the polypeptide level, in which case it
advantageously comprises the steps of
(a) obtaining a sample of cellular material from a subject;
(b) screening said sample with a ligand according to the invention; and
(c) detecting one or more mutant ErbB2 polypeptides in said sample.
In a preferred embodiment, the invention provides a method for treating a
patient suffering from
a tumour, comprising the steps of:
(a) determining if the tumour is ErbB2-dependent; and
(b) treating patients having ErbB2 dependent tumours with an inhibitor of
ErbB2 activity.
As provided by the present invention, ErbB2 dependency can be determined by
observing a
mutation in a ErbB2. Advantageously, the mutation is a mutation as set forth
above. Preferred
inhibitors of ErbB2 activity include Herceptin , Omnitarg and small molecule
ErbB2
inhibitors, for example inhibitors as set forth in US Patent Application
Publication
2003/0171386.
Accordingly, the invention further provides a method for determining whether a
patient is
susceptible to therapy with Herceptin or Omnitarg , comprising the steps of:
(a) determining whether the patient is suffering from an ErbB2 dependent
tumour; and
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(b) administering Herceptin and/or Omnitarg to patients suffering from ErbB2
dependent tumours.
Brief Description of the Figures
Figure 1 Shows a partial sequence of ErbB2, indicating the location and nature
of some
of the insertion mutations observed.
Figure 2 is a CLUSTAL W (1.82) sequence alignment of EGFR, ErbB2, KIT and
PDGFRA. Highlighted regions indicate the position and amino acids affected by
mutations.
PDGFRA and EGFR are in-frame deletions, EFGR missense in grey
KIT - both in-frame deletions and insertions
ERBB2 - in-frame insertions plus missense in underlined in purple
The position of the G-loop, AIK motif, Catalytic loop and DFG of the
activation segment
are boxed in yellow for orientation.
Figure 3 shows a series of tests for transforming activity of ErbB2 mutants in
cell-based
assays.
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Detailed description of the Invention
Unless defined otherwise, all technical and scientific terms used herein have
the same
meaning as commonly understood by one of ordinary skill in the art (e.g., in
cell culture,
5 molecular genetics, nucleic acid chemistry, hybridisation techniques and
biochemistry).
Standard techniques are used for molecular, genetic and biochemical methods.
See,
generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed.
(1989) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al.,
Short
Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.; as well
as
10 Guthrie et al., Guide to Yeast Genetics and Molecular Biology, Methods in
Enzymology,
Vol. 194, Academic Press, Inc., (1991), PCR Protocols: A Guide to Methods and
Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.),
McPherson et al.,
PCR Volume 1, Oxford University Press, (1991), Culture of Animal Cells: A
Manual of
Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),
and Gene
Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana
Press
Inc., Clifton, N.J.). These documents are incorporated herein by reference.
Definitions
The present application describes ErbB2 polypeptide mutants. As used herein,
the term
"ErbB2 polypeptide" is used to denote a polypeptide encoded by ErbB2/Her2/neu.
The
term "ErbB2" thus encompasses all known human ErbB2 homologues and variants,
as
well as other polypeptides which show sufficient homology to ErbB2 to be
identified as
ErbB2 homologues. The tem does not include EGFR, Her3 or Her4. Preferably,
ErbB2
is identified as a polypeptide having the sequence shown at NCBI accession no.
NM 004448.1, GI:4758297.
The term "ErbB2" preferably includes polypeptides which are 85%, 90%, 95%,
96%,
97%, 98% or 99% homologous to NM 004448.1. Homology comparisons can be
conducted by eye, or more usually, with the aid of readily available sequence
comparison
programs. These commercially available computer programs can calculate
percentage (%)
homology between two or more sequences.
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Percentage homology can be calculated over contiguous sequences, i.e. one
sequence is
aligned with the other sequence and each amino acid in one sequence directly
compared
with the corresponding amino acid in the other sequence, one residue at a
time. This is called
an "ungapped" alignment. Typically, such ungapped alignments are performed
only over a
relatively short number of residues (for example less than 50 contiguous amino
acids).
Although this is a very simple and consistent method, it fails to take into
consideration that,
for example, in an otherwise identical pair of sequences, one insertion or
deletion will cause
the following amino acid residues to be put out of alignment, thus potentially
resulting in a
large reduction in % homology when a global alignment is performed.
Consequently, most
sequence comparison methods are designed to produce optimal alignments that
take into
consideration possible insertions and deletions without penalising unduly the
overall
homology score. This is achieved by inserting "gaps" in the sequence alignment
to try to
maximise local homology.
However, these more complex methods assign "gap penalties" to each gap that
occurs in the
alignment so that, for the same number of identical amino acids, a sequence
aligmnent with
as few gaps as possible - reflecting higher relatedness between the two
compared sequences
- will achieve a higher score than one with many gaps. "Affine gap costs" are
typically used
that charge a relatively high cost for the existence of a gap and a smaller
penalty for each
subsequent residue in the gap. This is the most commonly used gap scoring
system. High
gap penalties will of course produce optimised alignments with fewer gaps.
Most aligmnent
programs allow the gap penalties to be modified. However, it is preferred to
use the default
values when using such software for sequence comparisons. For example when
using the
GCG Wisconsin Bestfit package (see below) the default gap penalty for amino
acid
sequences is -12 for a gap and -4 for each extension.
Calculation of maximum % homology therefore firstly requires the production of
an optimal
aligninent, taking into consideration gap penalties. A suitable computer
program for can-ying
out such an alignment is the GCG Wisconsin Bestfit package (University of
Wisconsin,
U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of
other
software than can perform sequence comparisons include, but are not limited
to, the
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BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et
czl.,
1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools.
Both
BLAST and FASTA are available for offline and online searching (see Ausubel et
al.,
1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit
program.
Although the final % homology can be measured in terms of identity, the
alignment
process itself is typically not based on an all-or-nothing pair comparison.
Instead, a scaled
similarity score matrix is generally used that assigns scores to each pairwise
comparison
based on chemical similarity or evolutionary distance. An example of such a
matrix
commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite
of
programs. GCG Wisconsin programs generally use either the public default
values or a
custom symbol comparison table if supplied (see user manual for further
details). It is
preferred to use the public default values for the GCG package, or in the case
of other
software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal aligmnent, it is possible to
calculate %
homology, preferably % sequence identity. The software typically does this as
part of the
sequence comparison and generates a numerical result.
A "fragment" of a polypeptide in accordance with the invention is a
polypeptide fragment
which encompasses the mutant amino acid(s) described in accordance with the
invention.
The fragment can be any length up to the full length of ErbB2 polypeptide; it
thus
encompasses ErbB2 polypeptides which have been truncated by a few amino acids,
as
well as shorter fragments. Advantageously, fragments are between about 1250
and about
5 amino acids in length; preferably about 5:';.to about 20 amino acids in
length;
advantageously, between about 10 and about 50 amino acids in length. Fragments
according to the invention are useful, inter crlia, for immunisation of
animals to raise
antibodies. Thus, fragments of polypeptides according to the invention
advantageously
comprise at least one antigenic determinant (epitope) characteristic of mutant
ErbB2 as
described herein. Whether a particular polypeptide fragment retains such
antigenic
properties can readily be determined by routine methods known in the art.
Peptides
composed of as few as six amino acid residues ore often found to evoke an
immune
response.
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A "nucleic acid" of the present invention is a nucleic acid which encodes a
human ErbB2
polypeptide as described above. The term moreover includes those
polynucleotides
capable of hybridising, under stringent hybridisation conditions, to the
naturally occurring
nucleic acids identified above, or the complement thereof.. "Stringent
hybridisation
conditions" refers to an overnight incubation at 42 C in a solution comprising
50%
formamide, 5x SSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium
phosphate
(pH 7.6), 5x Denhardt's solution, 10% dextran sulphate, and 20 pg/ml
denatured, sheared
salmon sperm DNA, followed by washing the filters in 0.1x SSC at about 65 C.
Although nucleic acids, as referred to herein, are generally natural nucleic
acids found in
nature, the term can include within its scope modified, artificial nucleic
acids having
modified backbones or bases, as are known in the art.
A nucleic acid encoding a fragment according to the invention can be the
result of nucleic
acid amplification of a specific region of a ErbB2 gene, incorporating a
mutation in
accordance with the present invention.
An "isolated" polypeptide or nucleic acid, as referred to herein, refers to
material removed
from its original environment (for example, the natural environment in which
it occurs in
nattire), and thus is altered by the hand of man from its natural state. For
example, an
isolated polynucleotide could be part of a vector or a composition of matter,
or could be
contained within a cell, and still be "isolated" because that vector,
composition of matter,
or particular cell is not the original environment of the polynucleotide.
Preferably, the
term "isolated" does not refer to genomic or cDNA libraries, whole cell total
or mRNA
preparations, genomic DNA preparations (including those separated by
electrophoresis
and transferred onto blots), sheared whole cell genomic DNA preparations or
other
compositions where the art demonstrates no distinguishing features of the
polypeptides/nucleic acids of the present invention.
The polypeptides according to the invention comprise one or more mutations.
"Mutations" includes amino acid addition, deletion or substitution;
advantageously, it
refers to amino acid substitutions or insertions in the form of insertions.
Such mutations
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at the polypeptide level are reflected at the nucleic acid level by addition,
deletion or
substitution of one or more nucleotides. Generally, such mutations do not
alter the
reading frame of the nucleic acid. Advantageously, the changes at the nucleic
acid level
are point mutations at one or two adjacent positions, or insertions.
The mutations in ErbB2 identified in the present invention occur naturally,
and have not
been intentionally induced in cells or tissue by the application of
carcinogens or other
tumourigenic factors. Thus, the mutations identified herein accurately reflect
natural
tumorigenesis in human tissues to in vivo. Their detection is thus a far
better basis for
diagnosis than the detection of mutations identified in rodents after
artificial chemical
tumour induction.
The mutations identified herein are somatic mutations.
A"somatic" mutation is a mutation which is not transmitted through the germ
line of an
organism, and occurs in somatic tissues thereof. Advantageously, a somatic
mutation is
one which is determined to be somatic though normal/tumour paired sample
analysis.
All amino acid and nucleotide numbering used herein starts from amino acid +1
of the
ErbB2 polypeptide or the first ATG of the nucleotide sequence encoding it.
"Amplification" reactions are nucleic acid reactions which result in specific
amplification
of target nucleic acids over non-target nucleic acids. The polymerase chain
reaction
(PCR) is a well known amplification reaction.
An "immunoglobulin" is one of a family of polypeptides which retain the
immunoglobulin fold characteristic of immunoglobulin (antibody) molecules,
which
contains two (3 sheets and, usually, a conserved disulphide bond. Members of
the
immunoglobulin superfamily are involved in many aspects of cellular and non-
cellular
interactions in vivo, including widespread roles in the immune system (for
example,
antibodies, T-cell receptor molecules and the like), involvement in cell
adhesion (for
example the ICAM molecules) and intracellular signalling (for example,
receptor
molecules, such as the PDGF receptor). The present invention is preferably
applicable to
antibodies, which are capable of binding to target antigens with high
specificity.
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"Antibodies" can be whole antibodies, or antigen-binding fragments thereof.
For
example, the invention includes fragments such as Fv and Fab, as well as Fab'
and F(ab')2,
and antibody variants such as scFv, single domain antibodies, Dab antibodies
and other
5 antigen-binding antibody-based molecules.
"Cancer" is used herein to refer to neoplastic growth arising from cellular
transformation
to a neoplastic phenotype. Such cellular transformation often involves genetic
mutation;
in the context of the present invention, transformation involves genetic
mutation by
10 alteration of one or more ErbB2 genes as described herein.
Methods for Detection of Nucleic Acids
15 The detection of mutant nucleic acids encoding ErbB2 can be employed, in
the context of
the present invention, to diagnose the presence or predisposition to cellular
transformation
and cancer. Since mutations in ErbB2 genes generally occur at the DNA level,
the
methods of the invention can be based on detection of mutations in genomic
DNA, as
well as transcripts and proteins themselves. It can be desirable to confirm
mutations in
genomic DNA by analysis of transcripts and/or polypeptides, in order to ensure
that the
detected mutation is indeed expressed in the subject.
Mutations in genomic nucleic acid are advantageously detected by techniques
based on
mobility shift in amplified nucleic acid fragments. For instance, Chen et al.,
Anal
Biochem 1996 Jul 15;239(1):61-9, describe the detection of single-base
mutations by a
competitive mobility shift assay. Moreover, assays based on the technique of
Marcelino
et al., BioTechniques 26(6): 1134-1148 (June 1999) are available commercially.
In a preferred example, capillary heteroduplex analysis may be used to detect
the
presence of mutations based on mobility shift of duplex nucleic acids in
capillary systems
as a result of the presence of mismatches.
Generation of nucleic acids for analysis from samples generally requires
nucleic acid
amplification. Many amplification methods rely on an enzymatic chain reaction
(such as
a polymerase chain reaction, a ligase chain reaction, or a self-sustained
sequence
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replication) or from the replication of all or part of the vector into which
it has been
cloned. Preferably, the amplification according to the invention is an
exponential
amplification, as exhibited by for example the polymerase chain reaction.
Many target and signal amplification methods have been described in the
literature, for
example, general reviews of these methods in Landegren, U., et al., Science
242:229-237
(1988) and Lewis, R., Genetic Engineering News 10:1, 54-55 (1990). These
amplification
methods can be used in the methods of our invention, and include polymerase
chain
reaction (PCR), PCR in situ, ligase amplification reaction (LAR), ligase
hybridisation,
Qbeta bacteriophage replicase, transcription-based amplification system (TAS),
genomic
amplification with transcript sequencing (GAWTS), nucleic acid sequence-based
amplification (NASBA) and in situ hybridisation. Primers suitable for use in
various
amplification techniques can be prepared according to methods known in the
art.
Polymerase Chain Reaction (PCR)
PCR is a nucleic acid amplification method described inter alia in U.S. Pat.
Nos.
4,683,195 and 4,683,202. PCR consists of repeated cycles of DNA polymerase
generated
primer extension reactions. The target DNA is heat denatured and two
oligonucleotides,
which bracket the target sequence on opposite strands of the DNA to be
amplified, are
hybridised. These oligonucleotides become primers for use with DNA polymerase.
The
DNA is copied by primer extension to make a second copy of both strands. By
repeating
the cycle of heat denaturation, primer hybridisation and extension, the target
DNA can be
amplified a million fold or more in about two to four hours. PCR is a
molecular biology
tool, which must be used in conjunction with a detection technique to
determine the
results of amplification. An advantage of PCR is that it increases sensitivity
by
amplifying the amount of target DNA by 1 million to 1 billion fold in
approximately 4
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hours. PCR can be used to amplify any known nucleic acid in a diagnostic
context (Mok
et al., (1994), Gynaecologic Oncology, 52: 247-252).
Self-Sustained Sequence Replication (3SR)
Self-sustained sequence replication (3SR) is a variation of TAS, which
involves the
isothermal amplification of a nucleic acid template via sequential rounds of
reverse
transcriptase (RT), polymerase and nuclease activities that are mediated by an
enzyme
cocktail and appropriate oligonucleotide primers (Guatelli et al. (1990) Proc.
Natl. Acad.
Sci. USA 87:1874). Enzymatic degradation of the RNA of the RNA/DNA
heteroduplex is
used instead of heat denaturation. RNase H and all other enzymes are added to
the
reaction and all steps occur at the same temperature and without further
reagent additions.
Following this process, amplifications of 106 to 109 have been achieved in one
hour at 42
oc.
Ligation Amplification (LAR/LAS)
Ligation amplification reaction or ligation amplification system uses DNA
ligase and four
oligonucleotides, two per target strand. This technique is described by Wu, D.
Y. and
Wallace, R. B. (1989) Genomics 4:560. The oligonucleotides hybridise to
adjacent
sequences on the target DNA and are joined by the ligase. The reaction is heat
denatured
and the cycle repeated.
Q(3 Replicase
In this technique, RNA replicase for the bacteriophage Q(3, which replicates
single-
stranded RNA, is used to amplify the target DNA, as described by Lizardi et
al. (1988)
Bio/Technology 6:1197. First, the target DNA is hybridised to a primer
including a T7
promoter and a Q(3 5' sequence region. Using this primer, reverse
transcriptase generates
a cDNA connecting the primer to its 5' end in the process. These two steps are
similar to
the TAS protocol. The resulting heteroduplex is heat denatured. Next, a second
primer
containing a Q(3 3' sequence region is used to initiate a second round of cDNA
synthesis.
This results in a double stranded DNA containing both 5' and 3' ends of the
Q(3
bacteriophage as well as an active T7 RNA polymerase binding site. T7 RNA
polymerase
then transcribes the double-stranded DNA into new RNA, which mimics the Q(3.
After
extensive washing to remove any unhybridised probe, the new RNA is eluted from
the
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target and replicated by Q(3 replicase. The latter reaction creates 107 fold
amplification in
approximately 20 minutes.
Alternative amplification technology can be exploited in the present
invention. For
example, rolling circle amplification (Lizardi et al., (1998) Nat Genet
19:225) is an
amplification technology available commercially (RCATTM) which is driven by
DNA
polymerase and can replicate circular oligonucleotide probes with either
linear or
geometric kinetics under isothermal conditions.
In the presence of two suitably designed primers, a geometric amplification
occurs via
DNA strand displacement and hyperbranching to generate 1012 or more copies of
each
circle in 1 hour.
If a single primer is used, RCAT generates in a few minutes a linear chain of
thousands of
tandemly linked DNA copies of a target covalently linked to that target.
A further technique, strand displacement amplification (SDA; Walker et czl.,
(1992)
PNAS (USA) 80:392) begins with a specifically defined sequence unique to a
specific
target. But unlike other techniques which rely on thermal cycling, SDA is an
isothermal
process that utilises a series of primers, DNA polymerase and a restriction
enzyme to
exponentially amplify the unique nucleic acid sequence.
SDA comprises both a target generation phase and an exponential amplification
phase.
In target generation, double-stranded DNA is heat denatured creating two
single-stranded
copies. A series of specially manufactured primers combine with DNA polymerase
(amplification primers for copying the base sequence and bumper primers for
displacing
the newly created strands) to form altered targets capable of exponential
amplification.
The exponential amplification process begins with altered targets (single-
stranded partial
DNA strands with restricted enzyme recognition sites) from the target
generation phase.
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An amplification primer is bound to each strand at its complementary DNA
sequence.
DNA polymerase then uses the primer to identify a location to extend the
primer from its
3' end, using the altered target as a template for adding individual
nucleotides. The
extended primer thus forms a double-stranded DNA segment containing a complete
restriction enzyme recognition site at each end.
A restriction enzyme is then bound to the double stranded DNA segment at its
recognition
site. The restriction enzyme dissociates from the recognition site after
having cleaved
only one strand of the double-sided segment, forming a nick. DNA polymerase
recognises
the nick and extends the strand from the site, displacing the previously
created strand.
The recognition site is thus repeatedly nicked and restored by the restriction
enzyme and
DNA polymerase with continuous displacement of DNA strands containing the
target
segment.
Each displaced strand is then available to anneal with amplification primers
as above. The
process continues with repeated nicking, extension and displacement of new DNA
strands, resulting in exponential amplification of the original DNA target.
Once the nucleic acid has been amplified, a number of techniques are available
for
detection of single base pair mutations. One such technique is Single Stranded
Confozmational Polymorphism (SSCP). SCCP detection is based on the aberrant
migration of single stranded mutated DNA compared to reference DNA during
electrophoresis. Mutation produces conformational change in single stranded
DNA,
resulting in mobility shift. Fluorescent SCCP uses fluorescent-labelled
primers to aid
detection. Reference and mutant DNA are thus amplified using fluorescent
labelled
primers. The amplified DNA is denatured and snap-cooled to produce single
stranded
DNA molecules, which are examined by non-denaturing gel electrophoresis.
Chemical mismatch cleavage (CMC) is based on the recognition and cleavage of
DNA
mismatched base pairs by a combination of hydroxylamine, osmium tetroxide and
piperidine. Thus, both reference DNA and mutant DNA are amplified with
fluorescent
labelled primers. The amplicons are hybridised and then subjected to cleavage
using
Osmium tetroxide, which binds to an mismatched T base, or Hydroxylamine, which
binds
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to mismatched C base, followed by Piperidine which cleaves at the site of a
modified
base. Cleaved fragments are then detected by electrophoresis.
Techniques based on restriction fragment polymorphisms (RFLPs) can also be
used.
5 Although many single nucleotide polymorphisms (SNPs) do not permit
conventional
RFLP analysis, primer-induced restriction analysis PCR (PIRA-PCR) can be used
to
introduce restriction sites using PCR primers in a SNP-dependent manner.
Primers for
PIRA-PCR which introduce suitable restriction sites can be designed by
computational
analysis, for example as described in Xiaiyi et al., (2001) Bioinformatics
17:838-839.
In an alternative embodiment, the present invention provides for the detection
of gene
expression at the RNA level. Typical assay formats utilising ribonucleic acid
hybridisation include nuclear run-on assays, RT-PCR and RNase protection
assays
(Melton et al., Nuc. Acids Res. 12:7035. Methods for detection which can be
employed
include radioactive labels, enzyme labels, chemiluminescent labels,
fluorescent labels and
other suitable labels.
RT-PCR is used to amplify RNA targets. In this process, the reverse
transcriptase enzyme
is used to convert RNA to complementary DNA (cDNA), which can then be
amplified
using PCR. This method has proven useful for the detection of RNA viruses. Its
application is otherwise as for PCR, described above.
Methods for Detection of Polypeptides
The invention provides a method wherein a protein encoded a mutant ErbB2 gene
is
detected. Proteins can be detected by protein gel assay, antibody binding
assay, or other
detection methods known in the art.
For example, therefore, mutant ErbB2 polypeptides can be detected by
differential
mobility on protein gels, or by other size analysis techniques such as mass
spectrometry,
in which the presence of mutant amino acids can be determined according to
molecular
weight. Peptides derived from mutant ErbB2 polypeptides, in particular, as
susceptible to
differentiation by size analysis.
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Advantageously, the detection means is sequence-specific, such that a
particular point
mutation can accurately be identified in the mutant ErbB2 polypeptide. For
example,
polypeptide or RNA molecules can be developed which specifically recognise
mutant
ErbB2 polypeptides in vivo or in vitro.
For example, RNA aptamers can be produced by SELEX. SELEX is a method for the
in
vitro evolution of nucleic acid molecules with highly specific binding to
target molecules.
It is described, for example, in U.S. patents 5654151, 5503978, 5567588 and
5270163, as
well as PCT publication WO 96/38579, each of which is specifically
incorporated herein
by reference.
The SELEX method involves selection of nucleic acid aptamers, single-stranded
nucleic
acids capable of binding to a desired target, from a library of
oligonucleotides. Starting
from a library of nucleic acids, preferably comprising a segment of randomised
sequence,
the SELEX method includes steps of contacting the library with the target
under
conditions favourable for binding, partitioning unbound nucleic acids from
those nucleic
acids which have bound specifically to target molecules, dissociating the
nucleic
acid-target complexes, ainplifying the nucleic acids dissociated from the
nucleic
acid-target complexes to yield a ligand-enriched library of nucleic acids,
then reiterating
the steps of binding, partitioning, dissociating and amplifying through as
many cycles as
desired to yield highly specific, high affmity nucleic acid ligands to the
target molecule.
SELEX is based on the principle that within a nucleic acid library containing
a large
number of possible sequences and structures there is a wide range of binding
affinities for
a given target. A nucleic acid library comprising, for example a 20 nucleotide
randomised segment can have 420 structural possibilities. Those which have the
higher
affinity constants for the target are considered to be most likely to bind.
The process of
partitioning, dissociation and amplification generates a second nucleic acid
library,
enriched for the higher binding affinity candidates. Additional rounds of
selection
progressively favour the best ligands until the resulting library is
predominantly
composed of only one or a few sequences. These can then be cloned, sequenced
and
individually tested for binding affinity as pure ligands.
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Cycles of selection and amplification are repeated until a desired goal is
achieved. In the
most general case, selection/amplification is continued until no significant
improvement
in binding strength is achieved on repetition of the cycle. The iterative
selection/amplification method is sensitive enough to allow isolation of a
single sequence
variant in a library containing at least 1014 sequences. The method could, in
principle, be
used to sample as many as about 1018 different nucleic acid species. The
nucleic acids of
the library preferably include a randomised sequence portion as well as
conserved
sequences necessary for efficient amplification. Nucleic acid sequence
variants can be
produced in a number of ways including synthesis of randomised nucleic acid
sequences
and size selection from randomly cleaved cellular nucleic acids. The variable
sequence
portion can contain fully or partially random sequence; it can also contain
subportions of
conserved sequence incorporated with randomised sequence. Sequence variation
in test
nucleic acids can be introduced or increased by mutagenesis before or during
the
selection/amplification iterations and by specific modification of cloned
aptamers.
Antibodies
ErbB2 polypeptides or peptides derived therefrom can be used to generate
antibodies for
use in the present invention. The ErbB2 peptides used preferably comprise an
epitope
which is specific for a mutant ErbB2 polypeptide in accordance with the
invention.
Polypeptide fragments which function as epitopes can be produced by any
conventional
means (see, for example, US 4,631,211) In the present invention, antigenic
epitopes
preferably contain a sequence of at least 4, at least 5, at least 6, at least
7, more preferably
at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at
least 14, at least 15,
at least 20, at least 25, at least 30, at least 40, at least 50. and, most
preferably, between
about 15 to about 30 amino acids. Preferred polypeptides comprising
immunogenic or
antigenic epitopes are at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95 or
100 amino acid residues in length.
Antibodies can be generated using antigenic epitopes of ErbB2 polypeptides
according to
the invention by immunising animals, such as rabbits or mice, with either free
or carrier-
coupled peptides, for instance, by intraperitoneal and/or intradermal
injection of
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emulsions containing about 100 g of peptide or carrier protein and Freund's
adjuvant or
any other adjuvant known for stimulating an immune response. Several booster
injections can be needed, for instance, at intervals of about two weeks, to
provide a useful
titre of anti-peptide antibody which can be detected, for example, by ELISA
assay using
free peptide adsorbed to a solid surface. The titre of anti-peptide antibodies
in serum from
an immunised animal can be increased by selection of anti-peptide antibodies,
for
instance, by adsorption to the peptide on a solid support and elution of the
selected
antibodies according to methods well known in the art.
The ErbB2 polypeptides of the present invention, and immunogenic and/or
antigenic
epitope fragments thereof can be fused to other polypeptide sequences. For
example, the
polypeptides of the present invention can be fused with immunoglobulin
domains.
Chimeric proteins consisting of the first two domains of the human CD4-
polypeptide and
various domains of the constant regions of the heavy or light chains of
mammalian
immunoglobulins have been shown to possess advantageous properties in vivo
(see, for
example, EP 0394827; Traunecker et al., (1988) Nature, 331: 84-86). Enhanced
delivery
of an antigen across the epithelial barrier to the immune system has been
demonstrated
for antigens (such as insulin) conjugated to an FeRn binding partner such as
IgG or Fc
fragments (see, for example, WO 96/22024 and WO 99/04813).
Moreover, the polypeptides of the present invention can be fused to marker
sequences,
such as a peptide which facilitates purification of the fused polypeptide. In
preferred
embodiments, the marker amino acid sequence is a hexa-histidine peptide, such
as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA,
91311),
among others, many of which are commercially available. As described in Gentz
et al.,
Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), for instance, hexa-histidine
provides for
convenient purification of the fusion protein. Another peptide tag useful for
purification,
the "HA" tag, corresponds to an epitope derived from the influenza
hemagglutinin protein
(Wilson et al., (1984) Cell 37: 767. Thus, any of these above fusions can be
engineered
using the nucleic acids or the polypeptides of the present invention.
In a preferred embodiment, the invention provides antibodies which
specifically
recognise ErbB2 mutants as described herein.
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Antibodies as described herein are especially indicated for diagnostic
applications.
Accordingly, they can be altered antibodies comprising an effector protein
such as a label.
Especially preferred are labels which allow the imaging of the distribution of
the antibody
in vivo. Such labels can be radioactive labels or radioopaque labels, such as
metal
particles, which are readily visualisable within the body of a patient.
Moreover, they can
be fluorescent labels or other labels which are visualisable on tissue
Recombinant DNA technology can be used to improve the antibodies of the
invention.
Thus, chimeric antibodies can be constructed in order to decrease the
immunogenicity
thereof in diagnostic or therapeutic applications. Moreover, immunogenicity
can be
minimised by humanising the antibodies by CDR grafting [see European Patent
Application 0 239 400 (Winter)] and, optionally, framework modification [EP 0
239 400;
Riechmann, L. et al., Nature, 332, 323-327, 1988; Verhoeyen M. et al.,
Science, 239,
1534-1536, 1988; Kettleborough, C. A. et al., Protein Engng., 4, 773-783,
1991; Maeda,
H. et al., Human Antibodies and Hybridoma, 2, 124-134, 1991; Gorman S. D. et
al., Proc.
Natl. Acad. Sci. USA, 88, 4181-4185, 1991; Tempest P. R. et al.,
Bio/Technology, 9,
266-271, 1991; Co, M. S. et al., Proc. Natl. Acad. Sci. USA, 88, 2869-2873,
1991; Carter,
P. et al., Proc. Natl. Acad. Sci. USA, 89, 4285-4289, 1992; Co, M. S. et al.,
J. Immunol.,
148, 1149-1154, 1992; and, Sato, K. et al., Cancer Res., 53, 851-856, 1993].
Antibodies as described herein can be produced in cell culture. Recombinant
DNA
technology can be used to produce the antibodies according to established
procedure, in
bacterial or preferably mammalian cell culture. The selected cell culture
system
optionally secretes the antibody product, although antibody products can be
isolated from
non-secreting cells.
Therefore, the present invention includes a process for the production of an
antibody
according to the invention comprising culturing a host, e.g. E. coli, an
insect cell or a
mammalian cell, which has been transfonned with a hybrid vector comprising an
expression cassette comprising a promoter operably linked to a first DNA
sequence
encoding a signal peptide linked in the proper reading frame to a second DNA
sequence
encoding said antibody protein, and isolating said protein.
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Multiplication of hybridoma cells or mammalian host cells in vitro is carried
out in
suitable culture media, which are the customary standard culture media, for
example
Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium, optionally
5 replenished by a mammalian serum, e.g. foetal calf serum, or trace elements
and growth
sustaining supplements, e.g. feeder cells such as nornnal mouse peritoneal
exudate cells,
spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin,
low density
lipoprotein, oleic acid, or the like. Multiplication of host cells which are
bacterial cells or
yeast cells is likewise carried out in suitable culture media known in the
art, for example
10 for bacteria in medium LB, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x
YT,
or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or
Complete Minimal Dropout Medium.
In vitro production provides relatively pure antibody preparations and allows
scale-up to
15 give large amounts of the desired antibodies. Techniques for bacterial
cell, yeast or
mammalian cell cultivation are known in the art and include homogeneous
suspension
culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or
immobilised or
entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose
microbeads or
ceramic cartridges.
Large quantities of the desired antibodies can also be obtained by multiplying
mammalian
cells in vivo. For this purpose, hybridoma cells producing the desired
antibodies are
injected into histocompatible mammals to cause growth of antibody-producing
tumours.
Optionally, the animals are primed with a hydrocarbon, especially mineral oils
such as
pristane (tetramethyl-pentadecane), prior to the injection. After one to three
weeks, the
antibodies are isolated from the body fluids of those mammals. For example,
hybridoma
cells obtained by fusion of suitable myeloma cells with antibody-producing
spleen cells
from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0
that
produce the desired antibodies are injected intraperitoneally into Balb/c mice
optionally
pre-treated 'with pristane, and, after one to two weeks, ascitic fluid is
taken from the
animals.
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The foregoing, and other, techniques are discussed in, for example, Kohler and
Milstein,
(1975) Nature 256:495-497; US 4,376,110; Harlow and Lane, Antibodies: a
Laboratory
Manual, (1988) Cold Spring Harbor, incorporated herein by reference.
Techniques for
the preparation of recombinant antibody molecules is described in the above
references
and also in, for example, EP 0623679; EP 0368684 and EP 0436597, which are
incorporated herein by reference.
The cell culture supernatants are screened for the desired antibodies,
preferentially by an
enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a
radioimmunoassay.
For isolation of the antibodies, the immunoglobulins in the culture
supernatants or in the
ascitic fluid can be concentrated, e.g. by precipitation with ammonium
sulphate, dialysis
against hygroscopic material such as polyethylene glycol, filtration through
selective
membranes, or the like. If necessary and/or desired, the antibodies are
purified by the
customary chromatography methods, for exainple gel filtration, ion-exchange
chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity
chromatography, e.g. affinity chromatography with the target antigen, or with
Protein-A.
The invention further concerns hybridoma cells secreting the monoclonal
antibodies of
the invention. The preferred hybridoma cells of the invention are genetically
stable,
secrete monoclonal antibodies of the invention of the desired specificity and
can be
activated from deep-frozen cultures by thawing and recloning.
The invention, in a preferred embodiment, relates to the production of anti
mutant ErbB2
antibodies. Thus, the invention also concerns a process for the preparation of
a
hybridoma cell line secreting monoclonal antibodies according to the
invention,
characterised in that a suitable mammal, for example a Balb/c mouse, is
immunised with
a one or more PDGF polypeptides or antigenic fragments thereof, or an
antigenic carrier
containing a mutant ErbB2 polypeptide; antibody-producing cells of the
immunised
mammal are fused with cells of a suitable myeloma cell line, the hybrid cells
obtained in
the fusion are cloned, and cell clones secreting the desired antibodies are
selected. For
example spleen cells of Balb/c mice immunised with mutant ErbB2 are fused with
cells of
the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag14, the obtained
hybrid cells
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are screened for secretion of the desired antibodies, and positive hybridoma
cells are
cloned.
Preferred is a process for the preparation of a hybridoma cell line,
characterised in that
Balb/c mice are immunised by injecting subcutaneously and/or intraperitoneally
between
1 and 100 g mutant ErbB2 and a suitable adjuvant, such as Freund's adjuvant,
several
times, e.g. four to six times, over several months, e.g. between two and four
months, and
spleen cells from the immunised mice are taken two to four days after the last
injection
and fused with cells of the myeloma cell line PAI in the presence of a fusion
promoter,
preferably polyethylene glycol. Preferably the myeloma cells are fused with a
three- to
twentyfold excess of spleen cells from the immunised mice in a solution
containing about
30 % to about 50 % polyethylene glycol of a molecular weight around 4000.
After the
fusion the cells are expanded in suitable culture media as described
hereinbefore,
supplemented with a selection medium, for example HAT medium, at regular
intervals in
order to prevent normal myeloma cells from overgrowing the desired hybridoma
cells.
The invention also concerns recombinant nucleic acids comprising an insert
coding for a
heavy chain variable domain and/or for a light chain variable domain of
antibodies
directed to mutant ErbB2 as described hereinbefore. By definition such DNAs
comprise
coding single stranded DNAs, double stranded DNAs consisting of said coding
DNAs
and of complementary DNAs thereto, or these complementary (single stranded)
DNAs
themselves.
Furthermore, DNA encoding a heavy chain variable domain and/or for a light
chain
variable domain of antibodies directed to mutant ErbB2 can be enzymatically or
chemically synthesised DNA having the authentic DNA sequence coding for a
heavy
chain variable domain and/or for the light chain variable domain, or a mutant
thereof. A
mutant of the authentic DNA is a DNA encoding a heavy chain variable domain
and/or a
light chain variable domain of the above-mentioned antibodies in which one or
more
amino acids are deleted or exchanged with one or more other amino acids.
Preferably said
modification(s) are outside the CDRs of the heavy chain variable domain and/or
of the
light chain variable domain of the antibody. Such a mutant DNA is also
intended to be a
silent mutant wherein one or more nucleotides are replaced by other
nucleotides with the
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new codons coding for the same amino acid(s). Such a mutant sequence is also a
degenerated sequence. Degenerated sequences are degenerated within the meaning
of the
genetic code in that an unlimited number of nucleotides are replaced by other
nucleotides
without resulting in a change of the amino acid sequence originally encoded.
Such
degenerated sequences can be useful due to their different restriction sites
and/or
frequency of particular codons which are preferred by the specific host,
particularly E.
coli, to obtain an optimal expression of the heavy chain murine variable
domain and/or a
light chain murine variable domain.
In this context, the term mutant is intended to include a DNA mutant obtained
by in vitro
mutagenesis of the authentic DNA according to methods known in the art.
For the assembly of complete tetrameric immunoglobulin molecules and the
expression
of chimeric antibodies, the recombinant DNA inserts coding for heavy and light
chain
variable domains are fused with the corresponding DNAs coding for heavy and
light
chain constant domains, then transferred into appropriate host cells, for
example after
incorporation into hybrid vectors.
The invention therefore also concerns recombinant nucleic acids comprising an
insert
coding for a heavy chain murine variable domain of an anti mutant ErbB2
antibody fused
to a human constant domain y, for example 71, 72, 73 or y4, preferably yl or
y4. Likewise
the invention concerns recombinant DNAs comprising an insert coding for a
light chain
murine variable domain of an anti mutant ErbB2 antibody directed to mutant
ErbB2 fused
to a human constant domain x or a,, preferably K.
In another embodiment the invention pertains to recombinant DNAs coding for a
recombinant polypeptide wherein the heavy chain variable domain and the light
chain
variable domain are linked by way of a spacer group, optionally comprising a
signal
sequence facilitating the processing of the antibody in the host cell and/or a
DNA coding
for a peptide facilitating the purification of the antibody and/or a cleavage
site and/or a
peptide spacer and/or an effector molecule.
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Antibodies and antibody fragments according to the invention are useful in
diagnosis.
Accordingly, the invention provides a composition for diagnosis comprising an
antibody
according to the invention.
In the case of a diagnostic composition, the antibody is preferably provided
together with
means for detecting the antibody, which can be enzymatic, fluorescent,
radioisotopic or
other means. The antibody and the detection means can be provided for
simultaneous,
simultaneous separate or sequential use, in a diagnostic kit intended for
diagnosis.
The antibodies of the invention can be assayed for immunospecific binding by
any
method known in the art. The immunoassays which can be used include but are
not
limited to competitive and non-competitive assay systems using techniques such
as
western blots, radioimmunoassays, ELISA, sandwich immunoassays,
immunoprecipitation assays, precipitin reactions, gel diffusion precipitin
reactions,
immunodiffiision assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays and protein A immunoassays.
Such assays are routine in the art (see, for example, Ausubel et al, eds,
1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York,
which is
incorporated by reference herein in its entirety). Exemplary immunoassays are
described
briefly below.
Immunoprecipitation protocols generally comprise lysing a population of cells
in a lysis
buffer such as RIPA buffer (1 % NP-40 or Triton X-100, 1% sodium deoxycholate,
0.1 %
SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2,1% Trasylol) supplemented
with
protein phosphatase and/or protease inhibitors (e. g., EDTA, PMSF, aprotinin,
sodium
vanadate), adding the antibody of interest to the cell lysate, incubating for
a period of
time (e. g., 1-4 hours) at 4 C, adding protein A and/or protein G sepharose
beads to the
cell lysate, incubating for about an hour or more at 4 C, washing the beads in
lysis buffer
and resuspending the beads in SDS/sample buffer. The ability of the antibody
of interest
to immunoprecipitate a particular antigen can be assessed by, e. g., western
blot analysis.
Western blot analysis generally comprises preparing protein samples,
electrophoresis of
the protein samples in a polyacrylamide gel (e. g., 8%-20% SDS-PAGE depending
on the
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molecular weight of the antigen), transferring the protein sample from the
polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane
in
blocking solution (e. g., PBS with 3% BSA or non-fat milk), washing the
membrane in
washing buffer (e. g., PBS-Tween 20), exposing the membrane to a primary
antibody (the
5 antibody of interest) diluted in blocking buffer, washing the membrane in
wasl7ing buffer,
exposing the membrane to a secondary antibody (which recognises the primary
antibody,
e. g., an antihuman antibody) conjugated to an enzymatic substrate (e. g.,
horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e. g., 32P or
125I) diluted in
blocking buffer, washing the membrane in wash buffer, and detecting the
presence of the
10 antigen.
ELISAs comprise preparing antigen, coating the well of a 96 well microtitre
plate with
the antigen, adding the antibody of interest conjugated to a detectable
compound such as
an enzymatic substrate (e. g., horseradish peroxidase or alkaline phosphatase)
to the well
15 and incubating for a period of time, and detecting the presence of the
antigen. In ELISAs
the antibody of interest does not have to be conjugated to a detectable
compound; instead,
a second antibody (which recognises the antibody of interest) conjugated to a
detectable
compound can be added to the well. Further, instead of coating the well with
the antigen,
the antibody can be coated to the well. In this case, a second antibody
conjugated to a
20 detectable compound can be added following the addition of the antigen of
interest to the
coated well.
The binding affinity of an antibody to an antigen and the off-rate of an
antibody-antigen
interaction can be determined by competitive binding assays. One example of a
25 competitive binding assay is a radioimmunoassay comprising the incubation
of labelled
antigen (e. g., 3H or 125I) with the antibody of interest in the presence of
increasing
amounts of unlabeled antigen, and the detection of the antibody bound to the
labelled
antigen. The affinity of the antibody of interest for a particular antigen and
the binding
off-rates can be determined from the data by scatchard plot analysis.
Competition with a
30 second antibody can also be determined using radioimmunoassays. In this
case, the
antigen is incubated with antibody of interest conjugated to a labelled
compound (e. g.,
3H or 1asI) in the presence of increasing amounts of an unlabeled second
antibody.
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Preparation of mutant ErbB2 polype tp ides
Mutant ErbB2 polypeptides in accordance with the present invention can be
produced by
any desired technique, including chemical synthesis, isolation from biological
samples
and expression of a nucleic acid encoding such a polypeptide. Nucleic acids,
in their turn,
can be synthesised or isolated from biological sources of mutant ErbB2.
The invention thus relates to vectors encoding a polypeptide according to the
invention,
or a fragment thereof. The vector can be, for example, a phage, plasmid,
viral, or
retroviral vector.
Nucleic acids according to the invention can be part of a vector containing
a'selectable
marker for propagation in a host. Generally, a plasmid vector is introduced in
a
precipitate, such as a calcium phosphate precipitate, or in a complex with a
charged lipid.
If the vector is a virus, it can be packaged in vitro using an appropriate
packaging cell line
and then transduced into host cells.
The nucleic acid insert is operatively linked to an appropriate promoter, such
as the phage
lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40
early and late
promoters and promoters of retroviral LTRs. Other suitable promoters are known
to
those skilled in the art. The expression constructs further contain sites for
transcription
initiation, termination, and, in the transcribed region, a ribosome binding
site for
translation. The coding portion of the transcripts expressed by the constructs
preferably
includes a translation initiating codon at the beginning and a termination
codon (UAA,
UGA or UAG) appropriately positioned at the end of the polypeptide to be
translated.
As indicated, the expression vectors preferably include at least one
selectable marker.
Such markers include dihydrofolate reductase, G418 or neomycin resistance for
eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance
genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts
include, but are not limited to, bacterial cells, such as E. coli,
Streptomyces and
Salmonella typhinaurium cells; fungal cells, such as yeast cells (e. g.,
Saccharomyces
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cerevisiae or Pichia pastoris); insect cells such as Drosophila S2 and
Spodoptera Sf9
cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant
cells.
Appropriate culture media and conditions for the above-described host cells
are known in
the art and available commercially.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9,
available
from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a,
pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a,
pKK2233, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among
preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG
available
from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
Preferred expression vectors for use in yeast systems include, but are not
limited to
pYES2, pYDI, pTEFI/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5,
pHIL-D2, pHIL-Sl, pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen,
Carlsbad, CA).
Introduction of the construct into the host cell can be effected by calcium
phosphate
transfection, DEAE-dextran mediated transfection, cationic lipid-mediated
transfection,
electroporation, transduction, infection, or other methods. Such methods are
described in
many standard laboratory manuals, such as Sambrook et al., referred to above.
A polypeptide according to the invention can be recovered and purified from
recombinant
cell cultures by well-known methods including am.monium sulphate or ethanol
precipitation, acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity
chromatography, hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is employed for
purification.
Polypeptides according to the present invention can also be recovered from
biological
sources, including bodily fluids, tissues and cells, especially cells derived
from tumour
tissue or suspected tumour tissues from a subject.
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In addition, polypeptides according to the invention can be chemically
synthesised using
techniques known in the art (for example, see Creighton, 1983, Proteins:
Structures and
Molecular Principles, W. H. Freeman & Co., N. Y., and Hunkapiller et al.,
Nature, 310:
105-111 (1984)). For example, a polypeptide corresponding to a fragment of a
mutant
ErbB2 polypeptide can be synthesised by use of a peptide synthesiser.
ErbB2 Mutations
Mutations in ErbB2 have been identified in human tumour cells. Table 1
describes the
location of these mutations and the tumours in which they were identified. The
mutations
are in the kinase domain of ErbB2. Most of the mutations can be confirmed as
somatic,
indicating that a paired normal/tumour sample was tested and the mutation
found only in
the tumour sample.
Table 1: ERBB2 mutations in primary tumours
Sample Tumour/Histology NucleotideT Amino AcidT
Lung Cancer
PD1353a NSCLC - adenocarcinoma 2322 ins/dup(GCATACGTGATG) ins774(AYVM)
PD0258a NSCLC - adenocarcinoma 2322 ins/dup(GCATACGTGATG) ins774(AYVM)
PD0317a NSCLC - adenocarcinoma 2322 ins/dup(GCATACGTGATG) ins774(AYVM)
PD0319a NSCLC - adenocarcinoma 2335 ins(CTGTGGGCT) ins779(VGS)
PD0270a NSCLC - adenocarcinoma TT2263-4CC L755P
Other
PD1487a Glioblastoma G2740A E914K
PD1403a Gastric cancer G2326A G776S
PD0888a Ovarian cancer A2570G N857S
tNumbering is relative to the A of the ATG/initiating methionine as nucleotide
one in
NCBI/RefSeq accession NM_004448.1
In addition, the following mutants have been identified in ErbB2 in cancer
patients:
stCE17-1157 PD0312a NSCLC adenocarcinoma Het A560G N187S
stCE17-1163 PD0293a NSCLC squamous ca Het C1157A A386D
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stCE17-1174 DV-90 NSCLC adenocarcinoma Het G2524A V8421
stCEl7-1379 PD0318a NSCLC adenocarcinoma Het C3647A A1216D
These variants are of unknown significance as they cannot be proven somatic
due to lack
of normal tissue. However, in a preferred embodiment, the invention moreover
comprises the above mutations in ErbB2 as indicated.
Compound Assa-ys
According to the present invention, mutant ErbB2 is used as a target to
identify compounds,
for example lead compounds for pharmaceuticals, which are capable of
modulating the
proliferative activity of mutant ErbB2. Accordingly, the invention relates to
an assay and
provides a method for identifying a compound or compounds capable, directly or
indirectly, of modulating the activity mutant ErbB2, comprising the steps of:
(a) incubating mutant ErbB2 with the compound or compounds to be assessed; and
(b) identifying those compounds which influence the activity of mutant ErbB2.
Mutant ErbB2 is as defined in the context of the present invention.
According to a first embodiment of this aspect invention, the assay is
configured to detect
polypeptides which bind directly to mutant ErbB2.
The invention therefore provides a method for identifying a modulator cell
proliferation,
comprising the steps of:
(a) incubating mutant ErbB2 with the compound or compounds to be assessed; and
(b) identifying those compounds which bind to mutant ErbB2.
Preferably, the method further comprises the step of:
(c) assessing the compounds which bind to mutant ErbB2 for the ability to
modulate cell viability or cell proliferation in a cell-based assay.
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Binding to mutant ErbB2 may be assessed by any technique known to those
skilled in the
art. Examples of suitable assays include the two hybrid assay system, which
measures
interactions in vivo, affinity chromatography assays, for example involving
binding to
polypeptides immobilised on a column, fluorescence assays in which binding of
the
5 compound(s) and mutant ErbB2 is associated with a change in fluorescence of
one or both
partners in a binding pair, and the like. Preferred are assays performed in
vivo in cells,
such as the two-hybrid assay.
In preferred embodiments, a nucleic acid encoding mutant ErbB2 is ligated into
a vector,
10 and introduced into suitable host cells to produce transformed cell lines
that express
mutant ErbB2. The resulting cell lines can then be produced for reproducible
qualitative
and/or quantitative analysis of the effect(s) of potential compounds affecting
mutant
ErbB2 function. Thus mutant ErbB2 expressing cells may be employed for the
identification of compounds, particularly low molecular weight compounds,
which
15 modulate the function of mutant ErbB2. Thus host cells expressing mutant
ErbB2 are
useful for drug screening and it is a further object of the present invention
to provide a
method for identifying compounds which modulate the activity of mutant ErbB2,
said
method comprising exposing cells containing heterologous DNA encoding mutant
ErbB2,
wherein said cells produce functional mutant ErbB2, to at least one compound
or mixture
20 of compounds or signal whose ability to modulate the activity of said
mutant ErbB2 is
sought to be determined, and thereafter monitoring said cells for changes
caused by said
modulation. Such an assay enables the identification of modulators, such as
agonists,
antagoni'sts and allosteric modulators, of mutant ErbB2. As used herein, a
compound or
signal that modulates the activity of mutant ErbB2 refers to a compound that
alters the
25 activity of mutant ErbB2 in such a way that the activity of mutant ErbB2 on
a target
thereof is different in the presence of the compound or signal (as compared to
the absence
of said compound or signal).
Cell-based screening assays can be designed by constructing cell lines in
which the
30 expression of a reporter protein, i.e. an easily assayable protein, such as
(3 -galactosidase,
chloramphenicol acetyltransferase (CAT) or luciferase, is dependent on the
activation of a
mutant ErbB2 substrate. For example, a reporter gene encoding one of the above
polypeptides may be placed under the control of an response element which is
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specifically activated by an ErbB2 target. Such an assay enables the detection
of
compounds that directly modulate mutant ErbB2 function, such as compounds that
antagonise phosphorylation of targets mutant ErbB2, or compounds that inhibit
or
potentiate other cellular functions required for the activity of mutant ErbB2.
Cells in
which wild-type, non-mutant ErbB2 is present provide suitable controls.
Alternative assay formats include assays which directly assess proliferative
responses in a
biological system. The constitutive expression of unregulated mutant ErbB2
results in an
proliferative phenotype in animal cells. Cell-based systems, such as 3T3
fibroblasts, may
be used to assess the activity of potential regulators of mutant ErbB2.
In a further preferred aspect, the invention relates to a method for
identifying a lead
compound for a pharmaceutical, comprising the steps of:
providing a purified mutant ErbB2 molecule;
incubating the mutant ErbB2 molecule with a substrate known to be
phosphorylated by mutant ErbB2 and a test compound or compounds; and
identifying the test compound or compounds capable of modulating the
phosphorylation of the substrate.
Optionally, the test compound(s) identified may then be subjected to in vivo
testing to
determine their effects on a mutant ErbB2 signalling pathway.
As used herein, "mutant ErbB2 activity" may refer to any activity of mutant
ErbB2,
including its binding activity, but in particular refers to the
phosphorylating activity of
mutant ErbB2 and/or the ability of mutant ErbB2 to dimerise with itself and/or
other
members of the ErbB family, such as EGFR, Her3 and Her4. Accordingly, the
invention
may be configured to detect the phosphorylation of target compounds by mutant
ErbB2,
and the modulation of this activity by potential therapeutic agents.
Examples of compounds which modulate the phosphorylating activity of mutant
ErbB2
include dominant negative mutants of ErbB2 itself. Such compounds are able to
compete
for the target of mutant ErbB2, thus reducing the activity of mutant ErbB2 in
a biological
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or artificial system. Thus, the invention moreover relates to compounds
capable of
modulating the phosphorylating activity of mutant ErbB2.
Compounds which influence the activity of mutant ErbB2 may be of almost any
general
description, including low molecular weight compounds, including organic
compounds
which may be linear, cyclic, polycyclic or a combination thereof, peptides,
polypeptides
including antibodies, or proteins. In general, as used herein, "peptides",
"polypeptides"
and "proteins" are considered equivalent.
Many compounds according to the present invention may be lead compounds useful
for
drug development. Useful lead compounds are especially antibodies and
peptides, and
particularly intracellular antibodies expressed within the cell in a gene
therapy context,
which may be used as models for the development of peptide or low molecular
weight
therapeutics. In a preferred aspect of the invention, lead compounds and
mutant ErbB2 or
other target peptides may be co-crystallised in order to facilitate the design
of suitable low
molecular weight compounds which mimic the interaction observed with the lead
compound.
Crystallisation involves the preparation of a crystallisation buffer, for
example by mixing
a solution of the peptide or peptide complex with a "reservoir buffer",
preferably in a 1:1
ratio, with a lower concentration of the precipitating agent necessary for
crystal
formation. For crystal formation, the concentration of the precipitating agent
is increased,
for example by addition of precipitating agent, for example by titration, or
by allowing
the concentration of precipitating agent to balance by diffusion between the
crystallisation
buffer and a reservoir buffer. Under suitable conditions such diffusion of
precipitating
agent occurs along the gradient of precipitating agent, for example from the
reservoir
buffer having a higher concentration of precipitating agent into the
crystallisation buffer
having a lower concentration of precipitating agent. Diffusion may be achieved
for
example by vapour diffusion techniques allowing diffusion in the common gas
phase.
Known techniques are, for example, vapour diffusion methods, such as the
"hanging
drop" or the "sitting drop" method. In the vapour diffusion method a drop of
crystallisation buffer containing the protein is hanging above or sitting
beside a much
larger pool of reservoir buffer. Alternatively, the balancing of the
precipitating agent can
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be achieved through a semipermeable membrane that separates the
crystallisation buffer
from the reservoir buffer and prevents dilution of the protein into the
reservoir buffer.
In the crystallisation buffer the peptide or peptide/binding partner complex
preferably has
a concentration of up to 30 mg/ml, preferably from about 2 mg/ml to about 4
mg/ml.
Formation of crystals can be achieved under various conditions which are
essentially
determined by the following parameters: pH, presence of salts and additives,
precipitating
agent, protein concentration and temperature. The pH may range from about 4.0
to 9Ø
The concentration and type of buffer is rather unimportant, and therefore
variable, e.g. in
dependence with the desired pH. Suitable buffer systems include phosphate,
acetate,
citrate, Tris, MES and HEPES buffers. Useful salts and additives include e.g.
chlorides,
sulphates and other salts known to those skilled in the art. The buffer
contains a
precipitating agent selected from the group consisting of a water miscible
organic solvent,
preferably polyethylene glycol having a molecular weight of between 100 and
20000,
preferentially between 4000 and 10000, or a suitable salt, such as a
sulphates, particularly
ammonium sulphate, a chloride, a citrate or a tartarate.
A crystal of a peptide or peptide/binding partner complex according to the
invention may
be chemically modified, e.g. by heavy atom derivatization. Briefly, such
derivatization is
achievable by soaking a crystal in a solution containing heavy metal atom
salts, or a
organometallic compounds, e.g. lead chloride, gold thiomalate, thimerosal or
uranyl
acetate, which is capable of diffusing through the crystal and binding to the
surface of the
protein. The location(s) of the bound heavy metal atom(s) can be determined by
X-ray
diffraction analysis of the soaked crystal, which information may be used e.g.
to construct
a three-dimensional model of the peptide.
A three-dimensional model is obtainable, for example, from a heavy atom
derivative of a
crystal and/or from all or part of the structural data provided by the
crystallisation.
Preferably building of such model involves homology modelling and/or molecular
replacement.
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The preliminary homology model can be created by a combination of sequence
alignment
with any ErbB/Her protein the structure of which is known, such as ErbB2
itself (Cho et
czl., Nature 421: 756-760, 2003), secondary structure prediction and screening
of
structural libraries. For example, the sequences of mutant ErbB2 and a
candidate peptide
can be aligned using a suitable software program.
Computational software may also be used to predict the secondary structure of
the peptide
or peptide complex. The peptide sequence may be incorporated into the mutant
ErbB2
structure. Structural incoherences, e.g. structural fragments around
insertions/deletions
can be modelled by screening a structural library for peptides of the desired
length and
with a suitable conformation. For prediction of the side chain conformation, a
side chain
rotamer library may be employed.
The final homology model is used to solve the crystal structure of the peptide
by
molecular replacement using suitable computer software. The homology model is
positioned according to the results of molecular replacement, and subjected to
further
refinement comprising molecular dynamics calculations and modelling of the
inhibitor
used for crystallisation into the electron density.
Assays for ErbB2 activity
The activity of mutant ErbB2 may be assayed, for example, by measuring kinase
activity
and through cellular transformation assays.
In a first embodiment, mutant forms of the receptor gene are isolated from the
tumour to
be assessed or constructed from sequence information derived from said tumour.
The
mutant ErbB2 gene(s) are transiently expressed in cells and then checked for
expression
by Western blot. The expression of the mutant and wild-type forms is then
assayed for its
effect on downstream signalling events.
Specifically, activation of the Ras/RAF/MEK/ERK pathway is assayed. This
involves
performing Ras activation assays using the "Ras-capture" approach (Marais et
al., (1998)
Science 280(5360):109-12). RAF, MEK and ERK assays are examined using
antibodies
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that recognise the phosphorylated and active forms and also by direct
immunoprecipitation kinase activity.
The ERK partway can be assayed as described, for example, in Karasarides M,
5 Chiloeches A, Hayward et al., Oncogene. 2004 Jun 21 [Epub ahead of print];
Wellbrock
et al., Cancer Res. 2004 Apr 1;64(7):2338-42; or Wan et al., Cell. 2004 Mar
19;116(6):855-67.
The assay may optionally be enhanced by examining transcription controls of
known
10 genes.
Other pathways that are known to be downstream of EGF signalling, such as the
P13-
kinase pathway, may also be exploited for measurement of ErbB2 activity. The
approach
is similar to the one described above.
In addition, long-term assays based on stable cell lines may be used. Stable
lines are
created and assessed for transformation by normal criteria in vitro (ability
to form
colonies, loss of contact inhibition, growth in soft agar) and also for the
ability to grow as
tumours in nude mice. Finally, more sophisticated assays can be performed,
such as
testing the ability of the cells to invade matrigel plugs and to migrate in
the absence of
growth factors.
For example, mutant ErbB2 may be transfected in to cell lines using a
transfection
reagent such as lipofectamine . In a example, the plasmid pEF/c-erbB2.6,
expressing
wild-type ErbB2 under the control of the EF1a promoter, is transfected into
NIH3T3
cells. Cells are also transfected with mutants of pEF/c-erbB2.6 comprising the
mutations
G776S, VGS and VYVM as described herein (see Figure 1). EGFR mutagenesis is
performed using the Quickchange II XL Site-Directed Mutagenesis Kit
(Stratagene).
Transfection experiments were performed using lipofectamine reagent
(Invitrogen) with
NIH3T3 cells in DMEM + 5% DCS. 2.5x105 cells per well of a six well dish are
plated
and incubated overnight.
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Transfection complexes are prepared on bacterial culture dishes. Each EGFR
(ErbB2)
vector is diluted with sterile PBS to 0.016ug/ul. 0.256ug of each EGFR vector
is used per
transfection (Total DNA per well 256ng). For each transfection, 13 1 of PBS is
mixed
with 3 1 of lipofectamine on a bacterial plate. 16 1 of the DNA mix is
combined with the
lipofectamine for 15 minutes at room temperature. While complexes are forming,
cells
are washed twice with serum free DMEM and 800u1 of serum free DMEM is added to
each well. 200u1 of serum free DMEM is added to each complexes
(DNA/lipofectamine
mix) and the total volume is added to the cells.
After 6 hours the media are removed, the cells washed twice in DMEM+5%DCS and
then
2ml of DMEM+5%DCS is added. The cells are incubated for 2days and then
harvested
by NP40 extraction buffer for western blotting. Figure 3A shows western blots
of two
transfection experiments using the above plasmids.
Passage 11 NIH3T3 cells transfected with the plasmids as above, plus a focus
formation
positive control (Ras) are used in a focus formation assay. Cells are
transfected with
800ng of each EGFR mutant plasmid. The Ras control is transfected at 100ng
together
with sufficient plink control vector (no insert in the polylinker) to give a
total of 800ng
total DNA.
After 24 hours, cells are trypsinised and split between two 10cm tissue
culture dishes
containing lOml of DMEM+5%DCS. The media on the cells are changed on day 5, 8,
13
and 15 and the assay is terminated on day 20 by fixing cells in 4%
formaldelyde for 30
minutes. Figure 3B shows the cell morphology observed after 8 days.
To count foci, cells were stained with 4% (w/v) Crystal Violet in 70% ethanol.
Only the
foci, which were 2mm in diameter were counted. Figure 3C shows the results of
the
focus formation assay; the crystal violet stains are shown in Figure 3D.
The focus formation assay results were the average of three independent
experiments.
The mutants according to the invention have potent transforming ability as
assessed in
vivo in the focus formation assay.
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Computational aspects of detection
The detection of mutant ErbB2 polypeptides and/or mutant ErbB2 nucleic acids
can be
automated to provide rapid massively parallel screening of sample populations.
Computerised methods for mutation detection are known in the art, and will
generally
involve the combination of a sequencing device, or other device capable of
detecting
sequence variation in polypeptides or nucleic acids, a data processing unit
and an output
device which is capable of displaying the result in a form interpretable by a
technician or
physician.
In a preferred aspect, therefore, the invention provides an automated method
for detecting
a mutation at a target sequence position in a nucleic acid derived from a
naturally-
occurring primary human tumour encoding a ErbB2 polypeptide, comprising:
sequencing a sample of an amplification product of the nucleic acid from the
naturally-occurring primary human tumour to provide a sample data set
specifying a
plurality of measured base pair identification data in a target domain
extending from a
start sequence position to an end sequence position;
determining presence or absence of the mutation in the sample conditional on
whether the measured base pair identification datum for the target sequence
position
corresponds to a reference base pair datum for the target sequence position;
and
generating an output indicating the presence or absence of the mutation in the
sample as established by the determining step.
Methods for sequencing and for detection of mutations in sequences are set
forth
above and generally known in the art. The invention makes use of such methods
in
providing an apparatus for carrying out the process of the invention, which
apparatus
comprises:
a sequence reading device operable to determine the sequence of a sample of a
nucleic acid to provide a sample data set specifying measured base pair
identification data
in a target domain extending from a start sequence position to an end sequence
position;
and
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a data analysis unit connected to receive the sample data set from the
sequencing
device and operable to determine presence or absence of the mutation in the
sample
conditional on whether the measured base pair identification datum for the
target
sequence position corresponds to a reference base pair datum for the target
sequence
position.
Suitable sequence reading devices include automated sequencers, RFLP-analysers
and
mobility shift analysis apparata. Advantageously, the sequence of an
amplification
product of the target nucleic acid is analysed, and the apparatus moreover
includes an
amplification device such as a PCR machine.
Preferably, the apparatus also comprises an output device operable to generate
an output
indicating the presence or absence of the mutation in the sample determined by
the data
analysis unit. For example, the output device can comprise at least one of: a
graphical
user interface; an audible user interface; a printer; a computer readable
storage medium;
and a computer interpretable carrier medium.
The invention can moreover be configured to detect the mutant ErbB2 protein
itself.
Thus, in a further aspect, the invention relates to an automated method for
detecting a
single amino acid mutation in a ErbB2 polypeptide from a naturally-occurring
primary
human tumour, comprising:
applying a marker to one or more target amino acids in a sample of the ErbB2
polypeptide;
reading the sample after applying the marker to determine presence or absence
of
the marker in the sample, thereby to indicate presence or absence of the
single amino acid
mutation in the sample; and
generating an output indicating the presence or absence of the single amino
acid
mutation in the sample as determined by the reading step.
The marker preferably comprises a ligand that binds differentially to a wild-
type ErbB2
polypeptide without single amino acid mutation and to a mutant ErbB2
polypeptide with
the mutation. Preferential binding to either form of ErbB2 is possible in the
context of
the invention.
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The invention moreover provides an apparatus for detecting an amino acid
mutation in a
ErbB2 polypeptide, comprising:
a protein marking device loaded with a marker and operable to apply a marker
to
one or more target ainino acids in a sample of the ErbB2 polypeptide; and
a marker reading device operable to determine presence or absence of the
marker
in the sample, thereby to indicate presence or absence of the single amino
acid mutation
in the sample.
The marker used can be an antibody, and the protein marking device can be
configured to
implement an ELISA process.
Advantageously, the protein marking device comprises a microarrayer which is
preferably configured to read the sample optically.
Preferably, the apparatus comprises an output device operable to generate an
output
indicating the presence or absence of the single amino acid mutation in the
sample as
determined by the marker reading device. Suitable output devices comprises at
least one
of: a graphical user interface; an audible user interface; a printer; a
computer readable
storage medium; and a computer interpretable carrier medium.
Uses of the Invention
The present invention provides novel mutants of ErbB2 polypeptides which are
useful in
the detection of neoplastic conditions, and the determination of prognoses for
subjects
suffering from such conditions as well as appropriate therapies for such
subjects. In
general, the presence of a mutation in ErbB2 as described herein is associated
with the
presence of adenocarcinoma of the lung.
In one aspect, the present invention provides a method for identifying
cancerous cells or
tissue (such as NSCLC), or of identifying cells or tissue which are
predisposed to
developing a neoplastic phenotype, comprising: amplifying at least part of an
ErbB2 gene
of the cells or tissue; analysing the amplification product to detect a
mutation in the
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ErbB2 gene as described herein; wherein a cell or tissue having one or more
ErbB2
mutations is categorised as being cancerous or being at an increased risk of
developing a
cancerous condition. Suitable amplification means include PCR and cloning.
5 In another embodiment, the present invention relates to a method for
determining a
therapeutic regime for a subject suffering from NSCLC. The method comprises:
amplifying the region of the ErbB2 gene as described above; analysing the
amplification
products for evidence of mutation as described above; and classifying a
subject having no
mutations in the ErbB2 gene as being less likely to respond to anti-ErbB2
therapy, and a
10 subject having said mutations as being more likely to respond to anti-ErbB2
therapy.
The techniques according to the invention can be automated, as required for
rapid
screening of samples for the identification of potentially cancerous
conditions. Generally,
an automated process will comprise automated amplification of nucleic acid
from tissue
15 or cell samples, detection of mutations in amplified nucleic acid, such as
by fluorescent
detection, and/or displaying the presence of mutations. Exemplary automated
embodiments are described above.
The identification of mutant ErbB2 according to the invention can thus be used
for
20 diagnostic purposes to detect, diagnose, or monitor diseases, disorders,
and/or conditions
associated with the expression of mutant ErbB2. In particular; the invention
is concerned
with the detection, diagnosis and/or monitoring of cancers associated with
mutant ErbB2
as set forth herein.
25 The invention provides a diagnostic assay for diagnosing cancer, comprising
(a) assaying
the expression of mutant ErbB2 in cells or body fluid of an individual using
one or more
antibodies specific to the ErbB2 mutant as defined herein. The presence of
mutant ErbB2
transcript in biopsy tissue from an individual can indicate a predisposition
for the
development of the disease, or can provide a means for detecting the disease
prior to the
30 appearance of actual clinical symptoms. A more definitive diagnosis of this
type allows
health professionals to employ appropriate therapies.
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Antibodies of the invention can be used to assay protein levels in a
biological sample
using classical immunohistological methods known to those of skill in the art
(e.g., see
Jalkanen, et al., (1985) J. Cell. Biol. 101:976-985; Jalkanen, et al., (1987)
J. Cell. Biol.
105:3087-3096). Other antibody-based methods useful for detecting protein gene
expression include immunoassays, such as the enzyme linked immunosorbent assay
(ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are
known in
the art and include enzyme labels, such as, glucose oxidase; radioisotopes,
such as iodine
(125I1121I), carbon (14C), sulphur (35S), tritium (3H), indium (11ZIn), and
technetium (99Tc);
luminescent labels, such as luminol; and fluorescent labels, such as
fluorescein and
rhodamine, and biotin.
Moreover, mutations in ErbB2 can be detected by analysis of nucleic acids, as
set forth
herein. For example, the presence of mutations can be detected by sequencing,
or by
SCCP analysis.
The present invention moreover provides kits that can be used in the above
methods. In
one embodiment, a kit comprises an antibody of the invention, preferably a
purified
antibody, in one or more containers. In a specific embodiment, the kits of the
present
invention contain a substantially isolated polypeptide comprising an epitope
which is
specifically immunoreactive with an antibody included in the kit. Preferably,
the kits of
the present invention further comprise a control antibody which does not react
with the
polypeptide of interest. In another specific embodiment, the kits of the
present invention
contain a means for detecting the binding of an antibody to a polypeptide of
interest (e. g.,
the antibody can be conjugated to a detectable substrate such as a fluorescent
compound,
an enzymatic substrate, a radioactive compound or a luminescent compound, or a
second
antibody which recognises the first antibody can be conjugated to a detectable
substrate).
In another specific embodiment of the present invention, the kit is a
diagnostic kit for use
in screening serum containing antibodies specific for mutant ErbB2
polypeptides as
described herein. Such a kit can include a control antibody that does not
react with the
mutant ErbB2 polypeptide. Such a kit can include a substantially isolated
polypeptide
antigen comprising an epitope which is specifically immunoreactive with at
least one
anti-ErbB2 antibody. Further, such a kit includes means for detecting the
binding of said
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antibody to the antigen (e. g., the antibody can be conjugated to a
fluorescent compound
such as fluorescein or rhodamine which can be detected by flow cytometry). In
specific
embodiments, the kit can include a recombinantly produced or chemically
synthesised
polypeptide antigen. The polypeptide antigen of the kit can also be attached
to a solid
support.
In an additional embodiment, the invention includes a diagnostic kit for use
in screening
serum containing antigens of the mutant ErbB2 polypeptide of the invention.
The
diagnostic kit includes a substantially isolated antibody specifically
immunoreactive with
polypeptide or polynucleotide antigens, and means for detecting the binding of
the
polynucleotide or polypeptide antigen to the antibody. In one embodiment, the
antibody is
attached to a solid support. In a specific embodiment, the antibody can be a
monoclonal
antibody. The detecting means of the kit can include a second, labelled
monoclonal
antibody. Alternatively, or in addition, the detecting means can include a
labelled,
competing antigen.
All publications mentioned in the above specification are herein incorporated
by
reference. Various modifications and variations of the described methods and
system of
the invention will be apparent to those skilled in the art without departing
from the scope
and spirit of the invention. Although the invention has been described in
connection with
specific preferred embodiments, it should be understood that the invention as
claimed
should not be unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention which are
apparent to
those skilled in molecular biology or related fields are intended to be within
the scope of
the following claims.
