Marker molecules associated with lung tumors
United States Patent: 7,355,025
Issued: April 8, 2008
Inventors: Coy; Johannes (Dossenheim,
DE), Hipfel; Rainer (Balingen, DE), Wasser; Birgit (Kornwestheim, DE)
Assignee: MTM Laboratories,
AG (Heidelberg, DE)
Appl. No.: 10/515,477
Filed: May 16, 2003
PCT Filed: May 16, 2003
PCT No.: PCT/EP03/50175
371(c)(1),(2),(4) Date: November
PCT Pub. No.: WO03/097871
PCT Pub. Date: November 27,
Executive MBA in Pharmaceutical Management, U. Colorado
The present invention relates to nucleic
acids and polypeptides associated with lung cancer. The invention is more
specifically related to a nucleic acids and the polypeptides transcribed
thereof, the expression of which is significantly altered in association
with lung cancer. The invention relates to a series of differentially
spliced transcripts of the gene disclosed herein, that are associated with
tumors of the respiratory tract. Furthermore the present invention
provides a method for early diagnosis, prognosis and monitoring of the
disease course and for therapy and vaccination of cell proliferative
disorders such as e.g. lung tumors.
Description of the
SUMMARY OF THE INVENTION
The present invention provides nucleic acids and polypeptides associated
with lung cancer. According to the present invention these molecules may
be used as molecular markers that allow for comprehensive detection of
cell proliferative disorders such as e.g. lung tumors even at early
The present invention thus provides polypeptides and nucleic acids, the
expression of which is significantly altered associated with lung tumors,
which allow for enhanced prognosis and diagnosis of diseases associated
with abnormalities of the growth of cells. Furthermore the nucleic acids
disclosed herein comprise transcripts arising from alternative splicing of
genes, that do not occur in normal tissue in the extent they may be found
in tumorous tissue.
In another aspect of the invention, the nucleic acids and/or polypeptides
disclosed herein alone or in combination with other molecules may be used
for therapy and/or vaccination of cell proliferative disorders such as
e.g. lung tumors.
Yet another aspect of the present invention are pharmaceutical
compositions containing polypeptides and/or polynucleotides disclosed
herein alone or combination with one or more other therapeutic or
diagnostic agents and/or carrier or adjuvant substances.
The present invention also provides kits such as diagnostic kits or
research kits for the detection of the polynucleotides or polypeptides
disclosed herein or comprising the polynucleotides or polypeptides
disclosed herein or combinations thereof.
During the experiments leading to the present invention a gene was
identified, the expression of which is associated with lung tumors. The
present invention furthermore is based on the inventors findings shown in
Examples 1 to 6, that the level of expression of nucleic acids as well as
of polypeptides transcribed from the marker gene presented herein in FIG.
1-11 (see Original Patent) in samples allows to diagnose and grade cell
proliferative disorders such as e.g. lung tumors, to predict the course of
the disease and to follow up the disease after initial therapy.
The present invention thus provides novel nucleic acids and polypeptides
associated with lung cancer.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect the method according to the present invention is especially
useful for early detection of cell proliferative disorders such as e.g.
lung tumors and for detection of disseminated tumor cells in the course of
diagnosis of minimal residual disease.
In another aspect of the present invention, the nucleic acids or
polypeptides disclosed herein may be used in the course of diagnosis of
disorders associated with abnormal proliferation of cells in samples such
as tumor resections, biopsies or the like. In this aspect the invention
provides a method, which allows to build a strategy for the therapy of
diseases according to their molecular properties. According to the present
invention, the level of said polypeptides and/or nucleic acids can be used
as a molecular marker for prognosis, monitoring and the design of a
strategy of tumor therapeutics.
It is yet another aspect of the present invention to provide methods for
identification of molecules binding to the nucleic acids and polypeptides
of the present invention as well as of activators and inhibitors of the
expression of the genes of the present invention. Also a method for the
identification of drug candidates for the therapy of proliferative
disorders is provided.
The present invention provides tumor associated nucleic acids and
polypeptides characterized by the sequences given in FIG. 1-11.
Marker molecules as used in the present invention my comprise nucleic
acids and polynucleotides. On the level of nucleic acids the marker
molecules may be DNA or RNA comprising genomic DNA, cDNA, and RNA such as
mRNA or hnRNA. In one preferred embodiment of the invention, nucleic acids
arising from particular differential splicing events may be marker
Expression as used according to the present invention may comprise for
example expression of proteins. The transcription to RNA and thus the
level of mRNA may also be understood to be expression according to the
The expression of a compound is said to be significantly altered according
to the present invention, if the level of expression differs by more than
30%. The alteration of the expression may comprise for example elevated
expression or reduced expression of said compound. Another aspect of the
altered expression may be an alteration in a way, that the compound is
expressed under non wild-type circumstances. This may comprise, that the
compound is for example expressed in situations that naturally suppress
the expression, or is not expressed in situations that naturally induce
the expression of the compound.
Alteration of the expression as used herein may also comprise an
alteration in the transcription pattern of a gene. E.g. the alteration of
the transcription pattern may comprise alternative splicing of the gene.
The alterations in the transcription pattern may influence the
polypeptides translated from the altered transcripts or may be restricted
to untranslated regions. The alteration in the transcription pattern of a
gene may comprise use of novel exons in the transcripts, deletions of
exons in the transcripts or the variation in the ratios of different
splicing variants in cells. Thus alterations in transcriptional patterns
of genes as used herein may comprise the production of nucleic acids such
as e.g. mRNA, cDNA etc. containing additional stretches of nucleic acid
sequences compared to wild type nucleic acids occurring in control
tissues. Alternatively the nucleic acids produced by alternative splicing
patterns may produce nucleic acids missing stretches of nucleic acid
sequences present in wild type polynucleotides. The presence of additional
stretches may occur simultaneously with the absence of original
sequence-stretches in single transcripts. Alterations in the expression of
genes as used in the context of the present invention may also comprise an
alteration in the level of expression of splicing variants of genes. This
may include increased or decreased expression of particular splicing
variants as well as expression of variants not present in wild type tissue
or the absence of expression of splicing variants present in wild type
tissue. In one embodiment, the alteration of the expression of the
splicing variants may comprise the alteration of the ratios of different
splicing variants in said tissue.
Nucleic acids as used in the context of the present invention are
preferably polynucleotides or fragments thereof. Preferred polynucleotides
comprise at least 20 consecutive nucleotides, preferably at least 30
consecutive nucleotides and more preferably at least 45 consecutive
nucleotides, which are identical, share sequence homology or encode for
identical, or homologous polypeptides, compared to the polypeptides
associated with the proliferative disorders disclosed herein. The nucleic
acids according to the present invention may also be complementary to any
of said polynucleotides. Polynucleotides may for example include
single-stranded (sense or antisense) or double-stranded molecules, and may
be DNA (genomic, cDNA or synthetic) or RNA. RNA molecules comprise as well
hnRNA (containing introns) as mRNA (not containing introns). According to
the present invention the polynucleotides may also be linked to any other
molecules, such as support materials or detection marker molecules, and
may, but need not, contain additional coding or non-coding sequences.
The polynucleotides according to the present invention may be native
sequences or variants thereof. The variants may contain one or more
substitutions, additions, deletions and/or insertions such that the
immunogenicity of the encoded polypeptide is not diminished, relative to
the respective native proteins. The variants show preferably 70%, more
preferably at least 80% and most preferably at least 90% of sequence
identity to the native nucleic acid molecules used in the methods
according to the present invention. Methods for determination of sequence
similarity are known to those of skill in the art.
One example for detecting the similarity of sequences can be carried out
using the FastA and/or BlastN bioinformatics software accessible on the
HUSAR server of the DKFZ Heidelberg.
Furthermore nucleic acids according to the present invention are all
polynucleotides, which hybridise to probes specific for the sequences
disclosed herein under stringent conditions. Stringent conditions applied
for the hybridisation reaction are known to those of ordinary skill in the
art and may be applied as described in Sambrook et al. Molecular cloning:
A Laboratory Manual, 2.sup.nd Edition, 1989.
The present invention also provides polynucleotides, which due to the
degeneracy of the genetic code encode the polypeptides natively encoded by
the disclosed nucleic acids while not showing the percentage of sequence
homology as described above within the nucleic acid sequence. Such nucleic
acids might arise by changing the codons present in the disclosed
sequences by degenerate codons and so preparing a synthetic nucleic acid.
The nucleotide sequences according to the present invention may be joined
to a variety of other nucleic acid sequences using the known recombinant
DNA techniques. The sequences may for example be cloned into any of a
variety of cloning vectors, such as plasmids, phagemids, lambda phage
derivatives and cosmids. Furthermore vectors such as expression vectors,
replication vectors, probe generation vectors and sequencing vectors may
be joined with the sequences disclosed herein. Sequences of special
interest, that could be cloned to the nucleic acids according to the
present invention are for example non coding sequences and regulatory
sequences including promoters, enhancers and terminators.
In a preferred embodiment, polynucleotides may be formulated such that
they are able to enter mammalian cells and to be expressed in said cells.
Such formulations are especially useful for therapeutic purposes. The
expression of nucleic acid sequences in target cells may be achieved by
any method known to those skilled in the art. The nucleic acids may for
example be joined to elements that are apt to enable their expression in a
host cell. Such elements may comprise promoters or enhancers, such as CMV-,
SV40-, RSV-, metallothionein I- or polyhedrin-promotors respectively CMV-
or SV40-enhancers. Possible methods for the expression are for example
incorporation of the polynucleotides into a viral vector including
adenovirus, adeno-associated virus, retrovirus, vaccinia virus or pox
virus. Viral vectors for the purpose of expression of nucleic acids in
mammalian host cells may comprise pcDNA3, pMSX, pKCR, pEFBOS, cDM8, pCEV4
etc. These techniques are known to those skilled in the art.
Other formulations for administration in therapeutic purposes include
colloidal dispersion systems such as for example macromolecule complexes,
microspheres, beads, micelles and liposomes.
Generally, by means of conventional molecular biological processes it is
possible (see, e.g., Sambrook et al., supra) to introduce different
mutations into the nucleic acid molecules of the invention. As a result
the inventive lung tumor associated polypeptides or polypeptids related
thereto with possibly modified biological properties are synthesized. One
possibility is the production of deletion mutants in which nucleic acid
molecules are produced by continuous deletions from the 5'- or 3'-terminal
of the coding DNA sequence and that lead to the synthesis of polypeptids
that are shortened accordingly. Another possibility is the introduction of
single-point mutation at positions where a modification of the amino aid
sequence influences, e.g., the proliferation specific properties. By this
method muteins can be produced, for example, that possess a modified
Km-value or that are no longer subject to the regulation mechanisms that
normally exist in the cell, e.g. with regard to allosteric regulation or
covalent modification. Such muteins might also be valuable as
therapeutically useful antagonists of the inventive lung tumor associated
For the manipulation in prokaryotic cells by means of genetic engineering
the nucleic acid molecules of the invention or parts of these molecules
can be introduced into plasmids allowing a mutagenesis or a modification
of a sequence by recombination of DNA sequences. By means of conventional
methods (cf. Sambrook et al., supra), bases can be exchanged and natural
or synthetic sequences can be added. In order to link the DNA fragments
with each other adapters or linkers can be added to the fragments.
Furthermore, manipulations can be performed that provide suitable cleavage
sites or that remove superfluous DNA or cleavage sites. If insertions,
deletions or substitutions are possible, in vitro mutagenesis, primer
repair, restriction or ligation can be performed. As analysis method
usually sequence analysis, restriction analysis and other biochemical or
molecular biological methods are used.
The polypeptides encoded by the various variants of the nucleic acid
molecules of the invention show certain common characteristics, such as
activity in the regulation of cell proliferation and differentiation,
molecular weight, immunological reactivity or conformation or physical
properties like the electrophoretical mobilty, chromatographic behavior,
sedimentation coefficients, solubility, spectroscopic properties,
stability, pH optimum, temperature optimum.
The invention furthermore relates to vectors containing the inventive lung
tumor associated nucleic acid molecules. Preferably, they are plasmids,
cosmids, viruses, bacteriophages and other vectors usually used in the
field of genetic engineering. Vectors suitable for use in the present
invention include, but are not limited to the T7-based dual expression
vectors (expression in prokaryotes and in eucaryotes) for expression in
mammalian cells and baculovirus-derived vectors for expression in insect
cells. Preferably, the nucleic acid molecule of the invention is
operatively linked to the regulatory elements in the recombinant vector of
the invention that guarantee the transcription and synthesis of an mRNA in
prokaryotic and/or eukaryotic cells that can be translated. The nucleotide
sequence to be transcribed can be operably linked to a promoter like a T7,
metallothionein I or polyhedrin promoter.
In a further embodiment, the present invention relates to recombinant host
cells transiently or stably containing the nucleic acid molecules or
vectors of the invention. A host cell is understood to be an organism that
is capable to take up in vitro recombinant DNA and, if the case may be, to
synthesize the polypeptids encoded by the nucleic acid molecules of the
invention. Preferably, these cells are prokaryotic or eukaryotic cells,
for example mammalian cells, bacterial cells, insect cells or yeast cells.
The host cells of the invention are preferably characterized by the fact
that the introduced nucleic acid molecule of the invention either is
heterologous with regard to the transformed cell, i.e. that it does not
naturally occur in these cells, or is localized at a place in the genome
different from that of the corresponding naturally occurring sequence.
A further embodiment of the invention relates to a polypeptide exhibiting
a biological property of the inventive lung tumor associated marker and
being encoded by the nucleic acid molecules of the invention, as well as
to methods for their production whereby, e.g., a host cell of the
invention is cultivated under conditions allowing the synthesis of the
polypeptide and the polypeptide is subsequently isolated from the
cultivated cells and/or the culture medium. Isolation and purification of
the recombinantly produced polypeptide may be carried out by conventional
means including preparative chromatography and affinity and immunological
separations using, e.g., an antibody directed against the inventive lung
tumor associated marker proteins, or, e.g., can be substantially purified
by the one-step method described in Smith and Johnson, Gene 67; 31-40
(1988). These polypeptides, however, not only comprise recombinantly
produced polypeptides but include isolated naturally occurring
polypeptides, synthetically produced polypeptides, or polypeptides
produced by a combination of these methods. Means for preparing such
polypeptides or related polypeptides are well understood in the art. These
polypeptides are preferably in a substantially purified form.
The production of a polypeptide according to the present invention may for
example be carried out in a cell free in vitro transcription and/or
translation system. Such systems are known to those of ordinary skill in
the art. One example may comprise an intro translation system as provided
by Roche molecular Biochemicals' Rapid translation System.
Polypeptides as used in the present invention comprise at least an
immunogenic portion of the inventive lung tumor associated marker proteins
disclosed herein. The polypeptides may be of any length. Immunogenic
portion as used above is a portion of a protein, that is recognized by a
B-cell and/or T-cell surface antigen receptor. The immunogenic portions
comprise at least 10 amino acid residues, more preferably at least 20
amino acid residues of the protein associated with a lung tumor. In a
preferred embodiment of the present invention, particular domains of the
proteins, such as for example transmembrane domains or N-terminal leader
sequences have been deleted. The immunogenic portions according to the
present invention react with antisera or specific antibodies in the same
or nearly same intensity as the native full length proteins.
The immunogenic portions are generally identified using the techniques
well known in the art. Possible techniques are for example screening of
the polypeptides for the ability to react with antigen-specific
antibodies, antisera and/or T-cell lines or clones.
The polypeptides associated with lung tumors according to the present
invention comprise also variants of the native proteins. These variants
may differ from the native protein in one or more alterations such as
substitutions, deletions, additions and/or insertions. The
immunoreactivity of the variants according to the present invention is not
substantially diminished compared to the native proteins. In a preferred
embodiment of the invention the immunoreactivity is diminished less than
50% in a more preferred embodiment the immunoreactivity is diminished less
than 20% compared to the native polypeptides.
In a preferred embodiment variants may be deficient in one or more
portions, such as for example N-terminal leader sequences, transmembrane
domains or small N- and/or C-terminal sequences. The variants exhibit 70%,
more preferably at least 90% and most preferably at least 95% identity to
the polypeptides disclosed according to the present invention.
The variants of the present invention are preferably conservative
substitutions, so that the amino acids changed are substituted for amino
acids with similar properties. The properties concerned may include
polarity, charge, solubility, hydrophobicity, hydrophilicity and/or
amphipathic nature of the amino acid residues. The variants disclosed
herein may also comprise additional terminal leader sequences, linkers or
sequences, which enable synthesis, purification or stability of the
polypeptides in an easier or more comfortable way.
The polypeptides according to the present invention comprise also
polypeptides that are fusion or chimeric polypeptides comprising the amino
acid sequence encoded by the nucleic acid sequence of the inventive lung
tumor associated marker disclosed herein. The polypeptides may be fused to
any suitable amino acid sequences. These sequences may for example
comprise antigenic fragments, receptors, enzymes, toxins, chelating
epitopes, etc. In a preferred embodiment of the present invention the
amino acid sequences, that are fused to the disclosed polypeptides are
tags useful in the purification or recovery of the polypeptides such as
e.g. his-tags or myc-tags. The amino acid sequences fused together may be
directly linked or may be separated by any linker or spacer sequences
suitable in the particular purpose.
The polypeptides and polynucleotides according to the present invention
are isolated. This means that the molecules are removed from their
original environment. Naturally occurring proteins are isolated if they
are separated from some or all of the materials, which coexist in the
natural environment. Polynucleotides are isolated for example if they are
cloned into vectors.
Furthermore, the present invention provides binding agents such as
antibodies and antigen-binding fragments, that specifically bind to the
proteins associated with a lung tumors disclosed herein.
The term binding agent comprises a variety of substances such as
oligopeptides, antibodies, peptdiomimetic molecules comprising antigen
binding oligopeptides, nucleic acids, carbohydrates, organic compounds,
etc. Antibody according to the present invention preferably relates to
antibodies which consist essentially of pooled monoclonal antibodies with
different epitopic specificities, as well as distinct monoclonal antibody
preparations. Monoclonal antibodies are made from an antigen containing
fragments of the polypeptides of the invention by methods well known to
those skilled in the art (see, e.g., Kohler et al., Nature 256 (1975),
495). As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab)
is meant to include intact molecules as well as antibody fragments (such
as, for example, Fab and F(ab') 2 fragments) which are capable of
specifically binding to protein. Fab and f(ab')2 fragments lack the Fc
fragment of intact antibody, clear more rapidly from the circulation, and
may have less non-specific tissue binding than an intact antibody. (Wahl
et al., J. Nucl. Med. 24: 316-325 (1983)). Thus, these fragments are
preferred, as well as the products of a FAB or other immunoglobulin
expression library. Moreover, antibodies of the present invention include
chimerical, single chain, and humanized antibodies.
Binding agents according to the present invention may for example be
employed for the inhibition of the activity of the inventive lung tumor
associated marker polypeptides disclosed herein. In this respect the term
"binding agents" relates to agents specifically binding to the
polypeptides transcribed from the novel lung tumor associated nucleic
acids and thus inhibiting the activity of said polypeptide. Such binding
agents may for example comprise nucleic acids (DNA, RNA, PNA etc.),
polypeptides (antibodies, receptors, antigenic fragments, oligopeptides),
carbohydrates, lipids, organic or inorganic compounds (metal-ions, sulfur
compounds, boranes, silicates, reducing agents, oxidizing agents). The
binding agents may preferably interact with the polypeptide by binding to
epitopes, that are essential for the biological activity. The interaction
may be reversible or irreversibly. The binding may be non-covalent or even
covalent binding to the polypeptide. Furthermore the binding agents may
introduce alterations to the polypeptide, that alter or diminish the
biological activity of the inventive polypeptide.
For certain purposes, e.g. diagnostic methods, the antibody or binding
agent of the present invention may be detectably labelled, for example,
with a radioisotope, a bioluminescent compound, a chemiluminescent
compound, a fluorescent compound, a metal chelate, or an enzyme. The
nucleic acid of the present invention may be detectably labelled, for
example, with a radioisotope, a bioluminescent compound, a
chemiluminescent compound, a fluorescent compound, a metal chelate,
biotin, digoxygenin, or an enzyme. Furthermore any method suitable for the
detection of the intermolecular interaction may be employed.
The antibody or antigen-binding agent is said to react specifically, if it
reacts at a detectable level with a protein disclosed herein, and does not
significantly react with other proteins. The antibodies according to the
present invention may be monoclonal or polyclonal antibodies. Other
molecules capable of binding specifically may be for example
antigen-binding fragments of antibodies such as Fab fragments, RNA
molecules or polypeptides. According to the present invention binding
agents may be used isolated or in combination. By means of combination it
is possible to achieve a higher degree of sensitivity.
The antibodies useful for the methods according to the present invention
may comprise further binding sites for either therapeutic agents or other
polypeptides or may be coupled to said therapeutic agents or polypeptides.
Therapeutic agents may comprise drugs, toxins, radio-nuclides and
derivatives thereof. The agents may be coupled to the binding agents
either directly or indirectly for example by a linker or carrier group.
The linker group may for example function in order to enable the coupling
reaction between binding agent and therapeutic or other agent or the
linker may act as a spacer between the distinct parts of the fusion
molecule. The linker may also be cleavable under certain circumstances, so
as to release the bound agent under said conditions. The therapeutic
agents may be covalently coupled to carrier groups directly or via a
linker group. The agent may also be non-covalently coupled to the carrier.
Carriers that can be used according to the present invention are for
example albumins, polypeptides, polysaccharides or liposomes.
The antibody used according to the present invention may be coupled to one
or more agents. The multiple agents coupled to one antibody may be all of
the same species or may be several different agents bound to one antibody.
The invention also relates to a transgenic non-human animal such as
transgenic mouse, rats, hamsters, dogs, monkeys, rabbits, pigs, C. elegans
and fish such as torpedo fish comprising a nucleic acid molecule or vector
of the invention, preferably wherein said nucleic acid molecule or vector
may be stably integrated into the genome of said non-human animal,
preferably such that the presence of said nucleic acid molecule or vector
leads to the expression of the inventive lung tumor associated marker
polypeptide (or related polypeptide) of the invention, or may otherwise be
transiently expressed within the non-human animal. Said animal may have
one or several copies of the same or different nucleic acid molecules
encoding one or several forms of the inventive lung tumor associated
marker polypeptide or mutant forms thereof. This animal has numerous
utilities, including as a research model for the regulation of cell
proliferation and differentiation and therefore, presents a novel and
valuable animal in the development of therapies, treatment, etc. for
diseases caused by deficiency or failure of the inventive lung tumor
associated marker protein involved in the development of cell
proliferative disorders, e.g., lung tumors. Accordingly, in this instance,
the non-human mammal is preferably a laboratory animal such as a mouse or
Preferably, the transgenic non-human animal of the invention further
comprises at least one inactivated wild type allele of the corresponding
gene encoding the inventive lung tumor associated polypeptide. This
embodiment allows for example the study of the interaction of various
mutant forms of the inventive lung tumor associated marker polypeptides on
the onset of the clinical symptoms of disease associated with the
regulation of cell proliferation and differentiation. All the applications
that have been herein before discussed with regard to a transgenic animal
also apply to animals carrying two, three or more transgenes. It might be
also desirable to inactivate the inventive lung tumor associated marker
protein expression or function at a certain stage of development and/or
life of the transgenic animal. This can be achieved by using, for example,
tissue specific, developmental and/or cell regulated and/or inducible
promoters which drive the expression of, e.g., an antisense or ribozyme
directed against the RNA transcript encoding the inventive lung tumor
associated marker encoding mRNA; see also supra. A suitable inducible
system is for example tetracycline-regulated gene expression as described,
e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. 89 USA (1992),
5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62). Similar,
the expression of the mutant inventive lung tumor associated protein may
be controlled by such regulatory elements.
Furthermore, the invention also relates to a transgenic mammalian cell
which contains (preferably stably integrated into its genome or
transiently introduced) a nucleic acid molecule according to the invention
or part thereof, wherein the transcription and/or expression of the
nucleic acid molecule or part thereof leads to reduction of the synthesis
of an inventive lung tumor associated marker protein. In a preferred
embodiment, the reduction is achieved by an anti-sense, sense, ribozyme,
co-suppression and/or dominant mutant effect. "Antisense" and "antisense
nucleotides" means DNA or RNA constructs which block the expression of the
naturally occurring gene product. In another preferred embodiment the
native nucleic acid sequence coding for the inventive lung tumor
associated marker polypeptide may be altered or substituted by a variant
of said nucleic acid sequence, e.g. by means of recombination, thus
rendering the inventive lung tumor associated marker gene non functional.
Thus an organism lacking the inventive lung tumor associated marker
polypeptide activity may be produced according to knock out experiments.
The provision of the nucleic acid molecule according to the invention
opens up the possibility to produce transgenic non-human animals with a
reduced level of the inventive lung tumor associated marker protein as
described above and, thus, with a defect in the regulation of cell
proliferation and differentiation. Techniques how to achieve this are well
known to the person skilled in the art. These include, for example, the
expression of antisense-RNA, ribozymes, of molecules which combine
antisense and ribozyme functions and/or of molecules which provide for a
co-suppression effect. When using the antisense approach for reduction of
the amount of the inventive lung tumor associated marker proteins in
cells, the nucleic acid molecule encoding the antisense-RNA is preferably
of homologous origin with respect to the animal species used for
transformation. However, it is also possible to use nucleic acid molecules
which display a high degree of homology to endogenously occurring nucleic
acid molecules encoding an inventive lung tumor associated marker protein.
In this case the homology is preferably higher than 80%, particularly
higher than 90% and still more preferably higher than 95%. The reduction
of the synthesis of a polypeptide according to the invention in the
transgenic mammalian cells can result in an alteration in, e.g.,
degradation of endogenous proteins. In transgenic animals comprising such
cells this can lead to various physiological, developmental and/or
Thus, the present invention also relates to transgenic non-human animals
comprising the above-described transgenic cells. These may show, for
example, a deficiency in regulation of cell proliferation and/or
differentiation compared to wild type animals due to the stable or
transient presence of a foreign DNA resulting in at least one of the
following features: (a) disruption of (an) endogenous gene(s) encoding the
inventive lung tumor associated marker; (b) expression of at least one
antisense RNA and/or ribozyme against a transcript comprising a nucleic
acid molecule of the invention; (c) expression of a sense and/or
non-translatable mRNA of the nucleic acid molecule of the invention; (d)
expression of an antibody of the invention; (e) incorporation of a
functional or non-functional copy of the regulatory sequence of the
invention; or (f) incorporation of a recombinant DNA molecule or vector of
Methods for the production of a transgenic non-human animal of the present
invention, preferably transgenic mouse, are well known to the person
skilled in the art. Such methods, e.g., comprise the introduction of a
nucleic acid molecule or vector of the invention into a germ cell, an
embryonic cell, stem cell or an egg or a cell derived therefrom. The
non-human animal can be used in accordance with a screening method of the
invention described herein and may be a non-transgenic healthy animal, or
may have a disorder, preferably a disorder caused by at least one mutation
in the inventive lung tumor associated marker protein. Such transgenic
animals are well suited for, e.g., pharmacological studies of drugs in
connection with mutant forms of the above described inventive lung tumor
associated marker polypeptide. Production of transgenic embryos and
screening of those can be performed, e.g., as described by A. L. Joyner
Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press.
The DNA of the embryonal membranes of embryos can be analyzed using, e.g.,
Southern blots with an appropriate probe, amplification techniques based
on nucleic acids (e.g. PCR) etc.; see supra.
Another aspect of the present invention is a pharmaceutical composition
for use in the treatment of disorders associated with abnormal cell
proliferation. The polypeptides, polynucleotides and binding agents (esp.
antibodies) according to the present invention may be incorporated into
pharmaceutical or immunogenic compositions.
The pharmaceutical compositions may be administered by any suitable way
known to those of skill in the art. The administration may for example
comprise injection, such as e.g., intracutaneous, intramuscular,
intravenous or subcutaneous injection, intranasal administration for
example by aspiration or oral administration. A suitable dosage to ensure
the pharmaceutical benefit of the treatment should be chosen according the
parameters, such as age, sex, body weight etc. of the patient, known to
those of skill in the art.
The pharmaceutical compositions comprise said compounds and a
physiologically acceptable carrier. The type of carrier to be employed in
the pharmaceutical compositions of this invention, will vary depending on
the mode of administration. For parenteral administration, such as
subcutaneous injection, the carrier preferably comprises water, saline,
alcohol, a lipid, a wax and/or a buffer. For oral administration, any of
the above carriers or a solid carrier, such as mannitol, lactose, starch,
magnesium stearate, sodium saccharine, talcum, cellulose, glucose,
sucrose, and/or magnesium carbonate, may be employed. Biodegradable
microspheres (e.g., polylactic glycolide) may also be employed as carriers
for the pharmaceutical compositions of this invention. Suitable
biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.
4,897,268 and 5,075,109.
A pharmaceutical composition or vaccine may for example contain DNA, that
codes for one or more polypeptides according to the present invention. The
DNA may be administered in a way that allows the polypeptides to be
generated in situ. Suitable expression systems are known to those skilled
in the art. In another embodiment of the invention the nucleic acids may
be for example anti-sense constructs. Pharmaceutical compositions may also
comprise nucleic acid molecules expressible in a mammalian or human host
system comprising a viral or other expression system for example an
adenoviral vector system.
The nucleic acid may also be administered as a naked nucleic acid. In this
case appropriate physical delivery systems, which enhance the uptake of
nucleic acid may be employed, such as coating the nucleic acid onto
biodegradable beads, which are efficiently transported into the cells.
Administration of naked nucleic acids may for example be useful for the
purpose of transient expression within a host or host cell.
Alternatively the pharmaceutical compositions may comprise one or more
polypeptides. The polypeptides incorporated into pharmaceutical
compositions may be the inventive lung tumor associated polypeptide.
Optionally the polypeptide may be administered in combination with one or
more other known polypeptides such as for example enzymes, antibodies,
regulatory factors, such as cyclins, cyclin-dependent kinases or CKIs, or
Polypeptides of the present invention or fragments thereof, that comprise
an immunogenic portion of an inventive lung tumor associated protein, may
be used in immunogenic compositions, wherein the polypeptide e.g.
stimulates the patient's own immune response to tumor cells. A patient may
be afflicted with disease, or may be free of detectable disease.
Accordingly, the compounds disclosed herein may be used to treat cancer or
to inhibit the development of cancer. The compounds may be administered
either prior to or following a conventional treatment of tumors such as
surgical removal of primary tumors, treatment by administration of
radiotherapy, conventional chemotherapeutic methods or any other mode of
treatment of the respective cancer or its precursors.
Immunogenic compositions such as vaccines may comprise one or more
polypeptides and a non-specific immune-response enhancer, wherein the
non-specific immune response enhancer is capable of eliciting or enhancing
an immune response to an exogenous antigen. Any suitable immune-response
enhancer may be employed in the vaccines of this invention. For example,
an adjuvant may be included. Most adjuvants contain a substance designed
to protect the antigen from rapid catabolism, such as aluminium hydroxide
or mineral oil, and a non-specific stimulator of immune response, such as
lipid A, Bordetella pertussis or Mycobacterium tuberculosis. Such
adjuvants are commercially available as, for example, Freund's Incomplete
Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.) and
Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.).
Pharmaceutical compositions and vaccines may also contain other epitopes
of tumor antigens, either incorporated into a fusion protein as described
above (i.e., a single polypeptide that contains multiple epitopes) or
present within a separate polypeptide.
Disorders characterized by abnormal cell proliferation, as used in the
context of the present invention, may comprise for example neoplasms such
as benign and malignant tumors, carcinomas, sarcomas, leukemias, lymhomas
or dysplasias. Tumors may comprise tumors of the head and the neck, tumors
of the respiratory tract, tumors of the gastrointestinal tract, tumors of
the urinary system, tumors of the reproductive system, tumors of the
endocrine system, tumors of the central and peripheral nervous system,
tumors of the skin and its appendages, tumors of the soft tissues and
bones, tumors of the lymphopoietic and hematopoietic system, breast
cancer, colorectal cancer, anogenital cancer etc.
In one preferred embodiment of the invention, the disorders are lung
tumors. Lung tumors according to the present invention comprise conditions
of the respiratory tract characterized by abnormal growth properties of
cells or tissues compared to the growth properties of normal control cells
or tissues. The growth of the cells or tissues may be for example
abnormally accelerated or may be regulated abnormally. Abnormal regulation
as used above may comprise any form of presence or absence of non-wild
type responses of the cells or tissues to naturally occurring growth
regulating influences. The abnormalities in growth of the cells or tissues
may be for example neoplastic or hyperplastic. In one preferred embodiment
of the invention the lung tumors are cancers or precancerours conditions
of the respiratory tract.
A sample according to the method of the present invention is any sample,
that may contain cells, tissues or body liquids. Furthermore any sample
potentially containing the marker molecules to be detected may be a sample
according to the present invention. Such samples are e.g. blood, plasma,
serum, swabs, washes, sputum, cell- and tissue-samples or biopsies.
Biopsies as used in the context of the present invention may comprise e.g.
resection samples of tumors, tissue samples prepared by endoscopic means
or needle biopsies. Furthermore any sample potentially containing the
marker molecules to be detected may be a sample according to the present
The method for detection of the level of the polynucleotides or
polypeptides according to the present invention is any method, which is
suited to detect very small amounts of specific molecules in samples. The
detection reaction according to the present invention may be for example
detection either on the level of nucleic acids or on the level of
polypeptides. The detection may either be a detection of the level of
polypeptides or nucleic acids in cells in total or in cell lysates or a
detection of the level of polypeptides or nucleic acids in distinct
subcellular regions. The methods for determining the subcellular
distribution of compounds are known to those of skill in the art. In one
embodiment of the invention the detection of the marker molecules may
comprise the detection of particular splicing variants. In another
embodiment of the present invention the detection method may comprise the
detection of methylation of nucleic acid molecules in samples.
Applicable formats for the detection reaction according to the present
invention may be blotting techniques, such as Western-Blot, Southern-blot,
Northern-blot. The blotting techniques are known to those of ordinary
skill in the art and may be performed for example as electro-blots,
semidry-blots, vacuum-blots or dot-blots. Furthermore immunological
methods for detection of molecules may be applied, such as for example
immunoprecipitation or immunological assays, such as ELISA, RIA, lateral
flow assays etc.
Methods for detection of methylation of nucleic acids are known to those
of skill in the art and may comprise for example methods employing
chemical pre-treatment of nucleic acids with e.g. sodium bisulphite,
permanganate or hydrazine, and subsequent detection of the modification by
means of specific restriction endonucleases or by means of specific probes
e.g. in the course of an amplification reaction. The detection of
methylation may furthermore be performed using methylation specific
In one preferred embodiment of the invention, the detection of the level
of marker molecules is carried out by detection of the level of nucleic
acids coding for the marker molecules or fragments thereof present in the
sample. The means for detection of nucleic acid molecules are known to
those skilled in the art. The procedure for the detection of nucleic acids
can for example be carried out by a binding reaction of the molecule to be
detected to complementary nucleic acid probes, proteins with binding
specificity for the nucleic acids or any other entities specifically
recognizing and binding to aid nucleic acids. This method can be performed
as well in vitro as directly in-situ for example in the course of a
detecting staining reaction. Another way of detecting the marker molecules
in a sample on the level of nucleic acids performed in the method
according to the present invention is an amplification reaction of nucleic
acids, which can be carried out in a quantitative manner such as for
example PCR, LCR or NASBA.
In another preferred embodiment of the invention the detection of the
level of marker molecules is carried out by determining the level of
expression of a protein. The determination of the marker molecules on the
protein level may for example be carried out in a reaction comprising a
binding agent specific for the detection of the marker molecules. These
binding agents may comprise for example antibodies and antigen-binding
fragments, bifunctional hybrid antibodies, peptidomimetics containing
minimal antigen-binding epitopes etc. The binding agents may be used in
many different detection techniques for example in western-blot, ELISA,
lateral flow assay, latex-agglutination, immunochromatographic strips or
immuno-precipitation. Generally binding agent based detection may be
carried out as well in vitro as directly in situ for example in the course
of an immuno-cytochemical staining reaction. Any other method suitable for
determining the amount of particular polypeptides in solutions of
biological samples, such as biochemical, chemical, physical or physico-chemical
methods, can be used according to the present invention.
In one preferred embodiment of the invention, the level of markers in
significantly elevated compared to a non tumorous test sample. In this
case the marker is overexpressed in the sample. In another preferred
embodiment of the present invention the level of the marker is lowered
compared to a non tumorous test sample. In a third embodiment there is no
detectable expression of the marker at all in the test sample unlike in a
control sample. In yet another embodiment there is detectable level of non
wild-type marker molecules. Non wild-Type marker molecules may comprise
any marker molecules that deviate in sequence or structure from the
structure or sequence, that is functional in wild type tissue not affected
by a cell proliferative disease. Wild type sequences or structures are the
sequences or structures predominantly present in normal cells or tissues.
In one preferred embodiment of the invention the level of particular
splicing variants of the marker gene is altered in the test samples
compared to the wild type tissue. This may lead to altered levels of
splicing variants, new splicing variants, neo-peptides, altered ratios of
different splicing variants of genes.
The detection of the level of molecular markers according to the present
invention may be the detection of the level of single marker molecules in
separated reaction mixtures as well as the detection of a combination of
markers simultaneously. The combination may comprise the molecular markers
disclosed herein and additionally further marker molecules useful for the
detection of tumors.
The detection may be carried out in solution or using reagents fixed to a
solid phase. The detection of one or more molecular markers may be
performed in a single reaction mixture or in two or separate reaction
mixtures. The detection reactions for several marker molecules may for
example be performed simultaneously in multi-well reaction vessels. The
markers characteristic for the lung tumors disclosed herein may be
detected using reagents that specifically recognise these molecules.
Simultaneously one or more further markers may be detected using reagents,
that specifically recognize them. The detection reaction for each single
marker may comprise one or more reactions with detecting agents either
recognizing the initial marker molecules or preferably recognizing
molecules used to recognize other molecules. Such reaction may e.g.
comprise the use of primary and secondary and further antibodies. The
detection reaction further may comprise a reporter reaction indicating the
level of the inventive polypeptides associated with lung tumors. The
reporter reaction may be for example a reaction producing a coloured
compound, a bioluminescence reaction, a fluorescence reaction, generally a
radiation emitting reaction etc.
In one preferred embodiment, the detection of tissues expressing marker
gene products is carried out in form of molecular imaging procedures. The
respective procedures are known to those of ordinary skill in the art.
Imaging methods for use in the context of the present invention may for
example comprise MRI, SPECT, PET and other methods suitable for in vivo
In one embodiment the method may be based on the enzymatic conversion of
inert or labelled compounds to molecules detectable in the course of
molecular imaging methods by the marker molecules. In another embodiment
the molecular imaging method may be based on the use of compounds carrying
a suitable label for in vivo molecular imaging, such as radio isotopes,
metal ions etc., specifically binding to marker molecules in vivo.
In a preferred embodiment of the invention, these compounds are non-toxic
compounds and may be eliminated from the circulation of organisms, such as
humans, in a time span, that allows for performing the detection of label
accumulated in tumor tissue overexpressing the respective marker gene. In
another preferred embodiment of the invention compounds are used for
molecular imaging, for which clearance from the circulation is not
relevant for performing the molecular imaging reaction. This may be for
example due to low background produced by the circulating molecules etc.
The compounds for use in molecular imaging methods are administered in
pharmaceutical acceptable form in compositions that may additionally
comprise any other suitable substances, such as e.g. other diagnostically
useful substances, therapeutically useful substances, carrier substances
or the like.
The marker molecules disclosed according to the present invention may be
used for diagnosis, monitoring of the disease course and prognosis in cell
proliferative disorders such as e.g. lung tumors.
Diagnosis of disorders associated with the expression of the inventive
gene as used herein may for example comprise the detection of cells or
tissues affected by abnormal growth. In one preferred embodiment diagnosis
means the primary detection of a disease in an organism or sample.
According to the present invention the method for diagnosis of disorders
such as tumors may be applied in routine screening tests for preventive
aspects in order to detect said disease at an early stage of the onset of
the disorder. In another preferred embodiment the diagnostic method may be
used to determine the minimal residual disease of a tumor after primary
therapy. In this respect the method of the invention may be applied to
determine cells in body samples displaying abnormal expression of marker
molecules according to the present invention, characteristic for lung
tumors. Thus a spread of affected cells may be detected in body liquids.
In one embodiment of the invention, the methods disclosed herein may be
used for the detection and identification of metastases. The method may be
applied either for detection of metastases in body tissues or organs by
the detection methods described herein, or the metastases may be diagnoses
with respect to prognosis and prediction of disease course.
Monitoring of the disease course may comprise determining the levels of
marker molecules at different time points, comparing the levels at the
different time points and assessing a diagnosis about the progression of
the disease over the covered period of time. Thus monitoring may enable
for assessment of prognosis and/or for design of an adequate therapy for a
Prognosis of the disease course of a cell proliferative disorders such as
e.g. lung tumors according to the present invention may comprise
determining the level of expression of one or more marker molecules,
comparing the levels with data from subsequent studies in a database and
prognosticating the disease course from said comparison. In a preferred
embodiment the method may comprise the detection of the levels of a set of
marker molecules, the distinct levels of which may characterize distinct
stages in the course of the disease. In a further embodiment of the
invention the combination of the levels of a combination of markers may be
an indicator for the prognosis of the further disease course and may build
the basis for design of an adequate therapy.
The present invention further provides kits for use in e.g. research or
diagnostic methods. Such kits may contain two or more components for
performing a scientific or diagnostic assay. Components may be compounds,
reagents, containers and/or equipment. One component may be an antibody or
fragment thereof that specifically binds to a polypeptide associated with
lung tumors. Additionally the kit may contain reagents, buffers or others
known in the art as necessary for performing the diagnostic assay.
Alternatively the research kit or diagnostic kit may contain nucleotide
probes or primers for the detection of DNA or RNA. Such a kit should
contain appropriate additional reagents and buffers known in the art.
A kit according to present invention comprises: a) reagents for the
detection of the molecular marker molecules, b) the reagents and buffers
commonly used for carrying out the detection reaction, such as buffers,
detection-markers, carrier substances and others, and c) a marker sample
for carrying out a positive control reaction.
The reagent for the detection of the marker includes any agent capable of
binding to the marker molecule. Such reagents may include proteins,
polypeptides, nucleic acids, glycoproteins, proteoglycans, polysaccharides
The sample for carrying out a positive control may comprise for example
nucleic acids in applicable form, such as solution or salt, peptides in
applicable form, tissue section samples or positive cells expressing the
molecules associated with lung tumors.
In a preferred embodiment of the invention, the detection of the marker
molecules is carried out on the level of polypeptides. In this embodiment,
the binding agents may be for example antibodies specific for the marker
molecules or fragments thereof.
In another embodiment of the test kit, the detection of the marker
molecule is carried out on the nucleic acid level. In this embodiment of
the invention the reagents for the detection may be for example nucleic
acid probes or primers complementary to said marker molecule nucleic
Another aspect of the present invention is to provide a method for therapy
and/or vaccination. According to the present invention a therapy of cell
proliferative disorders can be carried out using the inventive lung tumor
associated polypeptides and/or polynucleotides. The therapy may be for
example immunotherapy or somatic gene therapy.
The inventive lung tumor associated polypeptides and/or polynucleotides
may according to the present invention be used for vaccination against
cell proliferative disorders. Vaccination according to the present
invention may comprise administering an immunogenic compound to an
individual for the purpose of stimulating an immune response directed
against said immunogenic compound and thus immunizing said individual
against said immunogenic compound. Stimulating an immune response may
comprise inducing the production of antibodies against said compound as
well as stimulating cytotoxic T-cells. For the purpose of vaccination the
polypeptides, nucleic acids and binding agents according to the present
invention may be administered in a physiological acceptable form. The
composition to be administered to individuals may comprise one or more
antigenic components, physiologically acceptable carrier substances or
buffer solutions, immunostimulants and/or adjuvants. Adjuvants may
comprise for example Freund's incomplete adjuvant or Freund's complete
adjuvant or other adjuvants known to those of skill in the art.
The composition may be administered in any applicable way such as e.g.
intravenous, subcutaneous, intramuscular etc. The dosage of the
composition depends on the particular case and purpose of the vaccination.
It has to be adapted to parameters by the individual treated such as age,
weight, sex etc. Furthermore the type of the immune response to be
elicited has to be taken into account. In general it may be preferable if
an individual receives 100 .mu.g-1 g of a polypeptide according to the
present invention or 10.sup.6-10.sup.12 MOI of a recombinant nucleic acid,
containing a nucleic acid according to the present invention in a form
that may be expressed in situ.
Individuals for the purpose of vaccination may be any organisms containing
the inventive lung tumor associated polypeptides and/or polynucleotides
and being able to get affected by cell proliferative disorders.
Vaccination of individuals may be favourable e.g. in the case of altered,
non wild-type sequences or structure of marker molecules associated with
cell proliferative disorders.
Polypeptides disclosed herein may also be employed in adoptive
immunotherapy for the treatment of cancer. Adoptive immunotherapy may be
broadly classified into either active or passive immunotherapy. In active
immunotherapy, treatment relies on the in vivo stimulation of the
endogenous host immune system to react against tumors with the
administration of immune response-modifying agents (for example, tumor
vaccines, bacterial adjuvants, and/or cytokines).
In passive immunotherapy, treatment involves the delivery of biologic
reagents with established tumor-immune reactivity (such as effector cells
or antibodies) that can directly or indirectly mediate antitumor effects
and does not necessarily depend on an intact host immune system. Examples
of effector cells include T lymphocytes (for example, CD8+ cytotoxic
T-lymphocyte, CD4+ T-helper, tumor-infiltrating lymphocytes), killer cells
(such as Natural Killer cells, lymphokine-activated killer cells), B
cells, or antigen presenting cells (such as dendritic cells and
macrophages) expressing the disclosed antigens. The polypeptides disclosed
herein may also be used to generate antibodies or anti-idiotypic
antibodies (as in U.S. Pat. No. 4,918,164), for passive immunotherapy.
The predominant method of procuring adequate numbers of T-cells for
adoptive immunotherapy is to grow immune T-cells in vitro. Culture
conditions for expanding single antigen-specific T-cells to several
billion in number with retention of antigen recognition in vivo are well
known in the art. These in vitro culture conditions typically utilize
intermittent stimulation with antigen, often in the presence of cytokines,
such as IL-2, and non-dividing feeder cells. As noted above, the
immunoreactive polypeptides described herein may be used to rapidly expand
antigen-specific T cell cultures in order to generate sufficient number of
cells for immunotherapy. In particular, antigen-presenting cells, such as
dendritic, macrophage or B-cells, may be pulsed with immunoreactive
polypeptides or transfected with a nucleic acid sequence(s), using
standard techniques well known in the art. For example, antigen presenting
cells may be transfected with a nucleic acid sequence, wherein said
sequence contains a promoter region appropriate for increasing expression,
and can be expressed as part of a recombinant virus or other expression
system. For cultured T-cells to be effective in therapy, the cultured
T-cells must be able to grow and distribute widely and to survive long
term in vivo. Studies have demonstrated that cultured T-cells can be
induced to grow in vivo and to survive long term in substantial numbers by
repeated stimulation with antigen supplemented with IL-2 (see, for
example, Cheever, M., et al, "Therapy With Cultured T Cells: Principles
Revisited," Immunological Reviews, 157:177, 1997).
The polypeptides disclosed herein may also be employed to generate and/or
isolate tumor-reactive T-cells, which can then be administered to the
patient. In one technique, antigen-specific T-cell lines may be generated
by in vivo immunization with short peptides corresponding to immunogenic
portions of the disclosed polypeptides. The resulting antigen specific
CD8+ CTL clones may be isolated from the patient, expanded using standard
tissue culture techniques, and returned to the patient.
Alternatively, peptides corresponding to immunogenic portions of the
polypeptides of the invention may be employed to generate tumor reactive
T-cell subsets by selective in vitro stimulation and expansion of
autologous T-cells to provide antigen-specific T-cells which may be
subsequently transferred to the patient as described, for example, by
Chang et al. (Crit. Rev. Oncol. Hematol., 22(3), 213, 1996). Cells of the
immune system, such as T-cells, may be isolated from the peripheral blood
of a patient, using a commercially available cell separation system, such
as CellPro Incorporated's (Bothell, Wash.) CEPRATE.TM. system (see U.S.
Pat. Nos. 5,240,856; 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243).
The separated cells are stimulated with one or more of the immunoreactive
polypeptides contained within a delivery vehicle, such as a microsphere,
to provide antigen-specific T-cells. The population of tumor
antigen-specific T-cells is then expanded using standard techniques and
the cells are administered back to the patient.
In another embodiment, T-cell and/or antibody receptors specific for the
polypeptides can be cloned, expanded, and transferred into other vectors
or effector cells for use in adoptive immunotherapy.
In a further embodiment, syngeneic or autologous dendritic cells may be
pulsed with peptides corresponding to at least an immunogenic portion of a
polypeptide disclosed herein. The resulting antigen-specific dendritic
cells may either be transferred into a patient, or employed to stimulate
T-cells to provide antigen-specific T-cells, which may, in turn, be
administered to a patient. The use of peptide-pulsed dendritic cells to
generate antigen-specific T-cells and the subsequent use of such
antigen-specific T-cells to eradicate tumors in a murine model has been
demonstrated by Cheever et al, Immunological Reviews, 157:177, 1997.
Additionally, vectors expressing the disclosed nucleic acids may be
introduced into stem cells taken from the patient and clonally propagated
in vitro for autologous transplant back into the same patient.
Monoclonal antibodies of the present invention may also be used as
therapeutic compounds in order to diminish or eliminate tumors. The
antibodies may be used on their own (for instance, to inhibit metastases)
or coupled to one or more therapeutic agents. Suitable agents in this
regard include radio nuclides, differentiation inducers, drugs, toxins,
and derivatives thereof. Preferred radio nuclides include 90Y, 123, I125,
I131, I186Re, 188Re, 211At, and 212Bi. Preferred drugs include
methotrexate, and pyrimidine and purine analogs. Preferred differentiation
inducers include phorbol esters and butyric acid. Preferred toxins include
ricin, abrin, diptheria toxin, cholera toxin, gelonin, Pseudomonas
exotoxin, Shigella toxin, and pokeweed antiviral protein.
In one embodiment of the invention, the therapy of disorders characterized
by abnormal cell proliferation may comprise the administration of
antisense construct or ribozymes. The methods for administration of
ribozymes or antisense constructs are known to those of skill in the art.
The administration may take place as administration of naked nucleic acids
or as administration of nucleic acids that are suited for expression of
the relevant active products in situ.
In another embodiment of the invention the treatment of disorders may
comprise the administration of binding agents directed against the
inventive lung tumor associated molecules. These binding agents may for
example be coupled to other compounds such as toxins, enzymes,
In another embodiment of the invention, therapy of disorders associated
with abnormal expression of the presented inventive lung tumor associated
molecules may comprise the administration of antagonists or agonists of
the inventive lung tumor associated molecules, of binding partners of the
inventive lung tumor associated polypeptides of inhibitors or enhancer of
the expression of the inventive lung tumor associated polypeptides or of
drugs identifiable by assays involving the measurement of the activity of
the inventive lung tumor associated polypeptides. The methods for
identifying these substances are known to those of skill in the art.
An example for a method for identifying a binding partner of an inventive
lung tumor associated polypeptides (or related polypeptide) and/or
polynucleotide may comprise: (a) contacting the inventive lung tumor
associated polypeptide of the invention with a compound to be screened;
and (b) determining whether the compound affects an activity of the
The inventive lung tumor associated polypeptides may be used to screen for
proteins or other compounds that bind to the inventive lung tumor
associated polypeptides or for proteins or other compounds to which the
inventive lung tumor associated polypeptide binds. The binding of the
inventive lung tumor associated polypeptide and the molecule may activate
(agonist), increase, inhibit (antagonist), or decrease activity of the
inventive lung tumor associated polypeptide or the molecule bound.
Examples of such molecules include antibodies, oligonucleotides, proteins
(e.g., receptors), or small molecules.
Preferably, the molecule is closely related to the natural ligand of the
inventive lung tumor associated polypeptide, e.g., a fragment of the
ligand, or a natural substrate, a ligand, a structural or functional
mimetic; see, e.g., Coligan, Current Protocols in Immunology 1(2) (1991);
Chapter 5. Similarly, the molecule can be closely related to the natural
receptor to which the inventive lung tumor associated polypeptide might
bind, or at least, a fragment of the receptor capable of being bound by
the inventive lung tumor associated polypeptide (e.g., active site). In
either case, the molecule can be rationally designed using known
Preferably, the screening for these molecules involves producing
appropriate cells which express the inventive lung tumor associated
polypeptide, either as a secreted protein or on the cell membrane.
Preferred cells include cells from mammals, yeast, Drosophila, or E. coli.
Cells expressing the inventive lung tumor associated polypeptide (or cell
membrane containing the expressed polypeptide) are then preferably
contacted with a test compound potentially containing the molecule to
observe binding, stimulation, or inhibition of activity of the inventive
lung tumor associated polypeptide.
The assay may simply test binding of a candidate compound to the inventive
lung tumor associated polypeptide, wherein binding is detected by a label,
or in an assay involving competition with a labelled competitor. Further,
the assay may test whether the candidate compound results in a signal
generated by binding to the inventive lung tumor associated polypeptide.
Alternatively, the assay can be carried out using cell-free preparations,
polypeptide/molecule affixed to a solid support, chemical libraries, or
natural product mixtures. The assay may also simply comprise the steps of
mixing a candidate compound with a solution containing the inventive lung
tumor associated polypeptide, measuring the inventive lung tumor
associated polypeptide/molecule activity or binding, and comparing the
inventive lung tumor associated polypeptide/molecule activity or binding
to a standard.
Preferably, an ELISA assay can measure the inventive lung tumor associated
polypeptide level or activity in a sample (e.g., biological sample) using
a monoclonal or polyclonal antibody. The antibody can measure the
inventive lung tumor associated polypeptide level or activity by either
binding, directly or indirectly, to the inventive lung tumor associated
polypeptide or by competing with the inventive lung tumor associated
polypeptide for a substrate. All of these above assays can be used as
diagnostic or prognostic markers. The molecules discovered using these
assays can be used to treat disease or to bring about a particular result
in a patient (e.g., elimination of an epithelial tumor or stop of
progression of tumor growth) by activating or inhibiting the inventive
lung tumor associated molecule. Moreover, the assays can discover agents
which may inhibit or enhance the production of the inventive lung tumor
associated polypeptide from suitably manipulated cells or tissues.
Therefore, the invention includes a method of identifying compounds which
bind to the inventive lung tumor associated polypeptide comprising the
steps of: (a) incubating a candidate binding compound with a polypeptide
of the invention (the inventive lung tumor associated polypeptide); and
(b) determining if binding has occurred.
Moreover, the invention includes a method of identifying
activators/agonists or inhibitors/antagonists of a the inventive lung
tumor associated polypeptide comprising the steps of: (a) incubating a
candidate compound with a polypeptide of the invention; b) assaying a
biological activity, and (c) determining if a biological activity of the
polypeptide of the invention has been altered.
In a further embodiment, the present invention relates to method of
identifying and obtaining a drug candidate for therapy of a disorder
characterized by abnormal cell proliferation comprising the steps of (a)
contacting a lung tumor associated polypeptide of the present invention or
a cell expressing said polypeptide in the presence of components capable
of providing a detectable signal in response to altered regulation of cell
proliferation or to altered cell differentiation, with said drug candidate
to be screened under conditions to allow protein degradation, and (b)
detecting presence or absence of a signal or increase of the signal
generated from protein degradation, wherein the presence or increase of
the signal is indicative for a putative drug.
Experiments using animals or isolated cells or cell lines may be used to
examine the proliferative behavior of cells or tissues in dependence on
the inventive lung tumor associated polypeptide action. The same
procedures may be employed for the study of cell differentiation.
The drug candidate may be a single compound or a plurality of compounds.
The term "plurality of compounds" in a method of the invention is to be
understood as a plurality of substances which may or may not be identical.
Said compound or plurality of compounds may be chemically synthesized or
microbiologically produced and/or comprised in, for example, samples,
e.g., cell extracts from, e.g., plants, animals or microorganisms.
Furthermore, said compound(s) may be known in the art but hitherto not
known to be capable of suppressing or activating the inventive lung tumor
associated polypeptides. The reaction mixture may be a cell free extract
or may comprise a cell or tissue culture. Suitable set ups for the method
of the invention are known to the person skilled in the art and are, for
example, generally described in Alberts et al., Molecular Biology of the
Cell, third edition (1994) and in the appended examples. The plurality of
compounds may be, e.g., added to the reaction mixture, culture medium,
injected into a cell or otherwise applied to the transgenic animal. The
cell or tissue that may be employed in the method of the invention
preferably is a host cell, mammalian cell or non-human transgenic animal
of the invention described in the embodiments hereinbefore.
If a sample containing a compound or a plurality of compounds is
identified in the method of the invention, then it is either possible to
isolate the compound from the original sample identified as containing the
compound capable of suppressing or activating the inventive lung tumor
associated polypeptide, or one can further subdivide the original sample,
for example, if it consists of a plurality of different compounds, so as
to reduce the number of different substances per sample and repeat the
method with the subdivisions of the original sample. Depending on the
complexity of the samples, the steps described above can be performed
several times, preferably until the sample identified according to the
method of the invention only comprises a limited number of or only one
substance(s). Preferably said sample comprises substances of similar
chemical and/or physical properties, and most preferably said substances
Several methods are known to the person skilled in the art for producing
and screening large libraries to identify compounds having specific
affinity for a target. These methods include the phage-display method in
which randomized peptides are displayed from phage and screened by
affinity chromatography to an immobilized receptor; see, e.g., WO
91/17271, WO 92/01047, U.S. Pat. No. 5,223,409. In another approach,
combinatorial libraries of polymers immobilized on a chip are synthesized
using photolithography; see, e.g., U.S. Pat. No. 5,143,854, WO 90/15070
and WO 92/10092. The immobilized polymers are contacted with a labelled
receptor and scanned for label to identify polymers binding to the
receptor. The synthesis and screening of peptide libraries on continuous
cellulose membrane supports that can be used for identifying binding
ligands of the polypeptide of the invention and thus possible inhibitors
and activators is described, for example, in Kramer, Methods Mol. Biol. 87
(1998), 25-39. This method can also be used, for example, for determining
the binding sites and the recognition motifs in the polypeptide of the
invention. In like manner, the substrate specificity of the DnaK chaperon
was determined and the contact sites between human interleukin-6 and its
receptor; see Rudiger, EMBO J. 16 (1997), 1501-1507 and Weiergraber, FEBS
Lett. 379 (1996), 122-126, respectively. Furthermore, the above-mentioned
methods can be used for the construction of binding supertopes derived
from the polypeptide of the invention. A similar approach was successfully
described for peptide antigens of the anti-p24 (HIV-1) monoclonal
antibody; see Kramer, Cell 91 (1997), 799-809. A general route to
fingerprint analyses of peptide-antibody interactions using the clustered
amino acid peptide library was described in Kramer, Mol. Immunol. 32
(1995), 459-465. In addition, antagonists of the inventive lung tumor
associated polypeptide of the invention can be derived and identified from
monoclonal antibodies that specifically react with the polypeptide of the
invention in accordance with the methods as described in Doring, Mol.
Immunol. 31 (1994), 1059-1067.
More recently, WO 98/25146 described further methods for screening
libraries of complexes for compounds having a desired property,
especially, the capacity to agonize, bind to, or antagonize a polypeptide
or its cellular receptor. The complexes in such libraries comprise a
compound under test, a tag recording at least one step in synthesis of the
compound, and a tether susceptible to modification by a reporter molecule.
Modification of the tether is used to signify that a complex contains a
compound having a desired property. The tag can be decoded to reveal at
least one step in the synthesis of such a compound. Other methods for
identifying compounds which interact with the polypeptides according to
the invention or nucleic acid molecules encoding such molecules are, for
example, the in vitro screening with the phage display system as well as
filter binding assays or "real time" measuring of interaction using, for
example, the BIAcore apparatus (Pharmacia).
All these methods can be used in accordance with the present invention to
identify activators/agonists and inhibitors/antagonists of the lung tumor
associated polypeptide or related polypeptide of the invention.
Various sources for the basic structure of such an activator or inhibitor
can be employed and comprise, for example, mimetic analogues of the
polypeptide of the invention. Mimetic analogues of the polypeptide of the
invention or biologically active fragments thereof can be generated by,
for example, substituting the amino acids that are expected to be
essential for the biological activity with, e.g., stereoisomers, i.e.
D-amino acids; see e.g., Tsukida, J. Med. Chem. 40 (1997), 3534-3541.
Furthermore, in case fragments are used for the design of biologically
active analogs pro-mimetic components can be incorporated into a peptide
to reestablish at least some of the conformational properties that may
have been lost upon removal of part of the original polypeptide; see,
e.g., Nachman, Regul. Pept. 57 (1995), 359-370. Furthermore, the lung
tumor associated polypeptide of the invention can be used to identify
synthetic chemical peptide mimetics that bind to or can function as a
ligand, substrate, binding partner or the receptor of the polypeptide of
the invention as effectively as does the natural polypeptide; see, e.g.,
Engleman, J. Clin. Invest. 99 (1997), 2284-2292. For example, folding
simulations and computer redesign of structural motifs of the polypeptide
of the invention can be performed using appropriate computer programs (Olszewski,
Proteins 25 (1996), 286-299; Hoffman, Comput. Appl. Biosci. 11 (1995),
675-679). Computer modelling of protein folding can be used for the
conformational and energetic analysis of detailed peptide and protein
models (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp. Med.
Biol. 376 (1995), 37-45). In particular, the appropriate programs can be
used for the identification of interactive sites of the inventive lung
tumor associated polypeptide and its possible receptor, its ligand or
other interacting proteins by computer assistant searches for
complementary peptide sequences (Fassina, Immunomethods 5 (1994), 114-120.
Further appropriate computer systems for the design of protein and
peptides are described in the prior art, for example in Berry, Biochem.
Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci. 501 (1987),
1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from
the above-described computer analysis can be used for, e.g., the
preparation of peptide mimetics of the protein of the invention or
fragments thereof. Such pseudopeptide analogues of the natural amino acid
sequence of the protein may very efficiently mimic the parent protein (Benkirane,
J. Biol. Chem. 271 (1996), 33218-33224). For example, incorporation of
easily available achiral *-amino acid residues into a protein of the
invention or a fragment thereof results in the substitution of amide bonds
by polymethylene units of an aliphatic chain, thereby providing a
convenient strategy for constructing a peptide mimetic (Banerjee,
Biopolymers 39 (1996), 769-777). Superactive peptidomimetic analogues of
small peptide hormones in other systems are described in the prior art
(Zhang, Biochem. Biophys. Res. Commun. 224 (1996), 327-331). Appropriate
peptide mimetics of the protein of the present invention can also be
identified by the synthesis of peptide mimetic combinatorial libraries
through successive amide alkylation and testing the resulting compounds,
e.g., for their binding and immunological properties. Methods for the
generation and use of peptidomimetic combinatorial libraries are described
in the prior art, for example in Ostresh, Methods in Enzymology 267
(1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.
Furthermore, a three-dimensional and/or crystallographic structure of the
polypeptide of the invention can be used for the design of peptide mimetic
inhibitors of the biological activity of the polypeptide of the invention
(Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4
The structure-based design and synthesis of low-molecular-weight synthetic
molecules that mimic the activity of the native biological polypeptide is
further described in, e.g., Dowd, Nature Biotechnol. 16 (1998), 190-195;
Kieber-Emmons, Current Opinion Biotechnol. 8 (1997), 435-441; Moore, Proc.
West Pharmacol. Soc. 40 (1997), 115-119; Mathews, Proc. West Pharmacol.
Soc. 40 (1997), 121-125; Mukhija, European J. Biochem. 254 (1998),
It is also well known to the person skilled in the art, that it is
possible to design, synthesize and evaluate mimetics of small organic
compounds that, for example, can act as a substrate or ligand to the lung
tumor associated polypeptide of the invention or the related polypeptide.
For example, it has been described that D-glucose mimetics of hapalosin
exhibited similar efficiency as hapalosin in antagonizing multidrug
resistance assistance-associated protein in cytotoxicity; see Dinh, J.
Med. Chem. 41 (1998), 981-987.
The nucleic acid molecule of the invention can also serve as a target for
activators and inhibitors. Activators may comprise, for example, proteins
that bind to the mRNA of a gene encoding a the inventive lung tumor
associated polypeptide, thereby stabilizing the native conformation of the
mRNA and facilitating transcription and/or translation, e.g., in like
manner as Tat protein acts on HIV-RNA. Furthermore, methods are described
in the literature for identifying nucleic acid molecules such as an RNA
fragment that mimics the structure of a defined or undefined target RNA
molecule to which a compound binds inside of a cell resulting in
retardation of cell growth or cell death; see, e.g., WO 98/18947 and
references cited therein. These nucleic acid molecules can be used for
identifying unknown compounds of pharmaceutical and/or agricultural
interest, and for identifying unknown RNA targets for use in treating a
disease. These methods and compositions can be used in screening for novel
antibiotics, bacteriostatics, or modifications thereof or for identifying
compounds useful to alter expression levels of proteins encoded by a
nucleic acid molecule. Alternatively, for example, the conformational
structure of the RNA fragment which mimics the binding site can be
employed in rational drug design to modify known antibiotics to make them
bind more avidly to the target. One such methodology is nuclear magnetic
resonance (NMR), which is useful to identify drug and RNA conformational
structures. Still other methods are, for example, the drug design methods
as described in WO 95/35367, U.S. Pat. No. 5,322,933, where the crystal
structure of the RNA fragment can be deduced and computer programs are
utilized to design novel binding compounds which can act as antibiotics.
Some genetic changes lead to altered protein conformational states. For
example, some mutant the inventive lung tumor associated polypetides may
possess a tertiary structure that renders them far less capable of protein
degradation. Restoring the normal or regulated conformation of mutated
proteins is the most elegant and specific means to correct these molecular
defects, although it may be difficult. Pharmacological manipulations thus
may aim at restoration of wild-type conformation of the inventive lung
tumor associated polypeptide. Thus, the nucleic acid molecules and encoded
polypeptides of the present invention may also be used to design and/or
identify molecules which are capable of activating the wild-type function
of a the inventive lung tumor associated polypeptide or related
The compounds which can be tested and identified according to a method of
the invention may be expression libraries, e.g., cDNA expression
libraries, peptides, proteins, nucleic acids, antibodies, small organic
compounds, hormones, peptidomimetics, PNAs or the like (Milner, Nature
Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79
(1994), 193-198 and references cited supra). Furthermore, genes encoding a
putative regulator of the inventive lung tumor associated polypeptide
and/or which exert their effects up- or downstream the inventive lung
tumor associated polypeptide may be identified using, for example,
insertion mutagenesis using, for example, gene targeting vectors known in
the art. Said compounds can also be functional derivatives or analogues of
known inhibitors or activators. Such useful compounds can be for example
transacting factors which bind to the inventive lung tumor associated
polypeptide or regulatory sequences of the gene encoding it.
Identification of transacting factors can be carried out using standard
methods in the art (see, e.g., Sambrook, supra, and Ausubel, supra). To
determine whether a protein binds to the protein itself or regulatory
sequences, standard native gel-shift analyses can be carried out. In order
to identify a transacting factor which binds to the protein or regulatory
sequence, the protein or regulatory sequence can be used as an affinity
reagent in standard protein purification methods, or as a probe for
screening an expression library. The identification of nucleic acid
molecules which encode polypeptides which interact with the inventive lung
tumor associated polypeptides described above can also be achieved, for
example, as described in Scofield (Science 274 (1996), 2063-2065) by use
of the so-called yeast "two-hybrid system". In this system the polypeptide
encoded by a nucleic acid molecule according to the invention or a smaller
part thereof is linked to the DNA-binding domain of the GAL4 transcription
factor. A yeast strain expressing this fusion polypeptide and comprising a
lacZ reporter gene driven by an appropriate promoter, which is recognized
by the GAL4 transcription factor, is transformed with a library of cDNAs
which will express plant proteins or peptides thereof fused to an
activation domain. Thus, if a peptide encoded by one of the cDNAs is able
to interact with the fusion peptide comprising a peptide of an inventive
lung tumor associated polypeptide, the complex is able to direct
expression of the reporter gene. In this way the nucleic acid molecules
according to the invention and the encoded peptide can be used to identify
peptides and proteins interacting with the inventive lung tumor associated
protein. It is apparent to the person skilled in the art that this and
similar systems may then further be exploited for the identification of
inhibitors of the binding of the inventive lung tumor associated proteins.
Once the transacting factor is identified, modulation of its binding to or
regulation of expression of the inventive lung tumor associated
polypeptide can be pursued, beginning with, for example, screening for
inhibitors against the binding of the transacting factor to the protein of
the present invention. Activation or repression of the inventive lung
tumor associated proteins could then be achieved in animals by applying
the transacting factor (or its inhibitor) or the gene encoding it, e.g. in
an expression vector. In addition, if the active form of the transacting
factor is a dimer, dominant-negative mutants of the transacting factor
could be made in order to inhibit its activity. Furthermore, upon
identification of the transacting factor, further components in the
pathway leading to activation (e.g. signal transduction) or repression of
a gene involved in the control of the inventive lung tumor associated
polypeptide then can be identified. Modulation of the activities of these
components can then be pursued, in order to develop additional drugs and
methods for modulating the metabolism of protein degradation in animals.
Thus, the present invention also relates to the use of the two-hybrid
system as defined above for the identification of the inventive lung tumor
associated polypeptide or activators or inhibitors of the inventive lung
tumor associated polypeptide.
The compounds isolated by the above methods also serve as lead compounds
for the development of analogue compounds. The analogues should have a
stabilized electronic configuration and molecular conformation that allows
key functional groups to be presented to the inventive lung tumor
associated polypeptide or its possible receptor in substantially the same
way as the lead compound. In particular, the analogue compounds have
spatial electronic properties which are comparable to the binding region,
but can be smaller molecules than the lead compound, frequently having a
molecular weight below about 2 kD and preferably below about 1 kD.
Identification of analogue compounds can be performed through use of
techniques such as self-consistent field (SCF) analysis, configuration
interaction (CI) analysis, and normal mode dynamics analysis. Computer
programs for implementing these techniques are available; e.g., Rein,
Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss, New
York, 1989). Methods for the preparation of chemical derivatives and
analogues are well known to those skilled in the art and are described in,
for example, Beilstein, Handbook of Organic Chemistry, Springer edition
New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic
Synthesis, Wiley, New York, USA. Furthermore, said derivatives and
analogues can be tested for their effects according to methods known in
the art; see also supra. Furthermore, peptidomimetics and/or computer
aided design of appropriate derivatives and analogues can be used, for
example, according to the methods described above.
In a preferred embodiment of the above-described methods of the invention,
said cell is a cell of or, obtained by a method of the invention or is
comprised in the above-described transgenic non-human animal.
Once the described compound has been identified and obtained, it is
preferably provided in a therapeutically acceptable form.
The present invention provides methods for detection and treatment of
disorders characterized by abnormal cell proliferation, such as e.g.
cancers. In one aspect the present invention provides a method for the
detection of disorders characterized by abnormal cell proliferation, such
as e.g. cancers based on the determination of the presence or absence
and/or the level of expression of the inventive lung tumor associated gene
in biological samples. In a second aspect the present invention provides a
method for treatment of disorders characterized by abnormal cell
proliferation, such as e.g. cancers using the inventive lung tumor
associated gene products as therapeutically active agents. The invention
also provides for therapeutic methods based on the modulation of the
activity of the inventive lung tumor associated polypeptide. It is one
aspect of the invention to provide a method for rational tumor management
based on the detection of the inventive lung tumor associated gene
products in patient samples and the tailoring of a therapy correlated to
the detected overexpression of said gene products. Furthermore the present
invention provides for a research or diagnostic test kit for performing
the reactions involved in the detection of the presence or absence and/or
the level of overexpression of the inventive lung tumor associated gene.
Finally the present invention relates to pharmaceutical compositions
applicable in the treatment of disorders according to the present
Claim 1 of 12 Claims
1. An isolated nucleic acid comprising:
(a) SEQ ID NO: 1, 3, or 5, or (b) a nucleic acid molecule having a
sequence different from the sequence of the cDNA or mRNA of SEQ ID NO: 1,
3, or 5 due to the degeneracy of the genetic code of the cDNA or mRNA,
wherein said nucleic acid molecule encodes the amino acid sequence of SEQ
ID NO: 2, 4, or 6, respectively.
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