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Cancer Patent Abstract
The present invention relates to a novel polypeptide (BCMP 101)
compositions comprising the polypeptide, including vaccines, and
antibodies that are immunospecific for the polypeptide. The use
of the polypeptide in the diagnosis, prophylaxis and treatment of
cancer, in particular breast cancer is also provided.
Cancer Patent Claims
The invention claimed is:
1. An isolated polypeptide, said polypeptide comprising a material
selected from the group consisting of: a) an amino acid sequence
set out in SEQ ID NO: 1; and b) a fragment of SEQ ID NO: 1, said
fragment consisting of SEQ ID NO: 3 or SEQ ID NO: 10.
2. The isolated polypeptide of claim 1, wherein said isolated polypeptide
is operably linked to a second amino acid sequence to generate a
fusion polypeptide.
3. A composition comprising at least one polypeptide of claim 1
and at least one of pharmaceutically acceptable excipients, adjuvants,
carriers and diluents.
Cancer Patent Description
BACKGROUND OF THE INVENTION
The present invention relates to a novel polypeptide (BCMP 101)
compositions comprising the polypeptide, including vaccines, and
antibodies that are immunospecific for the polypeptide. The use
of the polypeptide in the diagnosis, prophylaxis and treatment of
cancer, in particular breast cancer is also provided.
Breast Cancer
Breast cancer is the most frequently diagnosed non-skin cancer
among women in the United States. It is second only to lung cancer
in cancer-related deaths. In the UK, breast cancer is by far the
commonest cancer in women, with 34,600 new cases in 1998 (Cancer
Research Campaign). Ninety-nine percent of breast cancers occur
in women. The risk of developing breast cancer steadily increases
with age; the lifetime risk of developing breast cancer is estimated
to be 1 in 8 for women in the US. The annual cost of breast cancer
treatment in the United States is approximately $10 billion (Fuqua,
et. al. 2000, American Association for Cancer Research, USA). Breast
cancer incidence has been rising over the past five decades, but
recently it has slowed. This may reflect a period of earlier detection
of breast cancers by mammography. A number of established factors
can increase a woman's risk of having the disease. These include
older age, history of prior breast cancer, significant radiation
exposure, strong family history of breast cancer, upper socioeconomic
class, nulliparity, early menarche, late menopause, or age at first
pregnancy greater than 30 years. Prolonged use of oral contraceptives
earlier in life appears to increase risk slightly. Prolonged postmenopausal
oestrogen replacement increases the risk 20 to 40%. It has been
speculated that a decrease in the age at menarche, changing birth
patterns, or a rise in the use of exogenous estrogens has contributed
to the increase in breast cancer incidence (Fuqua, et. al. 2000,
American Association for Cancer Research, USA).
Causes of Breast Cancer
Breast cancer is a heterogeneous disease. Although female hormones
play a significant role in driving the origin and evolution of many
breast tumours, there are a number of other recognised and unknown
factors involved. Perturbations in oncogenes identified include
amplification of the HER-2 and the epidermal growth factor receptor
genes, and over-expression of cyclin D1. Over-expression of these
oncogenes has been associated with a significantly poorer prognosis.
Similarly, genetic alterations or the loss of tumour suppressor
genes, such as the p53 gene, have been well documented in breast
cancer and are also associated with a poorer prognosis. Researchers
have identified two genes, called BRCA1 and BRCA2, which are predictive
of pre-menopausal familial breast cancer. Genetic risk assessment
is now possible, which may enhance the identification of candidates
for chemoprevention trials (Fuqua, et al. 2000, American Association
for Cancer Research, USA).
Diagnosis
Early diagnosis of breast cancer is vital to secure the most favourable
outcome for treatment. Many countries with advanced healthcare systems
have instituted screening programs for breast cancer. This typically
takes the form of regular x-ray of the breast (mammography) during
the 50-60 year old age interval where greatest benefit for this
intervention has been shown. Some authorities have advocated the
extension of such programs beyond 60 and to the 4049 age group.
Health authorities in many countries have also promoted the importance
of regular breast self-examination by women. Abnormalities detected
during these screening procedures and cases presenting as symptomatic
would typically be confirmed by aspiration cytology, core needle
biopsy with a stereotactic or ultrasound technique for nonpalpable
lesions, or incisional or excisional biopsy. At the same time other
information relevant to treatment options and prognosis, such as
oestrogen (ER) and progesterone receptor (PR) status would typically
be determined (National Cancer Institute, USA, 2000, Breast Cancer
PDQ).
Disease Staging and Prognosis
Staging of breast cancer is the key to choosing the optimum treatment
for each patient and to select those patients who will fare well
with less intensive forms of therapy from those for whom intensive
therapy is essential. Currently the process of staging involves
lump and axillary lymph node biopsies, combined with extensive histopathology.
Patients can be incorrectly staged with consequent over- or under-treatment.
As such, there is a need for new markers that can be correlated
with disease stage and used to reliably guide treatment decisions.
Such new markers would not only benefit patients and health care
providers by selecting the optimum treatment, but could provide
significant cost and time benefits in the histology lab.
Some breast tumours become refractory to treatments, as the cancer
cells develop resistance to chemotherapy drugs or lose their hormone
sensitivity, leading to recurrent or metastatic disease which is
often incurable. More recently, attention has focussed on the development
of immunological therapies, (Green, M. C., et al. Cancer Treat.
Rev. 26, 269-286 (2000); Davis, I. D., et al. Immunol. Cell Biol.
78, 179-195 (2000); Knuth, A., et al. Cancer Chemother Pharmacol.
46, S46-51 (2000); Shiku, H., et al. Cancer Chemother. Pharmacol.
46, S77-82 (2000); Saffran, D. C., et al. Cancer Metastasis Rev.
18, 437449 (1999)), such as cancer vaccines and monoclonal antibodies
(mAbs), as a means of initiating and targeting a host immune response
against tumour cells. In 1998 the FDA approved the use of Herceptin.TM.
(Stebbing, J., et al. Cancer Treat. Rev. 26, 287-290 (2000); Dillman,
R.O. Cancer Biother. Radiopharm. 14, 5-10 (1999); Miller, K. D.,
et al. Invest. New Drugs 17, 417427 (1999)), a mAb that recognises
the erbB2/HER2-neu receptor protein, as a treatment for metastatic
breast cancer. In combination with chemotherapy, Herceptinm.TM.
has been shown to prolong the time to disease progression, when
compared to patients receiving chemotherapy alone (Baselga, J.,
et al. Cancer Res. 58, 2825-2831 (1998)). Herceptinm.TM., however,
is only effective in treating the 10-20% of patients whose tumours
over-express the erbB2 protein. Thus, the identification of other
suitable targets or antigens for immunotherapy of breast cancer
has become increasingly important.
An ideal protein target for cancer immunotherapy should have a
restricted expression profile in normal tissues and be over-expressed
in tumours, such that the immune response will be targeted to tumour
cells and not against other organs. In addition, the protein target
should be exposed on the cell surface, where it will be accessible
to therapeutic agents. Tumour antigens have been identified for
a number of cancer types, by using techniques such as differential
screening of cDNA (Hubert, R. S., et al. Proc. Natl. Acad, Sci.
USA 96, 14523-14528 (1999); Lucas, S., et al. Int. J. Cancer 87,
55-60 (2000)), and the purification of cell-surface proteins that
are recognised by tumour-specific antibodies (Catimel, B., et al.
J. Biol. Chem. 271, 25664-25670 (1996)). Proteomics may be used
as an alternative approach to identifying breast tumour antigens
(EP 1208381, EP 1159618).
The present invention is based on the identification of a novel
protein (BCMP 101) as a target for cancer therapy and diagnosis.
BCMP 101 was identified and cloned from MDA-MB468 breast cancer
cell membranes. Expression of BCMP 101 in normal human tissue showed
that the highest levels of expression were found in mammary, kidney
and bladder tissue. Expression of BCMP 101 was elevated in kidney
cancer cell lines in comparison to normal tissues. Furthermore,
elevated levels of BCMP 101 gene expression were also observed in
tumour tissue from a set of seven matched normal and tumour samples
from breast cancer patients. The BCMP 101 sequence (FIG. 1, SEQ
ID NO: 1) matches GenBank entry CAD 10629--NSE2 protein [Homo sapiens]--a
Povel NS-containing protein), which was published after the priority
date of this application.
SUMMARY OF THE INVENTION
Thus, the present invention provides a polypeptide which: a) comprises
or consists of the amino acid sequence shown in FIG. 1 (SEQ ID NO:
1); b) is a derivative having one or more amino acid substitutions,
modifications, deletions or insertions relative to the amino acid
sequence shown in FIG. 1 (SEQ ID NO: 1); or c) is a fragment of
a polypeptide as defined in a) or b) above, which is at least ten
amino acids long.
In the present application, the term "polypeptides of the
invention" is used to refer to all polypeptides described in
a) to c) above.
Polypeptides of the invention may be in substantially pure, isolated
or recombinant form, and may be fused to other moieties. In particular,
fusions of the polypeptides of the invention with localisation-reporter
proteins such as the Green Fluorescent Protein (U.S. Pat. Nos. 5,625,048,
5,777,079, 6,054,321 and 5,804,387) or the DsRed fluorescent protein
(Matz, M. V. et al., Nature Biotech. 17:969-973.) are specifically
contemplated by the present invention. The polypeptides of the invention
may be provided in substantially pure form; that is to say, they
are free, to a substantial extent, from other polyeptides. Thus,
a polypeptide of the invention may be provided in a composition
in which it is the predominant component present (i.e. it is present
at a level of at least 50%; preferably at least 75%, at least 80%,
at least 85%, at least 90%, or at least 95%; when determined on
a weight/weight basis excluding solvents or carriers).
In order to more fully appreciate the present invention, polypeptides
within the scope of a)-c) above will now be discussed in greater
detail. It will be apparent to one skilled in the art that polypeptides
according to the invention include BCMP 101 (SEQ ID NO: 1), and
derivatives, fragments and modified forms thereof.
Polypeptides Within the Scope of a)
A polypeptide within the scope of a), may consist of the particular
amino acid sequence given in FIG. 1 (SEQ ID NO: 1) or may have an
additional N-terminal and/or an additional C-terminal amino acid
sequence relative to the sequence given in FIG. 1 (SEQ ID NO: 1).
Additional N-terminal or C-terminal sequences may be provided for
various reasons. Techniques for providing such additional sequences
are well known in the art.
Additional sequences may be provided in order to alter the characteristics
of a particular polypeptide. This can be useful in improving expression
or regulation of expression in particular expression systems. For
example, an additional sequence may provide some protection against
proteolytic cleavage. This has been done for the hormone Somatostatin
by fusing it at its N-terminus to part of the .beta. galactosidase
enzyme (Itakwa et al., Science 198: 105-63 (1977)).
Additional sequences can also be useful in altering the properties
of a polypeptide to aid in identification or purification. For example,
a fusion protein may be provided in which a polypeptide is linked
to a moiety capable of being isolated by affinity chromatography.
The moiety may be an antigen or an epitope and the affinity column
may comprise immobilised antibodies or immobilised antibody fragments
which bind to said antigen or epitope (desirably with a high degree
of specificity). The fusion protein can usually be eluted from the
column by addition of an appropriate eluant.
Additional N-terminal or C-terminal sequences may, however, be
present simply as a result of a particular technique used to obtain
a polypeptide of the present invention and need not provide any
particular advantageous characteristic to the polypeptide of the
present invention. Such polypeptides are within the scope of the
present invention.
Whatever additional N-terminal or C-terminal sequence is present,
it is preferred that the resultant polypeptide should exhibit the
immunological or biological activity of the polypeptide having the
amino acid sequence shown in FIG. 1 (SEQ ID NO: 1).
Polypeptides Within the Scope of b)
Turning now to the polypeptides defined in b) above, it will be
appreciated by the person skilled in the art that these polypeptides
are derivatives of the polypeptide given in a) above. Such derivatives
preferably exhibit the immunological or biological activity of the
polypeptide having the amino acid sequence shown in FIG. 1 (SEQ
ID NO: 1). It will be appreciated by one skilled in the art that
derivatives can include post-translational modifications, for example
but without limitation, phosphorylation, glycosylation and farnesylation.
Alterations in the amino acid sequence of a polypeptide, which
do not affect the function of a polypeptide, can occur. These include
amino acid deletions, insertions and substitutions and can result
from alternative splicing and/or the presence of multiple translation
start sites and/or stop sites. Polymorphisms may arise as a result
of the infidelity of the translation process. Thus changes in amino
acid sequence may be tolerated which do not affect the polypeptide's
biological or immunological function.
The skilled person will appreciate that various changes can often
be made to the amino acid sequence of a polypeptide which has a
particular activity to produce derivatives (sometimes known as variants
or "muteins") having at least a proportion of said activity,
and preferably having a substantial proportion of said activity.
Such derivatives of the polypeptides described in a) above are within
the scope of the present invention and are discussed in greater
detail below. They include allelic and non-allelic derivatives.
An example of a derivative of the polypeptide of the invention
is a polypeptide as defined in a) above, apart from the substitution
of one or more amino acids with one or more other amino acids. The
skilled person is aware that various amino acids have similar properties.
One or more such amino acids of a polypeptide can often be substituted
by one or more other such amino acids without eliminating a desired
activity of that polypeptide.
Thus, the amino acids glycine, alanine, valine, leucine and isoleucine
can often be substituted for one another (amino acids having aliphatic
side chains). Of these possible substitutions, it is preferred that
glycine and alanine are used to substitute for one another (since
they have relatively short side chains) and that valine, leucine
and isoleucine are used to substitute for one another (since they
have larger aliphatic side chains which are hydrophobic).
Other amino acids which can often be substituted for one another
include: phenylalanine, tyrosine and tryptophan (amino acids having
aromatic side chains); lysine, arginine and histidine (amino acids
having basic side chains); aspartate and glutamate (amino acids
having acidic side chains); asparagine and glutamine (amino acids
having amide side chains); cysteine and methionine (amino acids
having sulphur-containing side chains); and aspartic acid and glutamic
acid can substitute for phospho-serine and phospho-threonine, respectively
(amino acids with acidic side chains).
Substitutions of this nature are often referred to as "conservative"
or "semi-conservative" amino acid substitutions.
Amino acid deletions or insertions may also be made relative to
the amino acid sequence given in a) (SEQ ID NO: 1) above. Thus,
for example, amino acids which do not have a substantial effect
on the biological and/or immunological activity of the polypeptide,
or at least which do not eliminate such activity, may be deleted.
Such deletions can be advantageous since the overall length and
the molecular weight of a polypeptide can be reduced whilst still
retaining activity. This can enable the amount of polypeptide required
for a particular purpose to be reduced--for example, dosage levels
can be reduced.
Amino acid insertions relative to the sequence given in a) (SEQ
ID NO: 1) above can also be made. This may be done to alter the
properties of a polypeptide of the invention (e.g. to assist in
identification, purification or expression, as explained above in
relation to fusion proteins).
Amino acid changes relative to the sequence given in a) (SEQ ID
NO: 1) above can be made using any suitable technique e.g. by using
site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem.
253:6551).
It should be appreciated that amino acid substitutions or insertions
within the scope of the present invention can be made using naturally
occurring or non-naturally occurring amino acids. Whether or not
natural or synthetic amino acids are used, it is preferred that
only L- amino acids are present.
Whatever amino acid changes are made (whether by means of substitution,
modification, insertion or deletion), preferred polypeptides of
the present invention have at least 50% sequence identity with a
polypeptide as defined in a) above, more preferably the degree of
sequence identity is at least 75%. Sequence identities of at least
80%, at least 85%, at least 90%, at least 95%, at least 98% or at
least 99% are most preferred.
The term identity can be used to describe the similarity between
two polypeptide sequences. The degree of amino acid sequence identity
can be calculated using a program such as "bestfit" (Smith
and Waterman, Advances in Applied Mathematics, 482-489 (1981)) to
find the best segment of similarity between any two sequences. The
alignment is based on maximising the score achieved using a matrix
of amino acid similarities, such as that described by Schwarz and
Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.
O., Ed pp 353-358.
A software package well known in the art for carrying out this
procedure is the CLUSTAL program. It compares the amino acid sequences
of two polypeptides and finds the optimal alignment by inserting
spaces in either sequence as appropriate. The amino acid identity
or similarity (identity plus conservation of amino acid type) for
an optimal alignment can also be calculated using a software package
such as BLASTX. This program aligns the largest stretch of similar
sequence and assigns a value to the fit. For any one pattern comparison,
several regions of similarity may be found, each having a different
score. One skilled in the art will appreciate that two polypeptides
of different lengths may be compared over the entire length of the
longer fragment. Alternatively small regions may be compared. Normally
sequences of the same length are compared for a useful comparison
to be made.
Where high degrees of sequence identity are present there will
be relatively few differences in amino acid sequence. Thus for example
they may be less than 20, less than 10, or even less than 5 differences.
Polypeptides Within the Scope of c)
As discussed supra, it is often advantageous to reduce the length
of a polypeptide, provided that the resultant reduced length polypeptide
still has a desired activity or can give rise to useful antibodies.
Feature c) of the present invention therefore covers fragments of
polypeptides a) or b) above.
The skilled person can determine whether or not a particular fragment
has activity using the techniques disclosed above. Fragments are
at least 10 amino acids long, preferred fragments may be at least
20, at least 30, at least 40, at least 50, at least 75 or at least
100 amino acids long. Preferably, the fragments are less than 150
amino acids long.
As will be discussed below, the polypeptides of the invention will
find use in an immunotherapeutic approach to breast and/or kidney
cancer. The skilled person will appreciate that for the preparation
of one or more polypeptides of the invention, the preferred approach
will be based on recombinant DNA techniques.
A polypeptide of the invention may be useful as antigenic material,
and may be used in the production of vaccines for treatment or prophylaxis
of cancer, in particular breast cancer and/or kidney cancer. Such
material can be "antigenic" and/or "immunogenic".
Generally, "antigenic" is taken to mean that the polypeptide
is capable of being used to raise antibodies or indeed is capable
of inducing an antibody response in a subject. "Immunogenic"
is taken to mean that the polypeptide is capable of eliciting an
immune response in a subject. Thus, in the latter case, the polypeptide
may be capable of not only generating an antibody response but,
in addition, non-antibody based immune responses.
It is well known that is possible to screen an antigenic polypeptide
to identify epitopic regions, i.e. those regions which are responsible
for the polypeptide's antigenicity or immunogenicity. Methods well
known to the skilled person can be used to test fragments and/or
homologues and/or derivatives for antigenicity. Thus, the fragments
for use in the present invention may include one or more such epitopic
regions or be sufficiently similar to such regions to retain their
antigenic/immunogenic properties. Thus, for fragments for use according
to the present invention the degree of identity is perhaps irrelevant,
since they may be 100% identical to a particular part of a polypeptide
of the invention. The key issue may be that the fragment retains
the antigenic/immunogenic properties of the polypeptide from which
it is derived.
Homologues, derivatives and fragments may possess at least a degree
of the antigenicity/immunogenicity of the polypeptide from which
they are derived.
Thus, in a further aspect, the present invention provides the use
of a polypeptide of the invention in the production of a composition
for the treatment or prophylaxis of cancer, particularly breast
cancer and/or kidney cancer, wherein the composition is a vaccine.
The vaccine optionally comprises one or more suitable adjuvants.
Examples of adjuvants well-known in the art include inorganic gels,
such as aluminium hydroxide, and water-in-oil emulsions, such as
incomplete Freund's adjuvant. Other useful adjuvants will be well
known to the skilled person.
In yet further aspects, the present invention provides: (a) the
use of a polypeptide of the invention in the preparation of an immunogenic
composition, preferably a vaccine; (b) the use of such an immunogenic
composition in inducing an immune response in a subject; (c) a method
for the treatment or prophylaxis of cancer, particularly breast
and/or kidney cancer in a subject, or of vaccinating a subject against
cancer which comprises the step of administering to the subject
an effective amount of a polypeptide of the invention, preferably
as a vaccine; and (d) a method for monitoring/assessing breast and/or
kidney cancer treatment in a patient, which comprises the step of
determining the presence or absence and/or quantifying at least
one polypeptide, at least one nucleic acid molecule or at least
one antibody of the invention in a biological sample.
As will be discussed below, the polypeptides of the invention will
find use in an immunotherapeutic approach to cancer, particularly
breast cancer and/or kidney cancer. The skilled person will appreciate
that for the preparation of one or more such polypeptides, the preferred
approach will be based on recombinant DNA techniques. In addition,
nucleic acid molecules encoding the polypeptides or fragments thereof
may be used in their own right.
Thus, in a further aspect, the present invention provides an isolated
or recombinant nucleic acid molecule which: d) comprises or consists
of the DNA sequence shown in FIG. 1 (SEQ ID NO:2) or its RNA equivalent;
e) a sequence which codes for a derivative or fragment of a polypeptide
shown in FIG. 1 (SEQ ID NO:1); f) a sequence which is complementary
to the sequences of d) or e); g) a sequence which codes for the
same polypeptide, as the sequences of d), e) or f); or h) a sequence
which shows substantial identity with any of those of d), e), f)
and g).
These nucleic acid molecules are now discussed in greater detail.
The term identity can also be used to describe the similarity between
two individual DNA sequences. The `bestfit` program (Smith and Waterman,
Advances in applied Mathematics, 482-489 (1981)) is one example
of a type of computer software used to find the best segment of
similarity between two nucleic acid sequences, whilst the GAP program
enables sequences to be aligned along their whole length and finds
the optimal alignment by inserting spaces in either sequence as
appropriate. It is preferred if sequences which show substantial
identity with any of those of d), e), f) and g) have e.g. at least
50%, at least 75%, at least 80%, at least 85%, at least 90% or 95%
sequence identity.
It is preferred that if the nucleic acid molecule is a fragment
of the sequence given in FIG. 1 (SEQ ID NO: 2), that it does not
correspond to the following fragments: bp558-1054, bp80-565 or bp
45-547, according to the nucleotide numbering of FIG. 1 (SEQ ID
NO: 2).
In a further aspect, the present invention provides a method for
the prophylaxis and/or treatment of cancer, in particular breast
and/or kidney cancer, in a subject, which comprises administering
to said subject a therapeutically effective amount of at least one
nucleic acid as defined above.
In yet another aspect, the present invention provides the use of
at least one nucleic acid as defined above in the preparation of
a composition for use in the prophylaxis and/or treatment of cancer,
in particular breast and/or kidney cancer.
The polypeptides of the invention can be coded for by a large variety
of nucleic acid molecules, taking into account the well known degeneracy
of the genetic code. All of these molecules are within the scope
of the present invention. They can be inserted into vectors and
cloned to provide large amounts of DNA or RNA for further study.
Suitable vectors may be introduced into host cells to enable the
expression of polypeptides of the invention using techniques known
to the person skilled in the art.
The term `RNA equivalent` when used above indicates that a given
RNA molecule has a sequence which is complementary to that of a
given DNA molecule, allowing for the fact that in RNA `U` replaces
`T` in the genetic code. The nucleic acid molecule may be in isolated,
recombinant or chemically synthetic form.
Techniques for cloning, expressing and purifying polypeptides are
well known to the skilled person. DNA constructs can readily be
generated using methods well known in the art. These techniques
are disclosed, for example in J. Sambrook et al, Molecular Cloning
3.sup.rd Edition, Cold Spring Harbour Laboratory Press (2000); in
Old & Primrose Principles of Gene Manipulation 5th Edition,
Blackwell Scientific Publications (1994); and in Stryer Biochemistry
4th Edition, W H Freeman and Company (1995). Modifications of DNA
constructs and the polypeptides expressed such as the addition of
promoters, enhancers, signal sequences, leader sequences, translation
start and stop signals and DNA stability controlling regions, or
the addition of fusion partners may then be facilitated.
Normally the DNA construct will be inserted into a vector, which
may be of phage or plasmid origin. Expression of the polypeptide
is achieved by the transformation or transfection of the vector
into a host cell which may be of eukaryotic or prokaryotic origin.
Such vectors and suitable host cells form additional aspects of
the present invention.
The nucleotides of the present invention, including DNA and RNA,
and comprising a sequence encoding a polypeptide of the invention,
may be synthesised using methods known in the art, such as using
conventional chemical approaches or polymerase chain reaction (PCR)
amplification. The nucleotides of the present invention also permit
the identification and cloning of the gene encoding a polypeptide
as defined herein from any species, for instance by screening cDNA
libraries, genomic libraries or expression libraries.
Knowledge of the nucleic acid structure can be used to raise antibodies
and for gene therapy. Techniques for this are well-known by those
skilled in the art, as discussed in more detail herein.
By using appropriate expression systems, polypeptides of the invention
may be expressed in glycosylated or non-glycosylated form. Non-glycosylated
forms can be produced by expression in prokaryotic hosts, such as
E. coli.
Polypeptides comprising N-terminal methionine may be produced using
certain expression systems, whilst in others the mature polypeptide
will lack this residue. Preferred techniques for cloning, expressing
and purifying a polypeptide of the invention are summarised below:
Polypeptides may be prepared under native or denaturing conditions
and then subsequently refolded. Baculoviral expression vectors include
secretory plasmids (such as pACGP67 from Pharmingen), which may
have an epitope tag sequence cloned in frame (e.g. myc, V5 or His)
to aid detection and allow for subsequent purification of the polypeptide.
Mammalian expression vectors may include pCDNA3 and pSecTag (both
Invitrogen), and pREP9 and pCEP4 (Invitrogen). E. coli systems include
the pBad series (His tagged--Invitrogen) or pGex series (Pharmacia).
In addition to nucleic acid molecules coding for polypeptides of
the invention, referred to herein as "coding" nucleic
acid molecules, the present invention also includes complementary
nucleic acid molecules. Thus, for example, both strands of a double
stranded nucleic acid molecule are included within the scope of
the present invention (whether or not they are associated with one
another). Also included are mRNA molecules and complementary DNA
Molecules (e.g. cDNA molecules).
Nucleic acid molecules which can hybridise to any of the nucleic
acid molecules discussed above are also covered by the present invention.
Such nucleic acid molecules are referred to herein as "hybridising"
nucleic acid molecules. Hybridising nucleic acid molecules can be
useful as probes or primers, for example.
Desirably such hybridising molecules are at least 10 nucleotides
in length and preferably are at least 25 or at least 50 nucleotides
in length. The hybridising nucleic acid molecules preferably hybridise
to nucleic acids within the scope of d) (SEQ ID NO: 2), e), f),
g) or h) above specifically.
Desirably the hybridising molecules will hybridise to such molecules
under stringent hybridisation conditions. One example of stringent
hybridisation conditions is where attempted hybridisation is carried
out at a temperature of from about 35.degree. C. to about 65.degree.
C. using a salt solution which is about 0.9 molar. However, the
skilled person will be able to vary such conditions as appropriate
in order to take into account variables such as probe length, base
composition, type of ions present, etc. For a high degree of selectivity,
relatively stringent conditions are used to form the duplexes, such
as low salt or high temperature conditions. As used herein, "highly
stringent conditions" means hybridisation to filter-bound DNA
in 0.5 M NaHPO.sub.4, 7% sodium dodecyl sulphate (SDS), 1 mM EDTA
at 65.degree. C., and washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C. (Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular
Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley
& Sons, Inc., New York, at p. 2.10.3). For some applications,
less stringent conditions for duplex formation are required. As
used herein "moderately stringent conditions" means washing
in 0.2.times.SSC/0.1% SDS at 42.degree. C. (Ausubel et al., 1989,
supra). Hybridisation conditions can also be rendered more stringent
by the addition of increasing amounts of formamide, to destabilise
the hybrid duplex. Thus, particular hybridisation conditions can
be readily manipulated, and will generally be chosen depending on
the desired results. In general, convenient hybridisation temperatures
in the presence of 50% formamide are: 42.degree. C. for a probe
which is 95 to 100% identical to the fragment of a gene encoding
a polypeptide as defined herein, 37.degree. C. for 90 to 95% identity
and 32.degree. C. for 70 to 90% identity. In the preparation of
genomic libraries, DNA fragments are generated, some of which will
encode parts or the whole of a polypeptide as defined herein. The
DNA may be cleaved at specific sites using various restriction enzymes.
Alternatively, one may use DNAse in the presence of manganese to
fragment the DNA, or the DNA can be physically sheared, as for example,
by sonication. The DNA fragments can then be separated according
to size by standard techniques, including but not limited to agarose
and polyacrylamide gel electrophoresis, column chromatography and
sucrose gradient centrifugation. The DNA fragments can then be inserted
into suitable vectors, including but not limited to plasmids, cosmids,
bacteriophages lambda or T.sub.4, and yeast artificial chromosomes
(YACs). (See, for example, Sambrook et al., 2000, Molecular Cloning,
A Laboratory Manual, 3.sup.rd Ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA
Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol.
1, II; Ausubel F. M. et al., eds., 1989, Current Protocols in Molecular
Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley
& sons, Inc., New York). The genomic library may be screened
by nucleic acid hybridisation to labelled probe (Benton & Davis,
1977, Science 196:180; Grunstein & Hogness, 1975, Proc. Natl.
Acad. Sci. U.S.A. 72:3961).
Manipulation of the DNA encoding a polypeptide is a particularly
powerful technique for both modifying proteins and for generating
large quantities of protein for purification purposes. This may
involve the use of PCR techniques to amplify a desired nucleic acid
sequence. Thus the sequence data provided herein can be used to
design primers for use in PCR so that a desired sequence can be
targeted and then amplified to a high degree.
Typically, primers will be at least five nucleotides long and will
generally be at least ten nucleotides long (e.g. fifteen to twenty-five
nucleotides long). In some cases, primers of at least thirty or
at least thirty-five nucleotides in length may be used.
As a further alternative chemical synthesis may be used. This may
be automated. Relatively short sequences may be chemically synthesised
and ligated together to provide a longer sequence.
In addition to being used as primers and/or probes, hybridising
nucleic acid molecules of the present invention can be used as anti-sense
molecules to alter the expression of polypeptides of the present
invention by binding to complementary nucleic acid molecules. This
technique can be used in anti-sense therapy.
As used herein, an "antisense" nucleic acid refers to
a nucleic acid capable of hybridising by virtue of some sequence
complementarity to a portion of an RNA (preferably mRNA) encoding
a polypeptide of the invention. The antisense nucleic acid may be
complementary to a coding and/or non-coding region of a mRNA encoding
a polypeptide of the invention. Such antisense nucleic acids have
utility as compounds that inhibit expression, and can be used in
the treatment or prevention of cancer, in particular breast cancer
and/or kidney cancer.
In a specific embodiment, expression of a polypeptide of the invention
is inhibited by use of antisense nucleic acids. The present invention
provides the therapeutic or prophylactic use of nucleic acids comprising
at least six nucleotides that are antisense to a gene or cDNA encoding
a polypeptide of the invention.
A hybridising nucleic acid molecule of the present invention may
have a high degree of sequence identity along its length with a
nucleic acid molecule within the scope of d)-h) above (e.g. at least
50%, at least 75%, at least 80%, at least 85% or at least 90% or
95% sequence identity). As will be appreciated by the skilled person,
the higher the sequence identity a given single stranded nucleic
acid molecule has with another nucleic acid molecule, the greater
the likelihood that it will hybridise to a nucleic acid molecule
which is complementary to that other nucleic acid molecule under
appropriate conditions.
In view of the foregoing description the skilled person will appreciate
that a large number of nucleic acids are within the scope of the
present invention. Unless the context indicates otherwise, nucleic
acid molecules of the present invention may have one or more of
the following characteristics: 1) they may be DNA or RNA; 2) they
may be single or double stranded; 3) they may be provided in recombinant
form, e.g. covalently linked to a 5' and/or a 3' flanking sequence
to provide a molecule which does not occur in nature; 4) they may
be provided without 5' and/or 3' flanking sequences which normally
occur in nature; 5) they may be provided in substantially pure form.
Thus they may be provided in a form which is substantially free
from contaminating proteins and/or from other nucleic acids; and
6) they may be provided with introns or without introns (e.g. as
cDNA).
If desired, a gene encoding a polypeptide of the invention, a related
gene, or related nucleic acid sequences or subsequences, including
complementary sequences, can also be used in hybridisation assays.
A nucleotide encoding a polypeptide of the invention, or subsequences
thereof comprising at least 8 nucleotides, can be used as a hybridisation
probe. Hybridisation assays can be used for detection, prognosis,
diagnosis, or monitoring of conditions, disorders, or disease states,
associated with aberrant expression of genes encoding a polypeptide
as defined herein, or for differential diagnosis of patients with
signs or symptoms suggestive of cancer, in particular breast cancer
and/or kidney cancer. In particular, such a hybridisation assay
can be carried out by a method comprising contacting a patient sample
containing nucleic acid with a nucleic acid probe capable of hybridising
to a DNA or RNA that encodes a polypeptide of the invention, under
conditions such that hybridisation can occur, and detecting or measuring
any resulting hybridisation. Nucleotides can be used for therapy
of patients having cancer, in particular breast cancer and/or kidney
cancer, as described below.
In another embodiment, a preparation of oligonucleotides comprising
10 or more consecutive nucleotides complementary to a nucleotide
sequence encoding a polypeptide of the invention or fragment thereof
for use as vaccines for the treatment of cancer, in particular breast
cancer and/or kidney cancer. Such preparations may include adjuvants
or other vehicles.
In a specific embodiment, nucleic acids comprising a sequence encoding
a polypeptide of the invention, are administered to promote polypeptide
function by way of gene therapy. Gene therapy refers to administration
to a subject of an expressed or expressible nucleic acid. In this
embodiment, the nucleic acid produces its encoded protein that mediates
a therapeutic effect by promoting polypeptide function. Any of the
methods for gene therapy available in the art can be used according
to the present invention.
In a preferred aspect, the compound comprises a nucleic acid of
the invention, such as a nucleic acid encoding a polypeptide of
the invention, said nucleic acid being part of an expression vector
that expresses a polypeptide of the invention in a suitable host.
In particular, such a nucleic acid has a promoter operably linked
to the polypeptide coding region, said promoter being inducible
or constitutive (and, optionally, tissue-specific). In another particular
embodiment, a nucleic acid molecule is used in which the coding
sequences and any other desired sequences are flanked by regions
that promote homologous recombination at a desired site in the genome,
thus providing for intrachromosomal expression of the nucleic acid
(Koller & Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935;
Zijlstra et al., 1989, Nature 342:435438).
Delivery of the nucleic acid into a patient may be direct, in which
case the patient is directly exposed to the nucleic acid or nucleic
acid-carrying vector; this approach is known as in vivo gene therapy.
Alternatively, delivery of the nucleic acid into the patient may
be indirect, in which case cells are first transformed with the
nucleic acid in vitro and then transplanted into the patient; this
approach is known as ex vivo gene therapy.
As described herein, BCMP 101 is associated with cancer, in particular
breast and kidney cancer and as such provides a means of detection/diagnosis.
Thus, in another aspect, the present invention provides a method
of screening for and/or diagnosis of cancer, in particular breast
cancer and/or kidney cancer in a subject which comprises the step
of detecting and/or quantifying the amount of a polypeptide or nucleic
acid molecule of the invention in a biological sample obtained from
said subject. In a further embodiment, antibodies which recognise
the polypeptides of the invention are used to detect the amount
of a polypeptide as described herein in a biological sample.
In one embodiment, binding of antibody in tissue sections can be
used to detect aberrant polypeptide localisation or an aberrant
level of polypeptide. In a specific embodiment, antibody to a polypeptide
of the invention can be used to assay a patient tissue (e.g., a
breast biopsy) for the level of the polypeptide where an aberrant
level of polypeptide is indicative of cancer, in particular breast
cancer and/or kidney cancer. As used herein, an "aberrant level"
means a level that is increased compared with the level in a subject
free from cancer or a reference level.
Suitable immunoassays include, without limitation, competitive
and non-competitive assay systems using techniques such as western
blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion assays,
agglutination assays, complement-fixation assays, immunoradiometric
assays, fluorescent immunoassays and protein A immunoassays.
In another aspect, the present invention provides a method for
the prophylaxis and/or treatment of cancer, in particular breast
cancer and/or kidney cancer, in a subject, which comprises administering
to said subject a therapeutically effective amount of an antibody
which binds to at least one polypeptide of the invention.
A convenient means for such detection/quantifying will involve
the use of antibodies. Thus, the polypeptides of the invention also
find use in raising antibodies. Thus, in a further aspect, the present
invention provides antibodies, which bind to a polypeptide of the
invention and the use of these antibodies in the preparation of
a composition for use in the prophylaxis and/or treatment of cancer
in particular breast cancer and/or kidney cancer. In particular,
the preparation of vaccines and/or compositions comprising or consisting
of antibodies is a preferred embodiment of this aspect of the invention.
Preferred antibodies bind specifically to polypeptides of the present
invention so that they can be used to purify and/or inhibit the
activity of such polypeptides.
Thus, the polypeptide of the invention, may be used as an immunogen
to generate antibodies which immunospecifically bind such an immunogen.
Antibodies of the invention include, but are not limited to polyclonal,
monoclonal, bispecific, humanised or chimeric antibodies, single
chain antibodies, Fab fragments and F(ab') fragments, fragments
produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies,
and epitope-binding fragments of any of the above. The term "antibody"
as used herein refers to immunoglobulin molecules and immunologically
active portions of immunoglobulin molecules, i.e., molecules that
contain an antigen binding site that specifically binds an antigen.
The immunoglobulin molecules of the invention can be of any class
(e.g., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin
molecule.
In the production of antibodies, screening for the desired antibody
can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked
immunosorbent assay). For example, to select antibodies which recognise
a specific domain of a polypeptide of the invention, one may assay
generated hybridomas for a product which binds to a polypeptide
fragment containing such domain. For selection of an antibody that
specifically binds a first polypeptide homologue but which does
not specifically bind to (or binds less avidly to) a second polypeptide
homologue, one can select on the basis of positive binding to the
first polypeptide homologue and a lack of binding to (or reduced
binding to) the second polypeptide homologue.
For preparation of monoclonal antibodies (mAbs) directed toward
a polypeptide of the invention, any technique which provides for
the production of antibody molecules by continuous cell lines in
culture may be used. For example, the hybridoma technique originally
developed by Kohler and Milstein (1975, Nature 256:495497), as well
as the trioma technique, the human B-cell hybridoma technique (Kozbor
et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique
to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such
antibodies may be of any immunoglobulin class including IgG, IgM,
IgE, IgA, IgD and any subclass thereof. The hybridoma producing
the mAbs of the invention may be cultivated in vitro or in vivo.
In an additional embodiment of the invention, mAbs can be produced
in germ-free animals utilising known technology (PCT/US90/02545).
The mAbs include but are not limited to human mAbs and chimeric
mAbs (e.g., human-mouse chimeras). A chimeric antibody is a molecule
in which different portions are derived from different animal species,
such as those having a human immunoglobulin constant region and
a variable region derived from a murine mAb. (See, e.g., U.S. Pat.
No. 4,816,567; and U.S. Pat. No. 4,816,397) Humanised antibodies
are antibody molecules from non-human species having one or more
complementarity determining regions (CDRs) from the non-human species
and a framework region from a human immunoglobulin molecule. (See,
e.g., U.S. Pat. No. 5,585,089).
Chimeric and humanised mAbs can be produced by recombinant DNA
techniques known in the art, for example using methods described
in WO 87/02671; EP 184,187; EP 171,496; EP 173,494; WO 86/01533;
U.S. Pat. No. 4,816,567; EP 125,023; Better et al., 1988, Science
240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443;
Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc.
Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Canc. Res.
47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et
al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science
229:1202-1207; Oi et al., 1986, Bio/Techniques 4:214; U.S. Pat.
No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan
et al. (1988) Science 239:1534; and Beidler et al., 1988, J. Immunol.
141:4053-4060.
Completely human antibodies are particularly desirable for therapeutic
treatment of human patients. Such antibodies can be produced using
transgenic mice which are incapable of expressing endogenous immunoglobulin
heavy and light chain genes, but which can express human heavy and
light chain genes. The transgenic mice are immunised in the normal
fashion with a selected antigen, e.g., all or a portion of a polypeptide
of the invention. mAbs directed against the antigen can be obtained
using conventional hybridoma technology. The human immunoglobulin
transgenes harboured by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and somatic
mutation. Thus, using such a technique, it is possible to produce
therapeutically useful IgG, IgA, IgM and IgE antibodies. For an
overview of this technology for producing human antibodies, see
Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed
discussion of this technology for producing human antibodies and
human mAbs and protocols for producing such antibodies, see, e.g.,
U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806.
Completely human antibodies which recognise a selected epitope
can be generated using a technique referred to as "guided selection".
In this approach a selected non-human mAb, e.g., a mouse antibody,
is used to guide the selection of a completely human antibody recognising
the same epitope. (Jespers et al. (1994) Bio/technology 12:899-903).
The antibodies of the present invention can also be generated using
various phage display methods known in the art. In phage display
methods, functional antibody domains are displayed on the surface
of phage particles which carry the polynucleotide sequences encoding
them. In particular, such phage can be utilised to display antigen-binding
domains expressed from a repertoire or combinatorial antibody library
(e.g., human or murine). Phage expressing an antigen binding domain
that binds the antigen of interest can be selected or identified
with antigen, e.g., using labelled antigen or antigen bound or captured
to a solid surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulphide stabilised Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII protein.
Phage display methods that can be used to make the antibodies of
the present invention include those disclosed in Brinkman et al.,
J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods
184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958
(1994); Persic et al., Gene 187 9-18 (1997) Burton et al., Advances
in Immunology 57:191-280 (1994); PCT/GB91/01134; WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108.
As described in the above references, after phage selection, the
antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any desired
host, including mammalian cells, insect cells, plant cells, yeast,
and bacteria, e.g., as described in detail below. For example, techniques
to recombinantly produce Fab, Fab' and F(ab')2 fragments can also
be employed using methods known in the art such as those disclosed
in WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992);
and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science
240:1041-1043 (1988).
Examples of techniques which can be used to produce single-chain
Fvs and antibodies include those described in U.S. Pat. No. 4,946,778
and U.S. Pat. No. 5,258,498; Huston et al., Methods in Enzymology
203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra
et al., Science 240:1038-1040 (1988).
The invention further provides for the use of bispecific antibodies,
which can be made by methods known in the art. Traditional production
of full-length bispecific antibodies is based on the coexpression
of two immunoglobulin heavy chain-light chain pairs, where the two
chains have different specificities (Milstein et al., 1983, Nature
305:537-539). Because of the random assortment of immunoglobulin
heavy and light chains, these hybridomas (quadromas) produce a potential
mixture of 10 different antibody molecules, of which only one has
the correct bispecific structure. Purification of the correct molecule,
which is usually done by affinity chromatography steps, is rather
cumbersome, and the product yields are low. Similar procedures are
disclosed in WO 93/08829, and in Traunecker et al., 1991, EMBO J.
10:3655-3659.
According to a different and more preferred approach, antibody
variable domains with the desired binding specificities (antibody-antigen
combining sites) are fused to immunoglobulin constant domain sequences.
The fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3 regions.
It is preferred to have the first heavy-chain constant region (CH1)
containing the site necessary for light chain binding, present in
at least one of the fusions. DNAs encoding the immunoglobulin heavy
chain fusions and, if desired, the immunoglobulin light chain, are
inserted into separate expression vectors, and are co-transfected
into a suitable host organism. This provides for great flexibility
in adjusting the mutual proportions of the three polypeptide fragments
in embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yields. It is, however,
possible to insert the coding sequences for two or all three polypeptide
chains in one expression vector when the expression of at least
two polypeptide chains in equal ratios results in high yields or
when the ratios are of no particular significance.
In a preferred embodiment of this approach, the bispecific antibodies
are composed of a hybrid immunoglobulin heavy chain with a first
binding specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity)
in the other arm. It was found that this asymmetric structure facilitates
the separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations, as the presence of an immunoglobulin
light chain in only one half of the bispecific molecule provides
for a facile way of separation. This approach is disclosed in WO
94/04690. For further details for generating bispecific antibodies
see, for example, Suresh et al., Methods in Enzymology, 1986, 121:210.
The invention provides functionally active fragments, derivatives
or analogs of the anti-polypeptide immunoglobulin molecules. "Functionally
active" means that the fragment, derivative or analogue is
able to elicit anti-anti-idiotype antibodies (i.e., tertiary antibodies)
that recognise the same antigen that is recognised by the antibody
from which the fragment, derivative or analogue is derived. Specifically,
in a preferred embodiment the antigenicity of the idiotype of the
immunoglobulin molecule may be enhanced by deletion of framework
and CDR sequences that are C-terminal to the CDR sequence that specifically
recognises the antigen. To determine which CDR sequences bind the
antigen, synthetic peptides containing the CDR sequences can be
used in binding assays with the antigen by any binding assay method
known in the art.
The present invention provides antibody fragments such as, but
not limited to, F(ab')2 fragments and Fab fragments. Antibody fragments
which recognise specific epitopes may be generated by known techniques.
F(ab')2 fragments consist of the variable region, the light chain
constant region and the CH1 domain of the heavy chain and are generated
by pepsin digestion of the antibody molecule. Fab fragments are
generated by reducing the disulphide bridges of the F(ab').sub.2
fragments. The invention also provides heavy chain and light chain
dimers of the antibodies of the invention, or any minimal fragment
thereof such as Fvs or single chain antibodies (SCAs) (e.g., as
described in U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:42342;
Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and
Ward et al., 1989, Nature 334:544-54), or any other molecule with
the same specificity as the antibody of the invention. Single chain
antibodies are formed by linking the heavy and light chain fragments
of the Fv region via an amino acid bridge, resulting in a single
chain polypeptide. Techniques for the assembly of functional Fv
fragments in E. coli may be used (Skerra et al., 1988, Science 242:1038-1041).
In other embodiments, the invention provides fusion polypeptides
of the immunoglobulins of the invention (or functionally active
fragments thereof), for example in which the immunoglobulin is fused
via a covalent bond (e.g., a peptide bond), at either the N-terminus
or the C-terminus to an amino acid sequence of another polypeptide
(or portion thereof, preferably at least 10, 20 or 50 amino acid
portion of the polypeptide) that is not the immunoglobulin. Preferably
the immunoglobulin, or fragment thereof, is covalently linked to
the other polypeptide at the N-terminus of the constant domain.
As stated above, such fusion polypeptides may facilitate purification,
increase half-life in vivo, and enhance the delivery of an antigen
across an epithelial barrier to the immune system.
The immunoglobulins of the invention include analogues and derivatives
that are either modified, i.e., by the covalent attachment of any
type of molecule as long as such covalent attachment that does not
impair immunospecific binding. For example, but not by way of limitation,
the derivatives and analogues of the immunoglobulins include those
that have been further modified, e.g., by glycosylation, acetylation,
pegylation, phosphylation, amidation, derivatisation by known protecting/blocking
groups, proteolytic cleavage, linkage to a cellular ligand or other
protein, etc. Any of numerous chemical modifications may be carried
out by known techniques, including, but not limited to specific
chemical cleavage, acetylation, formylation, etc. Additionally,
the analogue or derivative may contain one or more non-classical
amino acids.
The foregoing antibodies can be used in methods known in the art
relating to the localisation and activity of the polypeptides of
the invention, e.g., for imaging or radioimaging these polypeptides,
measuring levels thereof in appropriate biological samples, in diagnostic
methods, etc. and for radiotherapy.
The antibodies of the invention can be produced by any method known
in the art for the synthesis of antibodies, in particular, by chemical
synthesis or by recombinant expression, and are preferably produced
by recombinant expression technique.
Recombinant expression of antibodies, or fragments, derivatives
or analogs thereof, requires construction of a nucleic acid that
encodes the antibody. If the nucleotide sequence of the antibody
is known, a nucleic acid encoding the antibody may be assembled
from chemically synthesised oligonucleotides (e.g., as described
in Kutmeier et al., 1994, BioTechniques 17:242), which, briefly,
involves the synthesis of overlapping oligonucleotides containing
portions of the sequence encoding antibody, annealing and ligation
of those oligonucleotides, and then amplification of the ligated
oligonucleotides by PCR.
Alternatively, the nucleic acid encoding the antibody may be obtained
by cloning the antibody. If a clone containing the nucleic acid
encoding the particular antibody is not available, but the sequence
of the antibody molecule is known, a nucleic acid encoding the antibody
may be obtained from a suitable source (e.g., an antibody cDNA library,
or cDNA library generated from any tissue or cells expressing the
antibody) by PCR amplification using synthetic primers hybridizable
to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide
probe specific for the particular gene sequence.
If an antibody molecule that specifically recognises a particular
antigen is not available (or a source for a cDNA library for cloning
a nucleic acid encoding such an antibody), antibodies specific for
a particular antigen may be generated by any method known in the
art, for example, by immunising an animal, such as a rabbit, to
generate polyclonal antibodies or, more preferably, by generating
monoclonal antibodies. Alternatively, a clone encoding at least
the Fab portion of the antibody may be obtained by screening Fab
expression libraries (e.g., as described in Huse et al., 1989, Science
246:1275-1281) for clones of Fab fragments that bind the specific
antigen or by screening antibody libraries (See, e.g., Clackson
et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad.
Sci. USA 94:4937).
Once a nucleic acid encoding at least the variable domain of the
antibody molecule is obtained, it may be introduced into a vector
containing the nucleotide sequence encoding the constant region
of the antibody molecule (see, e.g., WO 86/05807; WO 89/01036; and
U.S. Pat. No. 5,122,464). Vectors containing the complete light
or heavy chain for co-expression with the nucleic acid to allow
the expression of a complete antibody molecule are also available.
Then, the nucleic acid encoding the antibody can be used to introduce
the nucleotide substitution(s) or deletion(s) necessary to substitute
(or delete) the one or more variable region cysteine residues participating
in an intrachain disulphide bond with an amino acid residue that
does not contain a sulfhydyl group. Such modifications can be carried
out by any method known in the art for the introduction of specific
mutations or deletions in a nucleotide sequence, for example, but
not limited to, chemical mutagenesis, in vitro site directed mutagenesis
(Hutchinson et al., 1978, J. Biol. Chem. 253:6551), PCR based methods,
etc.
In addition, techniques developed for the production of "chimeric
antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci.
81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et
al., 1985, Nature 314:452454) by splicing genes from a mouse antibody
molecule of appropriate antigen specificity together with genes
from a human antibody molecule of appropriate biological activity
can be used. As described supra, a chimeric antibody is a molecule
in which different portions are derived from different animal species,
such as those having a variable region derived from a murine mAb
and a human antibody constant region, e.g., humanised antibodies.
Once a nucleic acid encoding an antibody of the invention has been
obtained, the vector for the production of the antibody may be produced
by recombinant DNA technology using techniques well known in the
art. Thus, methods for preparing the polypeptides of the invention
by expressing nucleic acid containing the antibody molecule sequences
are described herein. Methods which are well known to those skilled
in the art can be used to construct expression vectors containing
an antibody molecule coding sequences and appropriate transcriptional
and translational control signals. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic recombination. See, for example, the techniques described
in Sambrook et al. (1990, Molecular Cloning, A Laboratory Manual,
2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)
and Ausubel et al. (eds., 1998, Current Protocols in Molecular Biology,
John Wiley & Sons, NY).
The expression vector is transferred to a host cell by conventional
techniques and the transfected cells are then cultured by conventional
techniques to produce an antibody of the invention.
The host cells used to express a recombinant antibody of the invention
may be either bacterial cells such as E. coli, or, preferably, eukaryotic
cells, especially for the expression of whole recombinant antibody
molecule. In particular, mammalian cells such as Chinese hamster
ovary cells (CHO); in conjunction with a vector such as the major
intermediate early gene promoter element from human cytomegalovirus
is an effective expression system for antibodies (Foecking et al.,
1986, Gene 45: 101; Cockett et al., 1990, Bio/Technology 8:2).
A variety of host-expression vector systems may be utilised to
express an antibody molecule of the invention. Such host-expression
systems represent vehicles by which the coding sequences of interest
may be produced and subsequently purified, but also represent cells
which may, when transformed or transfected with the appropriate
nucleotide coding sequences, express the antibody molecule of the
invention in situ. These include but are not limited to microorganisms
such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing antibody coding sequences; yeast (e.g., Saccharomyces,
Pichia) transformed with recombinant yeast expression vectors containing
antibody coding sequences; insect cell systems infected with recombinant
virus expression vectors (e.g., baculovirus) containing the antibody
coding sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or transformed with recombinant plasmid expression
vectors (e.g., Ti plasmid) containing antibody coding sequences;
or mammalian cell systems (e.g., COS, CHO, BHK, HEK 293, 3T3 cells)
harbouring recombinant expression constructs containing promoters
derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the adenovirus late promoter;
the vaccinia virus 7.5K promoter).
In bacterial systems, a number of expression vectors may be advantageously
selected depending upon the use intended for the antibody molecule
being expressed. For example, when a large quantity of such a polypeptide
is to be produced, for the generation of pharmaceutical compositions
comprising an antibody molecule, vectors which direct the expression
of high levels of fusion polypeptide products that are readily purified
may be desirable. Such vectors include, but are not limited, to
the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO
J. 2:1791), in which the antibody coding sequence may be ligated
individually into the vector in frame with the lac Z coding region
so that a fusion polypeptide is produced; pIN vectors (Inouye &
Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,
1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may
also be used to express foreign polypeptides as fusion polypeptides
with glutathione S-transferase (GST). In general, such fusion polypeptides
are soluble and can easily be purified from lysed cells by adsorption
and binding to a matrix glutathione-agarose beads followed by elution
in the presence of free glutathione. The pGEX vectors are designed
to include thrombin or factor Xa protease cleavage sites so that
the cloned target gene product can be released from the GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis
virus (AcNPV) is used as a vector to express foreign genes. The
virus grows in Spodoptera frugiperda cells. The antibody coding
sequence may be cloned individually into non-essential regions (for
example the polyhedrin gene) of the virus and placed under control
of an AcNPV promoter (for example the polyhedrin promoter). In mammalian
host cells, a number of viral-based expression systems (e.g., an
adenovirus expression system) may be utilised.
As discussed above, a host cell strain may be chosen which modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Such modifications
(e.g., glycosylation) and processing (e.g., cleavage) of polypeptide
products may be important for the function of the polypeptide.
For long-term, high-yield production of recombinant antibodies,
stable expression is preferred. For example, cells lines that stably
express an antibody of interest can be produced by transfecting
the cells with an expression vector comprising the nucleotide sequence
of the antibody and the nucleotide sequence of a selectable (e.g.,
neomycin or hygromycin), and selecting for expression of the selectable
marker. Such engineered cell lines may be particularly useful in
screening and evaluation of agents that interact directly or indirectly
with the antibody molecule.
The expression levels of the antibody molecule can be increased
by vector amplification (for a review, see Bebbington and Hentschel,
The use of vectors based on gene amplification for the expression
of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic
Press, New York, 1987)). When a marker in the vector system expressing
antibody is amplifiable, increase in the level of inhibitor present
in culture of host cell will increase the number of copies of the
marker gene. Since the amplified region is associated with the antibody
gene, production of the antibody will also increase (Crouse et al.,
1983, Mol. Cell. Biol. 3:257).
The host cell may be co-transfected with two expression vectors
of the invention, the first vector encoding a heavy chain derived
polypeptide and the second vector encoding a light chain derived
polypeptide. The two vectors may contain identical selectable markers
which enable equal expression of heavy and light chain polypeptides.
Alternatively, a single vector may be used which encodes both heavy
and light chain polypeptides. In such situations, the light chain
should be placed before the heavy chain to avoid an excess of toxic
free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980,
Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the
heavy and light chains may comprise cDNA or genomic DNA.
Once the antibody molecule of the invention has been recombinantly
expressed, it may be purified by any method known in the art for
purification of an antibody molecule, for example, by chromatography
(e.g., ion exchange chromatography, affinity chromatography such
as with protein A or specific antigen, and sizing column chromatography),
centrifugation, differential solubility, or by any other standard
technique for the purification of polypeptides.
Alternatively, any fusion polypeptide may be readily purified by
utilising an antibody specific for the fusion polypeptide being
expressed. For example, a system described by Janknecht et al. allows
for the ready purification of non-denatured fusion polypeptides
expressed in human cell lines (Janknecht et al., 1991, Proc. Natl.
Acad. Sci. USA 88:8972-897). In this system, the gene of interest
is subcloned into a vaccinia recombination plasmid such that the
open reading frame of the gene is translationally fused to an amino-terminal
tag consisting of six histidine residues. The tag serves as a matrix
binding domain for the fusion polypeptide. Extracts from cells infected
with recombinant vaccinia virus are loaded onto Ni.sup.2+ nitriloacetic
acid-agarose columns and histidine-tagged proteins are selectively
eluted with imidazole-containing buffers.
In a preferred embodiment, antibodies of the invention or fragments
thereof are conjugated to a diagnostic or therapeutic moiety. The
antibodies can be used for diagnosis or to determine the efficacy
of a given treatment regimen. Detection can be facilitated by coupling
the antibody to a detectable substance. Examples of detectable substances
include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, radioactive nuclides,
positron emitting metals (for use in positron emission tomography),
and nonradioactive paramagnetic metal ions. See generally U.S. Pat.
No. 4,741,900 for metal ions which can be conjugated to antibodies
for use as diagnostics according to the present invention. Suitable
enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase,
or acetylcholinesterase; suitable prosthetic groups include streptavidin,
avidin and biotin; suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride and phycoerythrin; suitable luminescent
materials include luminol; suitable bioluminescent materials include
luciferase, luciferin, and aequorin; and suitable radioactive nuclides
include .sup.125I, .sup.131I, .sup.111In and .sup.99Tc.
Antibodies of the invention or fragments thereof can be conjugated
to a therapeutic agent or drug moiety to modify a given biological
response. The therapeutic agent or drug moiety is not to be construed
as limited to classical chemical therapeutic agents. For example,
the drug moiety may be a protein or polypeptide possessing a desired
biological activity. Such proteins may include, for example, a toxin
such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;
a polypeptide such as tumour necrosis factor, .alpha.-interferon,
.beta.-interferon, nerve growth factor, platelet derived growth
factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic
agent, e.g., angiostatin or endostatin; or, a biological response
modifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2
(IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating
factor (GM-CSF), granulocyte colony stimulating factor (G-CSF),
nerve growth factor (NGF) or other growth factor.
Techniques for conjugating such therapeutic moieties to antibodies
are well known, see, e.g., Arnon et al., "Monoclonal Antibodies
For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal
Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56
(Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For
Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody
Carriers Of Cytotoxic Agents In Cancer Therapy: A Review",
in Monoclonal Antibodies '84: Biological And Clinical Applications,
Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results,
And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody
In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection
And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985),
and Thorpe et al., "The Preparation And Cytotoxic Properties
Of Antibody-Toxin Conjugates", Immunol. Rev., 62:119-58 (1982).
Alternatively, an antibody can be conjugated to a second antibody
to form an antibody heteroconjugate as described in U.S. Pat. No.
4,676,980.
An antibody with or without a therapeutic moiety conjugated to
it can be used as a therapeutic that is administered alone or in
combination with cytotoxic factor(s) and/or cytokine(s).
Polyclonal antibodies can be raised by stimulating their production
in a suitable animal host (e.g. a chicken, mouse, rat, guinea pig,
rabbit, sheep, goat or monkey) when the polypeptide of the present
invention is injected into the animal. If necessary, an adjuvant
may be administered together with the polypeptide of the invention.
The antibodies can then be purified by virtue of their binding to
a polypeptide of the invention.
Monoclonal antibodies can be produced from hybridomas. These can
be formed by fusing myeloma cells and spleen cells which produce
the desired antibody in order to form an immortal cell line. This
is the well known Kohler & Milstein technique (Nature 256 52-55
(1975)).
A further aspect of the invention provides methods of screening
for agents that modulate (e.g., upregulate or downregulate) a characteristic
of, e.g., the expression or the enzymatic or binding activity, of
a polypeptide of the invention.
The invention provides methods for identifying active agents (e.g.,
chemical compounds, proteins, or peptides) that bind to a polypeptide
of the invention and/or have a stimulatory or inhibitory effect
on the expression or activity of a polypeptide of the invention.
Examples of candidate agents, include, but are not limited to, nucleic
acids (e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides,
peptidomimetics, agonists, antagonists, small molecules and other
drugs. Active agents can be obtained using any of the numerous suitable
approaches in combinatorial library methods known in the art, including:
biological libraries; spatially addressable parallel solid phase
or solution phase libraries; synthetic library methods requiring
deconvolution; the "one-bead one-compound" library method;
and synthetic library methods using affinity chromatography selection.
The biological library approach is limited to peptide libraries,
while the other four approaches are applicable to peptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer
Drug Des. 12:145; U.S. Pat. No. 5,738,996; and U.S. Pat. No. 5,807,683)
Examples of methods for the synthesis of molecular libraries can
be found in the art, for example in: DeWitt et al., 1993, Proc.
Natl. Acad. Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad.
Sci. USA 91:11422; Zuckermann et al., 1994, J. Med. Chem. 37:2678;
Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew.
Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem.
Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem. 37:1233.
Libraries of compounds may be presented, e.g., presented in solution
(e.g., Houghten, 1992, Bio/Techniques 13:412421), or on beads (Lam,
1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556),
bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698;
5,403,484; and 5,223,409), plasmids (Cull et al., 1992, Proc. Natl.
Acad. Sci. USA 89:1865-1869) or phage (Scott and Smith, 1990, Science
249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990,
Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J. Mol.
Biol. 222:301-310).
In one embodiment, agents that interact with (i.e., bind to) a
polypeptide of the invention are identified in a cell-based assay
system. In accordance with this embodiment, cells expressing a polypeptide
of the invention are contacted with a candidate agent or a control
agent and the ability of the candidate agent to interact with said
polypeptide is determined. If desired, this assay may be used to
screen a plurality (e.g. a library) of candidate agents. The cell,
for example, can be of prokaryotic origin (e.g., E. coli) or eukaryotic
origin (e.g., yeast or mammalian). Further, the cells can express
the polypeptide of the invention endogenously or be genetically
engineered to express said polypeptide. In some embodiments, the
polypeptide of the invention or the candidate agent is labelled,
for example with a radioactive label (such as .sup.32p, .sup.35S
or .sup.125I) or a fluorescent label (such as fluorescein isothiocyanate,
rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde
or fluorescamine) to enable detection of an interaction between
a polypeptide and a candidate agent. The ability of the candidate
agent to interact directly or indirectly with the polypeptide of
the invention can be determined by methods known to those of skill
in the art. For example, but without limitation, the interaction
between a candidate agent and a polypeptide of the invention can
be determined by flow cytometry, a scintillation assay, immunoprecipitation
or western blot analysis.
In another embodiment, agents that interact with (i.e., bind to)
a polypeptide of the invention are identified in a cell-free assay
system. In accordance with this embodiment, a native or recombinant
polypeptide of the invention is contacted with a candidate agent
or a control agent and the ability of the candidate agent to interact
with said polypeptide is determined. If desired, this assay may
be used to screen a plurality (e.g. a library) of candidate agents.
Preferably, the polypeptide is first immobilised, by, for example,
contacting the polypeptide with an immobilised antibody which specifically
recognises and binds it, or by contacting a purified preparation
of polypeptide with a surface designed to bind proteins. The polypeptide
may be partially or completely purified (e.g., partially or completely
free of other polypeptides) or part of a cell lysate. Further, the
polypeptide may be a fusion polypeptide comprising the polypeptide
of the invention and a domain such as glutathionine-S-transferase.
Alternatively, the polypeptide can be biotinylated using techniques
well known to those of skill in the art (e.g., biotinylation kit,
Pierce Chemicals; Rockford, Ill.). The ability of the candidate
agent to interact with the polypeptide can be can be duplicated
by methods known to those of skill in the art.
In another embodiment, a cell-based assay system is used to identify
active agents that bind to and/or modulate the activity of a protein,
such as an enzyme, or a biologically active portion thereof, which
is responsible for the production or degradation of the polypeptide
of the invention or is responsible for the post-translational modification
of the polypeptide. In a primary screen, a plurality (e.g., a library)
of agents are contacted with cells that naturally or recombinantly
express: (i) a polypeptide of the invention; and (ii) a protein
that is responsible for processing of the polypeptide in order to
identify compounds that modulate the production, degradation, or
post-translational modification of the polypeptide. If desired,
active agents identified in the primary screen can then be assayed
in a secondary screen against cells naturally or recombinantly expressing
the specific polypeptide of interest. The ability of the candidate
agent to modulate the production, degradation or post-translational
modification of a polypeptide can be determined by methods known
to those of skill in the art, including without limitation, flow
cytometry, a scintillation assay, immunoprecipitation and western
blot analysis.
In another embodiment, agents that competitively interact with
a polypeptide of the invention are identified in a competitive binding
assay. In accordance with this embodiment, cells expressing the
polypeptide are contacted with a candidate agent and as agent known
to interact with the polypeptide; the ability of the candidate agent
to competitively interact with the polypeptide is then determined.
Alternatively, agents that competitively interact with a polypeptide
of the invention are identified in a cell-free assay system by contacting
said polypeptide with a candidate agent and an agent known to interact
with the polypeptide. As stated above, the ability of the candidate
agent to interact with a polypeptide of the invention can be determined
by methods known to those of skill in the art. These assays, whether
cell-based or cell-free, can be used to screen a plurality (e.g.,
a library) of candidate agents.
In another embodiment, agents that modulate (i.e., upregulate or
down-regulate) the expression of a polypeptide of the invention
are identified by contacting cells (e.g., cells of prokaryotic origin
or eukaryotic origin) expressing the polypeptide with a candidate
agent or a control agent (e.g., phosphate buffered saline (PBS))
and determining the expression of the polypeptide, or mRNA encoding
the polypeptide. The level of expression of a selected polypeptide,
or mRNA encoding polypeptide, in the presence of the candidate agent
is compared to the level of expression of the polypeptide or mRNA
encoding the polypeptide in the absence of the candidate agent (e.g.,
in the presence of a control agent). The candidate agent can then
be identified as a modulator of the expression of the polypeptide
based on this comparison. For example, when expression of the polypeptide,
or mRNA encoding the polypeptide, is significantly greater in the
presence of the candidate agent than in its absence, the candidate
agent is identified as a stimulator of expression of the polypeptide,
or mRNA encoding the polypeptide. Alternatively, when expression
of the polypeptide, or mRNA encoding the polypeptide, is significantly
less in the presence of the candidate agent than in its absence,
the candidate agent is identified as an inhibitor of the expression
of the polypeptide or mRNA encoding the polypeptide. The level of
expression of a polypeptide of the invention or the mRNA that encodes
it can be determined by methods known to those of skill in the art
based on the present description. For example, mRNA expression can
be assessed by Northern blot analysis or RT-PCR, and protein levels
can be assessed by western blot analysis.
In another embodiment, active agents that modulate the activity
of a polypeptide of the invention are identified by contacting a
preparation containing the polypeptide, or cells (e.g., prokaryotic
or eukaryotic cells) expressing the polypeptide with a candidate
agent or a control agent and determining the ability of the candidate
agent to modulate (e.g., stimulate or inhibit) the activity of polypeptide.
The activity of a polypeptide can be assessed by detecting its effect
on a "downstream effector" for example, but without limitation,
induction of a cellular signal transduction pathway of the polypeptide,
detecting catalytic or enzymatic activity of the target on a suitable
substrate, detecting the induction of a reporter gene (e.g., a regulatory
element that is responsive to a polypeptide of the invention and
is operably linked to a nucleic acid encoding a detectable marker,
e.g., luciferase), or detecting a cellular response, for example,
cellular differentiation, or cell proliferation as the case may
be, based on the present description, techniques known to those
of skill in the art can be used for measuring these activities (see,
e.g., U.S. Pat. No. 5,401,639). The candidate agent can then be
identified as a modulator of the activity of a polypeptide of the
invention by comparing the effects of the candidate agent to the
control agent. Suitable control agents include phosphate buffered
saline (PBS) and normal saline (NS).
In another embodiment, active agents that modulate (i.e., upregulate
or downregulate) the expression, activity or both the expression
and activity of a polypeptide of the invention are identified in
an animal model. Examples of suitable animals include, but are not
limited to, mice, rats, rabbits, monkeys, guinea pigs, dogs and
cats. Preferably, the animal used represents a model of breast cancer
and/or kidney cancer. In accordance with this embodiment, the candidate
agent or a control agent is administered (e.g., orally, rectally
or parenterally such as intraperitoneally or intravenously) to a
suitable animal and the effect on the expression, activity or both
expression and activity of the polypeptide is determined. Changes
in the expression of a polypeptide can be assessed by any suitable
method described above, based on the present description.
In yet another embodiment, a polypeptide of the invention is used
as a "bait protein" in a two-hybrid assay or three-hybrid
assay to identify other proteins that bind to or interact with the
polypeptide (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993)
Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054;
Bartel et al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al.
(1993) Oncogene 8:1693-1696; and WO 94/10300). As those skilled
in the art will appreciate, such binding proteins are also likely
to be involved in the propagation of signals by the polypeptides
of the invention as, for example, upstream or downstream elements
of a signalling pathway involving the polypeptides of the invention.
Thus, the present invention provides assays for use in drug discovery
in order to identify or verify the efficacy of agents for treatment
or prevention of cancer, in particular breast cancer and/or kidney
cancer. Candidate agents can be assayed for their ability to modulate
levels of a polypeptide of the invention, in a subject having breast
cancer and/or kidney cancer. Active agents able to modulate levels
of a polypeptide of the invention in a subject having breast cancer
and/or kidney cancer towards levels found in subjects free from
breast cancer and/or kidney cancer, or to produce similar changes
in experimental animal models of breast cancer and/or kidney cancer,
can be used as lead agents for further drug discovery, or used therapeutically.
Expression of a polypeptide of the invention can be assayed by,
for example, immunoassays, gel electrophoresis followed by visualization,
detection of activity, or any other method taught herein or known
to those skilled in the art. Such assays can be used to screen candidate
drugs, in clinical monitoring or in drug development, where abundance
of a polypeptide of the invention can serve as a surrogate marker
for clinical disease.
One skilled in the art will also appreciate that a polypeptide
of the invention above may be used in a method for the structure-based
design of an agent, in particular a small molecule which acts to
modulate (e.g. stimulate or inhibit) the activity of said polypeptide,
said method comprising: 1) determining the three-dimensional structure
of said polypeptide; 2) deducing the three-dimensional structure
of the likely reactive or binding site(s) of the agent; 3) synthesizing
candidate agents that are predicted to react or bind to the deduced
reactive or binding site; and 4) testing whether the candidate agent
is able to modulate the activity of said polypeptide.
It will be appreciated that the method described above is likely
to be an iterative process.
This invention further provides novel active agents identified
by the above-described screening methods and uses thereof for treatments
as described herein.
As used herein, the term "active agent" refers to the
polypeptides of the invention and nucleic acid molecules encoding
the polypeptides, antibodies against the polypeptides and agents
which modulate the expression and/or activity of the polypeptides
of the invention. Preferably, the active agent is a small molecule.
As discussed herein, active agents of the invention find use in
the treatment or prophylaxis of breast and/or kidney cancer.
Thus, in an additional aspect, the present invention provides a
pharmaceutical composition comprising at least one active agent
of the invention, optionally together with one or more pharmaceutically
acceptable excipients, carriers or diluents. In one aspect, the
pharmaceutical composition is for use as a vaccine and so any additional
components will be acceptable for vaccine use. In addition, the
skilled person will appreciate that one or more suitable adjuvants
may be added to such vaccine preparations.
The composition will usually be supplied as part of a sterile,
pharmaceutical composition which will normally include a pharmaceutically
acceptable carrier. This pharmaceutical composition may be in any
suitable form, (depending upon the desired method of administering
it to a patient).
It may be provided in unit dosage form, will generally be provided
in a sealed container and may be provided as part of a kit. Such
a kit would normally (although not necessarily) include instructions
for use. It may include a plurality of said unit dosage forms.
The pharmaceutical composition may be adapted for administration
by any appropriate route, for example by the oral (including buccal
or sublingual), rectal, nasal, topical (including buccal, sublingual
or transdermal), vaginal or parenteral (including subcutaneous,
intramuscular, intravenous or intradermal) route. Such compositions
may be prepared by any method known in the art of pharmacy, for
example by admixing the active ingredient with the carrier(s) or
excipient(s) under sterile conditions.
Pharmaceutical compositions adapted for oral administration may
be presented as discrete units such as capsules or tablets; as powders
or granules; as solutions, syrups or suspensions (in aqueous or
non-aqueous liquids; or as edible foams or whips; or as emulsions).
Suitable excipients for tablets or hard gelatine capsules include
lactose, maize starch or derivatives thereof, stearic acid or salts
thereof.
Suitable excipients for use with soft gelatine capsules include
for example vegetable oils, waxes, fats, semi-solid, or liquid polyols
etc.
For the preparation of solutions and syrups, excipients which may
be used include for example water, polyols and sugars. For the preparation
of suspensions, oils (e.g. vegetable oils) may be used to provide
oil-in-water or water-in-oil suspensions.
Pharmaceutical compositions adapted for transdermal administration
may be presented as discrete patches intended to remain in intimate
contact with the epidermis of the recipient for a prolonged period
of time. For example, the active ingredient may be delivered from
the patch by iontophoresis as generally described in Pharmaceutical
Research, 3(6):318 (1986).
Pharmaceutical compositions adapted for topical administration
may be formulated as ointments, creams, suspensions, lotions, powders,
solutions, pastes, gels, sprays, aerosols or oils. When formulated
in an ointment, the active ingredient may be employed with either
a paraffinic or a water-miscible ointment base. Alternatively, the
active ingredient may be formulated in a cream with an oil-in-water
cream base or a water-in-oil base. Pharmaceutical compositions adapted
for topical administration to the eye include eye drops wherein
the active ingredient is dissolved or suspended in a suitable carrier,
especially an aqueous solvent. Pharmaceutical compositions adapted
for topical administration in the mouth include lozenges, pastilles
and mouth washes.
Pharmaceutical compositions adapted for rectal administration may
be presented as suppositories or enemas.
Pharmaceutical compositions adapted for nasal administration wherein
the carrier is a solid include a coarse powder having a particle
size for example in the range 20 to 500 microns which is administered
in the manner in which snuff is taken, i.e. by rapid inhalation
through the nasal passage from a container of the powder held close
up to the nose. Suitable compositions wherein the carrier is a liquid,
for administration as a nasal spray or as nasal drops, include aqueous
or oil solutions of the active ingredient.
Pharmaceutical compositions adapted for administration by inhalation
include fine particle dusts or mists which may be generated by means
of various types of metered dose pressurised aerosols, nebulizers
or insufflators.
Pharmaceutical compositions adapted for vaginal administration
may be presented as pessaries, tampons, creams, gels, pastes, foams
or spray compositions.
Pharmaceutical compositions adapted for parenteral administration
include aqueous and non-aqueous sterile injection solution which
may contain anti-oxidants, buffers, bacteriostats and solutes which
render the composition substantially isotonic with the blood of
the intended recipient; and aqueous and non-aqueous sterile suspensions
which may include suspending agents and thickening agents. Excipients
which may be used for injectable solutions include water, alcohols,
polyols, glycerine and vegetable oils, for example. The compositions
may be presented in unit-dose or multi-dose containers, for example
sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised)
condition requiring only the addition of the sterile liquid carried,
for example water for injections, immediately prior to use. Extemporaneous
injection solutions and suspensions may be prepared from sterile
powders, granules and tablets.
The pharmaceutical compositions may contain preserving agents,
solubilising agents, stabilising agents, wetting agents, emulsifiers,
sweeteners, colorants, odorants, salts (polypeptides of the present
invention may themselves be provided in the form of a pharmaceutically
acceptable salt), buffers, coating agents or antioxidants. They
may also contain therapeutically active agents in addition to the
polypeptide of the present invention.
Dosages of the active agent of the present invention can vary between
wide limits, depending upon the disease or disorder to be treated,
the age and condition of the individual to be treated, etc. and
a physician will ultimately determine appropriate dosages to be
used. This dosage may be repeated as often as appropriate. If side
effects develop the amount and/or frequency of the dosage can be
reduced, in accordance with normal clinical practice.
In a further aspect, the present invention provides a method for
the prophylaxis and/or treatment of breast and/or kidney cancer
in a subject, which comprises administering to said subject a therapeutically
effective amount of at least active agent of the invention.
In another aspect, the present invention provides the use of at
least one polypeptide or fragment thereof, nucleic acid molecule
or antibody of the invention in the preparation of a medicament
for use in the prophylaxis and/or treatment of breast and/or kidney
cancer. In particular, the preparation of vaccines and/or compositions
comprising or consisting of antibodies is a preferred embodiment
of this aspect of the invention.
In the context of the present invention, the biological sample
can be obtained from any source, such as a serum sample or a tissue
sample, e.g. breast or kidney tissue. When looking for evidence
of metastasis, one would look at major sites of breast metastasis
such as lymph nodes, liver, lung and/or bone and of kidney metastasis,
such as lymph nodes, lung and/or bone.
Preferred features of each aspect of the invention are as for each
of the other aspects mutatis mutandis. The prior art documents mentioned
herein are incorporated by reference to the fullest extent permitted
by law.
The invention will now be described with reference to the following
examples, which should not in any way be construed as limiting the
scope of the present invention. The examples refer to the figures
in which:
FIG. 1: shows the predicted amino acid sequence (SEQ ID NO: 1)
and the nucleic acid sequence (SEQ ID NO: 2) of BCMP 101. The tandem
mass spectrum is in bold and italicised. MALDI mass spectra are
in bold and underlined. The peptide sequence used to raise the polyclonal
antibody against BCMP 101 is shaded.
FIG. 2: shows tissue distribution of BCMP 101 mRNA. Levels of mRNA
in normal tissues (including kidney) and two kidney cancer cell
lines (Wilm's tumour cell line G401 and human embryonic kidney cell
line 293) were quantified by real time RT-PCR. mRNA levels are expressed
as the number of copies ng.sup.-1 cDNA.
FIG. 3: shows the expression of BCMP 101 mRNA in normal and tumour
breast tissues. Levels of BCMP 101 mRNA in matched normal and tumour
tissues from seven breast cancer patients were measured by real
time RT-PCR. mRNA levels are expressed as the number of copies ng.sup.-1
cDNA.
FIG. 4: compares expression of BCMP 101 mRNA in metastatic/non-metastatic
breast tumour tissues. A=Samples 1-23, which are derived from tumour
samples not involving metastasis to the lymph nodes. B=Samples 26-50,
which are derived from tumour samples involving metastases to variable
numbers of lymph nodes. C=8 samples from normal breast tissues (reduction
mammoplasties). mRNA levels are expressed as the number of copies
ng.sup.-1 cDNA. There is a statistically significant difference
between all tumour samples and normal samples (T-test, p<0.05).
FIG. 5: in situ RT PCR analysis of BCMP 101 expression in sections
of invasive ductal breast cancer tissue (upper panel), and consecutive
negative control section in which the BCMP 101 primers have been
replaced with primers to a control gene (Prostate Specific Antigen)
(lower panel). Note the high BCMP 101 expression (dark staining)
in a portion of epithelial hyperplasia (typical of breast carcinoma),
flanked with two arrowheads in the upper panel.
FIG. 6: cellular localisation of BCMP 101 in breast cancer cell
lines. Fluorescence microscopy showing expression of C-terminal
SuperGlo.TM. AFP-tagged BCMP101 in MDA-MB468 (A) and T47D (B) cell
lines. Membrane localisation is indicated by white arrowheads. Magnification
using.times.60 oil immersion objective.
FIG. 7: immunohistochemical localisation of BCMP 101 protein expression
in breast carcinoma tissue sections. BCMP 101 immunostaining in
carcinoma cells is indicated by arrowheads.
EXAMPLE 1
Identification and Cloning of BCMP 101
Protein BCMP 101 was isolated from MDA-MB468 cell membranes. The
breast carcinoma cell line MDA-MB-468 (ATCC:HTB-132) was cultured
and integral membranes were extracted with the Tx 114 detergent.
These were subsequently analysed by two-dimensional gel electrophoresis
as described in U.S. Pat. Nos. 6,064,754 and 6,278,794.
Mass Spectrometry
Proteins excised from the 2D gel were digested with trypsin and
analysed by MALDI-TOF-MS (Voyager STR, Applied Biosystems) using
a 337 nm wavelength laser for desorption and the reflectron mode
of analysis. Selected masses for BCMP 101 were further characterised
by tandem mass spectrometry using a QTOF-MS equipped with a nanospray
ion source, (Micromass UK Ltd.). Prior to MALDI analysis the samples
were desalted and concentrated using C18 Zip Tips.TM. (Millipore).
Samples for tandem MS were purified using a nano LC system (LC Packings)
incorporating C18 SPE material.
Using the SEQUEST search program (Eng et al., 1994, J. Am. Soc.
Mass Spectrom. 5:976-989), uninterpreted tandem mass sp |