HK1078615A - Compositions and methods for detection and treatment of proliferative abnormalities associated with overexpression of human transketolase like-1 gene - Google Patents

Description

Compositions and methods for diagnosing and treating proliferative disorders associated with overexpression of human transketolase like-1 gene

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The present invention relates to methods for treating and diagnosing disorders associated with aberrantly proliferating cells. In one aspect the invention relates to methods for the detection of overexpression of the human transketolase like-1 gene (transketolaselide-1 gene) in biological samples, particularly for the diagnosis of tumors and their prognostic stage. In another aspect the invention relates to methods for treating conditions associated with overexpression of the human transketolase like-1 gene. The methods of treatment may include gene therapy methods and methods of inhibiting or reducing the activity of a transketolase like-1 polypeptide.

Despite significant scientific and medical research efforts, neoplastic disease remains a major cause of human mortality. For example: in germany, over 34 million people develop cancer each year, and over 21 million die from cancer. Epithelial tumors account for the majority of cancers: lung cancer is the leading cause of cancer death in men and breast cancer in women. Colon cancer is the second leading cause of death in both men and women (Becker, N. and Wahrendorf, J., (1997) Atlas of cancer Mortality in the Federal republic of Germany 1981-1990, Springer-Verlag, Berlin, Heidelberg).

The main reason for this unsatisfactory situation is that most tumor diseases are diagnosed at a very advanced stage, when isolated tumor cells or small aggregates of tumor cells have been released from the primary tumor and distributed throughout the host organism and may have ultimately resulted in cryptic or overt metastatic disease. Early cancers, particularly precancers, do not usually produce any symptoms and are unknown to the patient.

To overcome this situation, more research effort and clinical planning is required to improve the early diagnosis of cancer and to develop a true prophylactic or therapeutic vaccination strategy to immunize a patient before the appearance of a definite cancer or after the excision of the cancer or its precursors to prevent the survival of disseminated isolated cancer cells (DTCs) that may have been released from the tumor prior to or during the initial surgical intervention.

For a few cancers, especially cervical cancer, an effective early diagnosis of cancer can be established. The subsequent reduction in the mortality rate of these particular tumors convincingly confirms the effectiveness of the early diagnostic protocol.

In summary, the diagnostic methods used to date are unfortunately very insensitive and, due to the lack of specificity, also risk producing false positive results. Furthermore, any conclusions regarding the malignancy grade of a tumor, the progression of a tumor and its metastatic potential cannot be predicted accurately using current diagnostic methods.

Therefore, the use of reliable diagnostic molecular markers is very useful for understanding the molecular basis of epithelial tumors, such as colon tumors, to distinguish benign from malignant tissue and for staging and segmentation of cancers, especially for metastatic cancer patients with poor prognosis. Such markers are also expected to be useful in developing new approaches to cancer therapy.

The understanding of the molecular events of normal cells transforming into invasive tumor cells of varying degrees, and the availability of appropriate experimental systems for the selection of cancer-associated genes, are absolutely prerequisites for the identification of such novel diagnostic markers and therapeutic drug targets.

Tumorigenesis is generally considered to be a complex, multi-stage process in which genetic changes and environmental factors are thought to deregulate the cellular processes that control cell proliferation and differentiation. This multi-stage process is well illustrated by the example of colon cancer, which typically develops over several decades and apparently requires the development of multiple genetic events (reviewed by Kinzler and Vogelstein, 1996, Cell 87, 159-170). This process results in additional somatic mutations due to the heritability of genetic alterations (caused by an obvious cause) and genomic instability (caused by genotoxic agents in the environment). Clearly, the decisive factors involved in tumor formation are not well understood and clearly depend on the different tumor types.

Therefore, the technical problem of the present invention is to provide means for diagnosis and treatment of epithelial cell tumors, so as to overcome the disadvantages of the existing diagnosis and treatment methods. The solution to the above technical problem is described by providing the embodiments in the claims.

The present invention is based on the discovery by the inventors that the human transketolase like-1 gene shown in SEQ. ID.1(cf. TKT-L1, TKR: NM-012253; Access number: X91817) is highly overexpressed in colon, pancreatic, lung and gastric cancer tissues compared to the levels in the respective normal control tissues. This is particularly important for diagnosis, since transketolase is not similarly overexpressed in tumor tissue.

Thus, tumor diagnostic methods may be based on detecting overexpression of a transketolase like-1 gene product in a biological sample. The course of the disease can be predicted, prognosis can be assessed, and the appropriate patient treatment regimen can be tailored to the patient based on the detection of the presence or absence and/or level of the transketolase like-1 gene product.

In addition, the present invention can provide a viable treatment for conditions associated with overexpression of a transketolase like-1 gene product. In one aspect, the invention provides methods of treating a disorder with a transketolase like-1 nucleic acid or polypeptide. In another aspect, the invention provides therapeutic methods based on reducing the enzymatic activity of a transketolase like-1 gene polypeptide. It is therefore an aspect of the present invention to provide methods for rational tumor management based on the detection of transketolase like-1 gene products in patient samples and for tailoring of treatment protocols corresponding to the detected overexpression of such gene products.

Finally, the present invention relates to diagnostic and research kits and pharmaceutical compositions for carrying out the methods disclosed herein.

Brief Description of Drawings

FIG. 1: detecting the overexpression of the transketolase sample-1 gene in the colon cancer by using an RT-PCR method; the induced expression of the transketolase like-1 gene in colon cancer tissues compared to control tissues is shown.

FIG. 2: detecting the overexpression of the transketolase sample-1 gene in the lung adenocarcinoma by using an RT-PCR method; the figure shows the induced expression of transketolase like-1 gene in lung adenocarcinoma tissue compared to control tissue.

FIG. 3: detecting the overexpression of transketolase like-1 gene in gastric cancer by using an RT-PCR method; the figure shows the induced expression of transketolase like-1 gene in gastric cancer tissues compared to control tissues.

FIG. 4: detecting the overexpression of transketolase in colon cancer by using an RT-PCR method; the induced expression of transketolase in colon cancer tissues compared to control tissues is shown.

FIG. 5: detecting the overexpression of transketolase in lung adenocarcinoma by using an RT-PCR method; the figure shows the induced expression of transketolase in lung adenocarcinoma tissue compared to control tissue.

FIG. 6: detecting the overexpression of transketolase in gastric cancer by using an RT-PCR method; the figure shows the induced expression of transketolase in gastric cancer tissues compared to control tissues.

FIG. 7: the DNA and amino acid sequence of tktl 1; the protein portion used for immunization to generate antibodies is indicated in bold letters; other polypeptides used for immunization of polypeptides to produce antibodies are underlined.

FIG. 8: immunohistochemical analysis of gastric cancer tissue (B) and corresponding normal tissue (a) with primary antibody against tktl 1. Strong overexpression of tktl1 protein was detected in 1666 patients' cancer.

FIG. 9: immunohistochemical analysis of gastric cancer patient # 1682 (a) and gastric cancer patient # 1697 (B) with primary antibody against tktl 1. Strong overexpression of tktl1 protein was detected in both the nucleus and cytoplasm of 1682 patient's carcinoma. Very strong overexpression of tktl1 protein was detected in both the nucleus and cytoplasm of 1697 patient's carcinoma.

FIG. 10: immunohistochemical analysis of 1699 gastric cancer patients with primary antibody against tktl 1. Panel A shows cancer with normal tissue. Where low expression of tktl1 was detected in normal tissues and very strong overexpression of tktl1 was detected in cancer tumor cells. Panel B is an enlarged view of the boundary of normal and tumor tissue. Tumor-specific granular staining can be detected.

FIG. 11: immunohistochemical analysis of 1698 gastric cancer patients with primary antibody against tktl 1. Panel A shows that strong overexpression of tktl1 protein was detected in both the nucleus and cytoplasm of gastric tumor cells. Low or no expression was detectable in the surrounding fibroblasts. Panel B is an enlarged view of the cancer cell area and surrounding connective tissue. Tumor-specific granular staining can be detected.

The present invention provides methods for the diagnosis and treatment of disorders characterized by abnormal cell proliferation, such as cancer.

A first aspect of the present invention is to provide a diagnostic method for a disorder characterized by abnormal cell proliferation, such as cancer, based on the determination of the presence and/or expression level of the human transketolase like-1 gene of the sequence shown as SEQ.ID.1(cf. TKT-L1, TKR: NM-012253; Accession number: X91817) in a biological sample.

In a second aspect of the invention, there is provided a method of treating a condition characterized by abnormal cell proliferation, such as cancer, with a human transketolase like-1 gene product as a therapeutically active agent.

A third aspect of the present invention is a research or diagnostic kit for carrying out a reaction involved in detecting the presence or absence and/or an overexpression level of a human transketolase like-1 gene.

A fourth aspect of the invention relates to a pharmaceutical composition useful in the treatment of a condition according to the invention.

The transketolase like-1 gene products mentioned herein may comprise polypeptides and nucleic acids encoded by the transketolase like-1 gene.

The polypeptides and polynucleotides used to carry out the method according to the invention are isolated. This means that these molecules are removed from their original environment. A naturally occurring protein is isolated if it is separated from some or all of the materials with which it coexists in its natural environment.

The polynucleotide is isolated, for example, if it is to be cloned into a vector.

The human transketolase like-1 nucleic acid molecule used for carrying out the method according to the invention may comprise a polynucleotide or a fragment thereof. Preferably, the polynucleotide may comprise at least 20 contiguous nucleotides, preferably at least 30 contiguous nucleotides, and more preferably at least 45 contiguous nucleotides which are identical or have sequence homology or encode identical or homologous polypeptides to the wild-type transketolase like-1 polypeptide, but not other transketolase like polypeptides or transketolases. The nucleic acid according to the invention may also be complementary or reverse complementary to any of the polynucleotides. For example, a polynucleotide may comprise a single-stranded (sense or antisense) or double-stranded molecule, which may be DNA (genomic, cDNA or synthetic DNA) or RNA. RNA molecules include hnRNA (including introns) and mRNA (not including introns). Polynucleotides according to the invention may also be linked to any other molecule, such as a support material or a detection marker molecule, and may, but need not, comprise additional coding or non-coding sequences.

The human transketolase like-1 polynucleotide used according to the invention may be the native sequence or a variant thereof. The variant may comprise one or more substitutions, additions, deletions and/or insertions such that the immunogenicity of the encoded polypeptide is not reduced relative to the native tumor protein. Variants are, for example, allelic variations of polynucleotides. An allelic variation as used herein is another (alternative) form of a gene, which may be caused by at least one mutation in a nucleic acid sequence. Alleles may result in altered mRNA or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or more allelic forms. Common mutations that occur in alleles are typically caused by natural deletions, additions or substitutions of nucleotides. Each type of alteration may occur alone or in combination with other alterations, or one or more times in a given sequence. Variants according to the invention exhibit preferably 70%, more preferably at least 80%, most preferably at least 90% sequence identity with the native nucleic acid molecule disclosed herein. Methods for determining sequence similarity are well known to those skilled in the art.

Sequence similarity determinations can be made, for example, using FastA and/or BlastN bioinformatics software provided by the HUSAR server of DKFZ Heidelberg.

The nucleic acids referred to in the present invention may be all polynucleotides which hybridize under stringent conditions to a probe specific for a transketolase like-1 sequence used herein. Stringent conditions for hybridization reactions are well known to those skilled in the art, and Molecular cloning, as in Sambrook et al, can also be used: a Laboratory Manual, 2nd Edition, 1989.

The invention also encompasses polynucleotides that encode polypeptides naturally encoded by human transketolase like-1 nucleic acids whose nucleic acid sequences do not exhibit the above-described percentages of sequence homology due to the degeneracy of the genetic code. Such nucleic acids can be produced, for example, by substituting codons of the disclosed sequences with degenerate codons to prepare synthetic nucleic acids. The preparation of such artificial nucleic acid sequences can be carried out by methods known to the person skilled in the art.

The human transketolase like-1 nucleotide sequence used in accordance with the invention may be linked to a variety of other nucleic acid sequences using known recombinant DNA techniques. For example, the sequences can be cloned into any cloning vector, such as plasmids, phagemids, lambda phage derivatives and cosmids. Moreover, vectors such as expression vectors, replication vectors, probe generation vectors, and sequencing vectors can all be linked to the sequences disclosed herein.

Sequences from which nucleic acids according to the invention can be cloned include coding sequences, non-coding sequences, and regulatory sequences including promoters, enhancers, and terminators. The human transketolase like-1 nucleic acid sequences disclosed herein may be present in association with other coding sequences. These sequences may encode a variety of proteins such as enzymes, receptors, antigens, immunogenic fragments or epitopes, binding proteins, and the like. The nucleic acid sequences may be directly linked or separated by a nucleic acid encoding a spacer or linker region. The nucleic acid sequences may be separated by a length of nucleic acid which is removable following transcription. Non-coding sequences that may be linked to the sequences disclosed herein may be, for example, promoter regions, enhancers, cis-regulatory elements, 5' untranslated regions, terminators, and the like.

In a preferred embodiment, the human transketolase like-1 polynucleotide may be formulated so that it is capable of entering and being expressed in prokaryotic or eukaryotic cells, such as mammalian cells. These formulations are useful, for example, for therapeutic purposes. Expression of the nucleic acid sequence in the target cell can be achieved using methods known to those skilled in the art. For example, the nucleic acid may be linked to elements that render it suitable for expression in a host cell. These elements may comprise promoters or enhancers, such as CMV-, SV40-, RSV-, metallothionein I-or polyhedrin promoters, e.g.CMV or SV40 enhancers. Possible methods for expression are, for example, incorporation of the polynucleotide into viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, vaccinia viruses or poxviruses. Viral vectors for the purpose of expressing nucleic acids in mammalian host cells may include pcDNA3, pMSX, pKCR, pEFBOS, cDM8, pCEV4, and the like. These techniques are known to those skilled in the art.

The human transketolase like-1 sequence fragment used herein may comprise oligonucleotides such as nucleic acid probes for hybridization, primers for amplification reactions or antisense nucleic acid constructs for antisense technology. The nucleic acid probe according to the invention may be any nucleic acid probe which is at least 80% identical to at least 15 contiguous nucleotide portions of the nucleic acid sequence of the human transketolase-like-1 gene, or which is complementary or reverse-complementary to this sequence, but which does not hybridize to other transketolase or transketolase-like sequences. The nucleic acid probes according to the invention are also characterized in that they can hybridize under stringent conditions to the nucleic acid sequences disclosed herein. The primer may be any nucleotide suitable for performing a specific amplification reaction. Thus, the primer used according to the present invention may be a nucleic acid oligomer having at least 15 contiguous nucleotides identical to at least 80% of the sequence of a nucleic acid sequence of a human transketolase like-1 gene, or complementary thereto in the reverse direction. Primers according to the invention can specifically hybridize to a sequence disclosed herein, or a portion thereof, but not to other transketolase or transketolase-like sequences, under conditions suitable for use in a nucleic acid amplification reaction process. Antisense oligonucleotides used herein can be nucleic acid molecules that are reverse complementary to a transcript of a disclosed coding sequence and which can bind to the transcript by base pairing and thereby inhibit or reduce expression of the coding sequence.

The nucleic acids used according to the invention may also be chemically pretreated nucleic acids. These chemically pretreated nucleic acids can comprise any of the nucleic acids disclosed herein that have been treated with a chemical agent suitable for producing a modification in the nucleic acid molecule. The modification may include, for example, specific modification of a specific base in a nucleic acid. The chemical treatment may comprise treatment with, for example, sodium bisulfite, hydrazine, or potassium permanganate. Particular sequences of interest in experiments employing chemically pretreated nucleic acids may contain, for example, coding or non-coding regions of the sequence. Examples of non-coding regions that may be chemically treated include the promoter region or CpG islands in the 5' UTR.

Human transketolase like-1 polypeptides used according to the invention may comprise amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds. Thus, a polypeptide comprising a portion of one of the above-described human transketolase like-1 proteins, e.g., a protein comprising the amino acid sequence of a human transketolase like-1 protein, may consist of the entire portion, or the portion may be present in a large polypeptide containing additional sequences. The additional sequences may be derived from a native protein or a heterologous protein, and such sequences may (but need not) be immunologically active and/or antigenic. As described in detail below, the polypeptide may be isolated from tumor tissue or prepared by synthetic or recombinant means.

A polypeptide exhibiting the biological properties of a human transketolase like-1 polypeptide as used herein is understood to be a polypeptide having at least one activity, such as the activity of an enzyme (transketolase activity), an interaction activity between proteins, an enzymatic sensitivity to the presence of thiamine, or an antigenic or immunogenic property, such as the ability to bind to an antibody directed against said polypeptide (i.e. comprising an immunogenic portion) of said human transketolase-1 polypeptide.

An immunogenic moiety as used herein refers to a portion of a protein that is recognized by a B cell and/or T cell surface antigen receptor. The immunogenic portion comprises at least 5 amino acid residues, more preferably at least 10 amino acid residues, and most preferably at least 15 amino acid residues of the proteins disclosed herein. In a preferred embodiment of the invention, specific domains of the protein, such as the transmembrane region or the N-terminal leader sequence, have been deleted.

The immunogenic portion according to the invention reacts with antisera or specific antibodies with the same or almost the same intensity as the native full-length protein. Immunogenic portions are generally identified using methods well known in the art. Possible methods are for example screening of the polypeptides for their ability to react with antigen-specific antibodies, anti-serum and/or T-cell lines or clones.

The transketolase-1-like polypeptides used according to the inventors also comprise variants of the native protein. These variants may differ from the native protein by one or more alterations such as substitutions, deletions, additions and/or insertions. The immunoreactivity and/or the biological activity of the variants according to the invention are not substantially reduced compared to the native protein. In a preferred embodiment of the invention the immunoreactivity and/or activity is reduced by less than 50%, in a more preferred embodiment by less than 20% compared to the native polypeptide. In another preferred embodiment of the invention, the variant of the polypeptide may be altered such that the activity of the native protein is increased, decreased or deleted. These variants are useful, for example, in the treatment of conditions associated with overexpression of the human transketolase like-1 gene. In a preferred embodiment, the variant may lack one or more portions, such as an N-terminal leader sequence, a transmembrane region, or a small N-and/or C-terminal sequence. The variant shows preferably 70%, more preferably at least 90% and most preferably at least 95% identity to the polypeptide disclosed according to the invention.

The variants used according to the invention preferably comprise conservative substitutions, so that the amino acids changed are amino acid substitutions of similar nature. The properties involved may include polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphiphilicity of the amino acid residues.

The variants used according to the invention may also comprise additional terminal leader sequences, linkers or sequences, making the synthesis, purification or stabilization of the polypeptide simple and easy.

The polypeptide variants used in accordance with the methods of the present invention may be prepared by conventional molecular biology methods (see, e.g., Sambrook et al, 1989, Experimental procedures for molecular cloningGuide, molecular cloning, A Laboratory Manual, 2^ndedition Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY). It is for example possible to introduce different mutations into the nucleic acid molecules of the invention. As a result, it is possible to synthesize a human transketolase like-1 polypeptide whose biological properties are likely to be improved. One possibility is to generate deletion mutants, the nucleic acid molecules of which are generated by successive deletions from the 5 'or 3' end of the coding DNA sequence, with the result that correspondingly shortened polypeptides are synthesized. Another possibility is to introduce single point mutations at positions where, for example, modifications of the amino acid sequence would affect the enzyme activity or regulation of the enzyme. By this method, it is possible to generate mutant proteins, for example proteins which have an altered Km value or which no longer comply with the normal regulatory mechanisms in the cell (for example those associated with allosteric regulation or covalent modification). Such muteins are useful, for example, as therapeutic compounds, e.g., antagonists.

The nucleic acid molecules used in the process of the invention or parts of these molecules can be introduced into plasmids by genetic engineering in prokaryotic cells, and sequence mutations or modifications can be obtained by recombination of DNA sequences. The bases may be replaced and natural or synthetic sequences added using conventional methods (see, Sambrook et al, supra). To ligate the DNA fragments to each other, a linker or linker may be added to the fragments. In addition, manipulations can be performed to provide appropriate cleavage sites or to remove excess DNA or cleavage sites. If insertions, deletions or substitutions are possible, in vitro mutations, primer repairs, restriction or ligation reactions are possible. As the analysis method, commonly used sequence analysis, restriction enzyme analysis and other biochemical or molecular biological analysis methods can be used.

The polypeptide may comprise a fused or chimeric polypeptide comprising a sequence disclosed herein. A fusion protein comprises an inventive polypeptide or a portion thereof or a variant of an inventive polypeptide or a portion thereof, together with any second and more polypeptides, such as another inventive polypeptide or a portion thereof or a variant of an inventive polypeptide or a portion thereof, and/or any heterologous polypeptide. The heterologous polypeptide may comprise an enzyme, receptor molecule, antigen, antigenic or immunogenic epitope or fragment thereof, antibody or fragment thereof, signal polypeptide or signaling polypeptide, and the like. Immunogenic proteins are for example proteins capable of eliciting a memory response. Examples of such proteins include tetanus, tuberculosis, and hepatitis proteins (see, e.g., Stoute et al New Engl. J. Med., 336: 8691 (1997)). In one embodiment of the invention it is possible to construct fusion peptides to enhance the detection or purification of polypeptides. For purification, tags, such as his-tags, myc-tags, and the like, may be added to the polypeptide. For detection, antigenic moieties, enzymes, chromogenic sequences, etc. may be fused to the polypeptide. The fusion protein of the invention may (but need not) include a linker peptide between the first and second polypeptides.

The nucleic acid sequences encoding the fusion proteins used in the present invention are constructed using known recombinant DNA techniques, and the nucleic acid sequences encoding the first and second polypeptides, respectively, are assembled into a suitable expression vector. The 3 'end of the nucleic acid sequence encoding the first polypeptide is linked, with or without a peptide linker, to the 5' end of the nucleic acid sequence encoding the second polypeptide to ensure proper sequence reading frame for translation of the two nucleic acid sequence mrnas into a single fusion protein, which retains the biological activity of the first and second polypeptides.

The peptide linker sequence may be used to separate the first and second polypeptides by a sufficient distance to ensure that each polypeptide folds into its secondary and tertiary structure. Such peptide linker sequences are introduced into the fusion protein using standard techniques well known in the art. An appropriate peptide linker sequence may be selected based on the following factors: (1) its ability to adopt a flexible extended conformation; (2) its ability to adopt secondary structures that may interact with functional epitopes of the first and second polypeptides; and (3) hydrophobic or charged residues that are not reactive with a functional epitope of a polypeptide. Preferably the peptide linker sequence comprises Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Amino acid sequences useful as linkers include those disclosed in Maratea et al, Gene 40: 39-46, 1985; murphy et al, proc.natl.acad.sci.usa 83: 8258-8262, 1986; U.S. Pat. No.4,935,233 and U.S. Pat. No.4,751,180. Linker sequences can be 1 to about 50 amino acids in length. Peptide sequences are not required when the first and second polypeptides have an optional N-terminal amino acid site that allows for separation of the functional domains and prevents steric hindrance.

Human transketolase like-1 polypeptide and nucleic acids encoding such polypeptide for use in the methods according to the invention may be isolated from tumor tissue by any method well known in the art. The nucleic acid sequence corresponding to the gene (or a portion thereof) encoding one of the tumor polypeptides of the invention can be isolated from a tumor cDNA library using subtractive techniques. The partial nucleic acid sequences thus obtained can be used as oligonucleotide primers for the design of Polymerase Chain Reactions (PCR) for amplification of full-length nucleic acid sequences from human genomic nucleic acid libraries or tumor cDNA libraries using techniques well known in the art (see, e.g., Mullis et al, Cold Spring Harbor Symp. Quant. biol. 51: 263, 1987; Erliched., PCR techniques, Stockton Press, NY, 1989). Sequence specific primers for use in such methods can be designed, purchased or synthesized based on the nucleotide sequences provided herein.

The human transketolase like-1 polypeptides used in the methods disclosed herein may also be produced by synthetic methods. In particular, the synthesized polypeptides are less than about 100 amino acids, usually less than about 50 amino acids, and can be produced using techniques well known to those skilled in the art. For example: such polypeptides may be added sequentially to a growing amino acid chain using any commercially available solid phase technique, such as Merrifield solid phase synthesis (see, e.g., Merrifield, J.Am.chem.Soc.85: 2149-S2146, 1963). Automated polypeptide synthesis equipment is commercially available from suppliers such as Perkinelmer/Applied BioSystems Division (Foster City, Calif.) and may be operated according to manufacturer's instructions.

The ligated nucleic acid sequences encoding the polypeptides used in the methods disclosed herein are ligated to appropriate transcriptional or translational regulatory elements known to those skilled in the art. The regulatory elements responsible for expression of the nucleic acid may be located, for example, 5 'to the nucleic acid sequence encoding the first polypeptide, within the coding sequence, or 3' to the nucleic acid sequence encoding the first or any additional polypeptides. The stop codon required for translation and transcription termination signals is located at the 3' end of the nucleic acid sequence encoding the second polypeptide.

The polypeptides used according to the method of the invention may be isolated. This means that these molecules can be removed from their original environment. A naturally occurring protein is isolated if it is separated from some or all of the materials that exist in the natural environment. The polynucleotide is isolated, for example, for cloning into a vector.

In certain preferred embodiments, which are described in more detail below, the polypeptides used in the methods disclosed herein can be prepared in an isolated, substantially pure form (i.e., the polypeptides are homogeneous as determined by amino acid composition and base sequence analysis). Preferably, the polypeptide is at least about 90% pure, more preferably at least about 95% pure, and most preferably at least about 99% pure. The substantially pure polypeptide may be used, for example, as a pharmaceutical composition.

In addition, the present invention uses a binding factor that specifically binds to a human transketolase like-1 protein. These binding factors may include, for example, antibodies and antigen binding fragments, bifunctional hybrid antibodies, peptidomimetics (peptidomimetics) comprising a minimal antigen binding epitope, and the like.

A specific response is said to be if the antibody or antigen binding factor reacts at a detectable level with the protein used herein and does not substantially react with other proteins. The antibody according to the invention may be a monoclonal or polyclonal antibody. The term antibody or monoclonal antibody as used herein is meant to include intact molecules and antibody fragments (e.g., Fab and F (ab') 2 fragments) that are capable of specifically binding to a protein. Fab and F (ab') 2 fragments are cleared more rapidly from the circulation without the Fc fragment of an intact antibody and may bind less to nonspecific tissues than an intact antibody (wahl et al. J.Nucl.Med.24: 316-325 (1983.) therefore, these fragments are more preferred as are the products of Fab or other immunoglobulin expression libraries.

Binding factors may be used according to the invention alone or in combination. High sensitivity can be obtained by the combined use. Antibody terms preferably relate to the substantial combination of monoclonal antibodies of different epitope specificities and different monoclonal antibody preparations.

Monoclonal Antibodies are made from the antigens comprising polypeptide fragments used in the present invention using any technique known to those skilled in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring harbor Laboratory, 1988). In one such technique, an immunogen comprising an antigenic polypeptide, or a synthetic portion thereof, is initially injected into any of a variety of mammals (e.g., mice, rats, rabbits, sheep, and goats). In this step, the polypeptide of the present invention can be used as an immunogen without modification. Alternatively, particularly for shorter polypeptides, a higher immune response may be elicited if the polypeptide is linked to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is injected into the animal host, preferably in combination with one or more boosters according to a predetermined schedule, and the animal is bled periodically. Polyclonal antibodies specific for a polypeptide may be purified from antisera, for example by affinity chromatography using the polypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for the antigenic polypeptide of interest can be prepared using K  hler and Milstein (Eur. J. Immunol.6: 511-519, 1976) and modified techniques thereof. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies with the specificity of interest (i.e., reactivity with the polypeptide of interest). These cell lines can be generated, for example, from spleen cells obtained from the immunized animals described above. Spleen cells are immortalized by, for example, fusion with a myeloma cell fusion partner, preferably a myeloma cell fusion partner that is syngeneic with the immunized animal. A variety of fusion techniques may be applied. For example, spleen cells and myeloma cells can be combined with a non-ionic detergent for several minutes and then seeded at low density in a selective medium that supports hybrid cells but not myeloma cell growth. Preferably, the selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, typically about 1 to 2 weeks, heterozygous clones can be observed. Single clones were selected and tested for binding activity against the polypeptide. Highly reactive and specific hybridomas are preferred.

Monoclonal antibodies can be isolated from the hybridoma clone growth supernatants. In addition, various techniques can be used to increase yield, such as injecting the hybridoma cell line into the peritoneal cavity of an appropriate vertebrate host, such as a mouse. The monoclonal antibodies can then be harvested from the ascites fluid or blood. Impurities may be removed by conventional methods such as chromatography, gel filtration, precipitation and extraction. The human transketolase like-1 polypeptide may be used in a purification process such as an affinity chromatography step.

Antibodies specific for human transketolase like-1 used according to the invention may comprise additional binding sites to bind to, or be coupled to, a therapeutic agent or other polypeptide. The therapeutic agent may comprise a drug, a toxin, a radionuclide and derivatives thereof. The agent may be coupled to the binding member, directly or indirectly, for example, via a linker or carrier group. The linker group may function, for example, to enable a coupling reaction of the binding agent with the therapeutic agent or other agent, or to act as a spacer between different parts of the fusion molecule. The linker may also be cleaved under certain conditions and thus release the binding factor under such conditions. The therapeutic agent may be covalently coupled to the carrier group, either directly or through a linker group. The therapeutic agent may also be non-covalently coupled to the carrier. According to the invention, for example, albumin, polypeptides, polysaccharides or liposomes can be used as carriers.

The human transketolase like-1 specific antibodies used according to the invention may be conjugated to one or more agents. Multiple agents conjugated to an antibody may be identical, or several different agents bind to an antibody.

The methods disclosed herein are applicable to all eukaryotes affected by conditions associated with overexpression of the transketolase like-1 gene. The subject used in the present invention may comprise, for example, a mammal, such as an agriculturally beneficial animal (cows, sheep, horses, pigs, etc.), a pet (cats, dogs, etc.), a research animal (rats, mice, hamsters, etc.), or a human.

The diagnosis referred to in the present invention may comprise determination of the level of the human transketolase like-1 gene product in a sample. Based on the measured levels of the human transketolase like-1 gene product in the sample, individuals can be divided into several subpopulations. The subpopulation may be generated based on clinical data relating to a particular level of a transketolase like-1 gene product determined in the sample, such as survival rate, disease recurrence, frequency of metastasis, and the like.

Based on these subpopulations, prognostic assessments can be made. Treatment regimens can be tailored to the individual affected by the tumor according to the subpopulation.

For example, overexpression of the transketolase like-1 gene and its enhanced activity in the pentose phosphate cycle in colon, stomach, pancreas and lung tumors suggests a mechanism by which thiamine (vitamin B1) promotes ribonuclease synthesis and tumor cell proliferation via the non-oxidative transketolase pathway. Therefore, thiamine uptake in cancer patients has a direct causal relationship with tumor growth rate and transketolase like-1 gene overexpression. This also provides background information that will help develop guidelines for the selection of therapeutics for use with anti-thiamine transketolase inhibitors in clinical settings. Clinical and experimental data indicate the increased utilization of thiamine by human tumors and its interfering effects on chemotherapy experiments. RNA ribose analysis showed that more than 90% of the ribose synthesis in cultured cervical and pancreatic cancer cells is contributed by glucose carbon atoms, and ribose is synthesized mainly through the thiamine-dependent transketolase-dependent pathway (> 70%). In several tumor models, anti-thiamine compounds significantly inhibited nucleic acid synthesis and tumor cell proliferation in vitro and in vivo. The role of the thiamine-dependent transketolase-dependent reaction in the production of ribose in tumor cells, which is the central process in de novo nucleic acid synthesis and purine salvage pathways, is rarely reported in the medical literature.

Because the thiamine-dependent transketolase pathway is the central pathway for providing phosphoribosyl phosphate to tumor nucleic acids, excessive thiamine supplementation may result in failure of therapeutic attempts to terminate cancer cell proliferation. The detection of colon and lung tumor subtypes with over-expression of transketolase like-1 gene provides an important step for the personalized treatment of tumors, and the method for limiting the administration of thiamine and the concomitant treatment of the tumors by using transketolase inhibitors becomes a more reasonable method for treating cancers.

Thus, based on the detection of TKT-L1 overexpression, new therapeutic strategies can be tailored to the specific biochemical response of the pentose phosphate cycle, treating cancer patients with hormones associated with glucose metabolism, cofactors controlling thiamine uptake, the non-oxidative transketolase pentose phosphate cycle response, or with anti-thiamine analogs.

Thus, in one embodiment of the invention, conditions characterized by overexpression of human transketolase like-1 can be treated based on the level of overexpression of the transketolase like-1 gene. The non-oxidized pentose phosphate cycle reaction is used for inhibiting glucose utilization pathway to selectively produce nucleic acid, and a strong control effect on cell cycle is achieved, so that a novel target point for tumor treatment is provided.

In one embodiment, treatment of a condition associated with overexpression of a transketolase like-1 gene may comprise limiting thiamine administration to affected individuals. In another embodiment, the treatment may comprise administration of a transketolase inhibitor, e.g., an anti-thiamine compound.

Monitoring may include detection of the level of the human transketolase like-1 gene product in samples taken at different time points, as well as detection of changes in such levels. The course of the disease can be followed based on the changes. The course of the disease can be used to select a treatment regimen for a particular individual.

Another aspect of the diagnosis and monitoring of the course of the disease according to the invention may comprise the detection of mild sequelae. This may involve, for example, the detection of the level of a human transketolase like-1 gene product in one or more body samples at one or more time points after initiation of treatment of an individual. Depending on the level of human transketolase like-1 gene product detected in the sample, an appropriate treatment regimen may be selected for a particular individual.

In another preferred embodiment, the diagnostic method is performed to detect disseminated tumor cells in the biological sample as a diagnosis of mild sequelae (MRD).

Conditions which are characterized by abnormal cell proliferation, as referred to herein, may include, for example, tumors, such as benign and malignant tumors, carcinomas, sarcomas, leukemias, lymphomas or dysplasias. Tumors may include head and neck tumors, respiratory tumors, gastrointestinal tumors, urological tumors, reproductive tumors, endocrine tumors, central and peripheral nervous system tumors, tumors of the skin and its appendages, soft tissue and bone tumors, lymphoid and hematopoietic tumors, and the like.

In a preferred embodiment, the tumor is, for example, cancer of the head and neck, respiratory tract, gastrointestinal tract, skin and its appendages, central and peripheral nervous system, urinary system, reproductive system, endocrine system, soft tissue and bone, lymphatic and hematopoietic systems. In the most preferred embodiment of the present invention, the cancer is neck cancer, colon cancer, stomach cancer, breast cancer, bladder cancer, and the like.

Tumors according to the present invention may comprise detectable lymph node affected tumors (lymph node positive tumors) and tumors that have no detectable spread to lymph nodes (lymph node negative tumors). In one embodiment of the invention, the gastrointestinal tumor is a tumor that does not have a detectable spread to lymph nodes.

The sample according to the method of the invention may comprise any sample containing cells or cell debris. The sample may comprise a clinically relevant sample, e.g. secretions such as gastric juice, bile or pancreatic juice, smears, body fluids such as serum, blood, plasma urine, semen, stool, biopsies or cell and tissue samples. The biopsy referred to in the present invention may comprise a tumor resection sample, a tissue sample prepared by endoscopic methods or a biopsy of an organ puncture. Furthermore, any sample which may contain the marker molecules to be detected may be used as a sample according to the present invention.

These samples may comprise, for example, whole cells, lysed cells, or any liquid containing proteins, peptides, or nucleic acids. Even a solid to which cells, cell debris or marker molecules (e.g.human transketolase-like-1 nucleic acids or human transketolase-like-1 proteins) can be attached may be used as a sample according to the invention. Such solids may include, for example, films, slides, beads, and the like.

The preparation of the sample may comprise, for example, the obtaining of a tissue sample, a body fluid sample, a cell debris sample from a patient. The preparation of a sample according to the invention may also comprise several further pre-processing steps of the sample, such as the preparation of a dissected specimen, the preparation of lysed cells, the preparation of a tissue array, the isolation of polypeptides or nucleic acids, the solid phase preparation of immobilized peptides or nucleic acids, or the preparation of beads, membranes or slides, which are coupled covalently or non-covalently to the molecule to be tested.

The method for detecting the level of a human transketolase like-1 gene product according to the present invention is any method suitable for detecting a minute amount of a specific biologically active molecule in a biological sample. The detection reaction according to the invention is a detection at the nucleic acid level or at the polypeptide level.

The detection can be carried out in solution or using reagents immobilized to a solid phase. The detection of one or more molecular markers, such as polypeptides or nucleic acids, may be performed in a single reaction mixture or in two or separate reaction mixtures. Alternatively, the detection reaction of several marker molecules may be performed, for example, simultaneously in a multi-well reaction dish. The characteristic markers of the human transketolase like-1 gene product can be detected by means of reagents which specifically recognize these molecules. The detection reaction of the labeled molecule may comprise one or more reactions with a detection reagent that recognizes the original labeled molecule or recognizes a previous molecule that was used to recognize another molecule.

The detection reaction may also comprise a reporter reaction indicative of the presence or absence and/or level of a human transketolase like-1 gene marker. The reporter reaction may be, for example, a reaction that produces a colored compound, a bioluminescent reaction, a fluorescent reaction, a general radioemission reaction, and the like. In a preferred embodiment, different marker molecules are recognised by the substance generating different reporter signals, and the result indicates that the signal of the marker molecule can be distinguished.

A suitable format for the detection reaction according to the invention may be a blotting technique such as Western blotting, Southern blotting, Northern blotting. Blotting techniques are well known to those skilled in the art and can be performed, for example, by electroblotting, semi-dry blotting, vacuum blotting, or dot blotting. Amplification reactions can also be applied, for example, to the detection of nucleic acid molecules. In addition, immunological methods can be used for molecular detection, for example immunoprecipitation or immunological assays, such as ELISA, RIA, lateral flow assay (lateral flow assay), immunocytochemistry, and the like.

In a preferred embodiment of the present invention, the detection of the level of the human transketolase like-1 gene product is performed by detecting the level of a nucleic acid encoding the human transketolase like-1 gene product or a fragment thereof present in the sample. Methods for the detection of nucleic acid molecules are well known to those skilled in the art. Nucleic acid detection methods can be accomplished, for example, by binding of the test molecule to a complementary nucleic acid probe, a protein that specifically binds nucleic acid, or any other entity that specifically recognizes and binds the nucleic acid. These methods can be performed in vitro or directly in situ, for example during a staining reaction. Another method for detecting the human transketolase like-1 gene product at the nucleic acid level in a sample according to the method of the present invention is a nucleic acid amplification reaction, which can be carried out in a quantitative manner, such as the polymerase chain reaction. In a preferred embodiment of the invention, real-time RT-PCR can be used to quantify the level of transketolase like-1 RNA in tumor samples.

In another preferred embodiment of the present invention, the level of the human transketolase like-1 gene product is detected by measuring the level of protein expression. The determination of the human transketolase like-1 gene product at the protein level may be accomplished, for example, by a reaction comprising an antibody specifically detecting the human transketolase like-1 protein. The antibodies can be used in a variety of different detection techniques, for example in Western blotting, ELISA or immunoprecipitation. Typically, antibody-based detection can be performed in vitro as well as directly in situ, e.g., during an immunohistochemical staining reaction. Any other method for determining the amount of a particular polypeptide in a biological sample may be used according to the present invention.

In a preferred embodiment of the invention, the level of the human transketolase like-1 gene product is significantly increased compared to the control test sample. In this case, the human transketolase like-1 gene is overexpressed in the sample.

An example of diagnosing a condition associated with expression of a human transketolase like-1 gene may include detecting autoantibodies directed against a polypeptide encoded by the human transketolase like-1 gene. The polypeptides used according to the methods of the invention may be used to detect the presence or absence of such antibodies in body fluids using methods known to those skilled in the art.

In a preferred embodiment, detection of tissue expressing a transketolase like-1 gene product may be performed as a form of molecular imaging method. Related methods are known to those skilled in the art. The imaging methods used herein may include, for example, MRI, SPECT, PET, and other methods suitable for in vivo imaging.

The method in one embodiment may be based on the enzymatic conversion of an inert or labeled compound into a detectable molecule by a transketolase like-1 molecule during the molecular imaging method. In another embodiment, the molecular imaging method may be based on the use of a label carrying a suitable compound such as a radioisotope, metal ion, or the like for molecular imaging in vivo to specifically bind to a transketolase like-1 molecule in vivo.

In a preferred embodiment of the invention, these compounds are non-toxic and are cleared from the circulation of an organism, such as a human, over a period of time, thereby allowing the detection of markers accumulated in tumor tissues overexpressing the transketolase like-1 gene. In another preferred embodiment of the invention, the clearance of the compound used for molecular imaging from the circulation is independent of the molecular imaging reaction. This may be due to, for example, low background generated by circulating molecules and the like. The compounds used in the molecular imaging methods are administered in a pharmaceutically acceptable form in a composition which may also contain any other suitable substances such as other diagnostic materials, therapeutic materials, carrier materials and the like.

Another aspect of the invention is a kit for performing the method according to the invention. The kit may be, for example, a diagnostic kit or a research kit.

Kits according to the invention comprise at least one reagent suitable for detecting the molecules disclosed herein. Furthermore, the kit according to the invention may comprise: a) reagents for detecting a human transketolase like-1 gene product; b) reagents and buffers commonly used to carry out detection reactions, such as buffers, detection labels, support materials, and the like; c) human transketolase like-1 sample as a positive control reaction.

The reagent for detecting the human transketolase like-1 gene may include any reagent capable of binding to the human transketolase like-1 molecule. Such agents may include proteins, polypeptides, nucleic acids, peptide nucleic acids, glycoproteins, proteoglycans, polysaccharides or lipids.

The human transketolase like-1 sample as a positive control may comprise, for example, a human transketolase like-1 nucleic acid or polypeptide or fragment thereof in a suitable form, e.g., in solution or salt form, a peptide in a suitable form, a tissue section sample, or positive cells.

In a preferred embodiment of the invention, the detection of the human transketolase like-1 gene product is carried out at the polypeptide level. In this embodiment, the binding agent can be, for example, an antibody specific for human transketolase like-1 or a fragment thereof.

In another preferred embodiment of the kit of the present invention, the detection of human transketolase like-1 is performed at the nucleic acid level. In this embodiment of the invention, the detection reagent may be, for example, a nucleic acid probe or a reverse-complementary primer of the above-described human transketolase like-1 nucleic acid.

In another aspect, the invention relates to the use of one or more compounds, such as nucleic acid molecules, recombinant vectors, polypeptides, antisense RNA sequences, ribozymes or antibodies, for the preparation of a pharmaceutical composition for the treatment of cancer, preferably colon cancer, pancreatic cancer, gastric cancer, lung cancer, according to the method of the invention.

The polypeptides, polynucleotides and binding agents used in the methods according to the invention may be incorporated into pharmaceutical or immunogenic compositions. The pharmaceutical composition comprises the compound described above and a physiologically acceptable carrier.

The pharmaceutical composition or vaccine may comprise, for example, DNA encoding one or more polypeptides according to the invention. The DNA may be introduced in a manner that allows for the in situ production of the polypeptide. Suitable expression systems are well known to those skilled in the art. Expression of the polypeptide may be, for example, permanent or transient. In the pharmaceutical compositions and/or vaccines, to provide for in situ expression of the polypeptide, the nucleic acid may be present in any suitable delivery system known to those skilled in the art, including nucleic acid expression systems, bacterial and viral expression systems. Suitable nucleic acid expression systems include nucleic acid regulatory sequences necessary for expression in the patient, such as appropriate promoters, terminators, and the like. Bacterial delivery systems may use, for example, bacteria that introduce epitopes that express cellular antigens on the cell surface. In a preferred embodiment, the nucleic acid is introduced by a viral expression system, such as vaccinia, retrovirus, or adenovirus, and may include the use of an apathogenic, replication-competent virus. Suitable systems are disclosed, for example, in: Fisher-Hoch et al, PNAS 86317-321, 1989; flexner et al, ann.n.y.acad sci.569: 86-103, 1989; flexner et al Vaccine 8: 17-21, 1990; U.S. Pat. nos.4,603,112, 4,769,330, and 5,017,487; WO 89/01973; pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; berkner, Biotechniques 6: 616-627, 1988; rosenfeld et al, Science 252: 431-434, 1991; kolls et al, PNAS 91: 215-; Kass-Eisler et al, PNAS 90: 11498. 11502, 1993; guzman et al, Circulation 88: 2838 2848, 1993; and Guzman et al, cir. Res.73: 1202-1207, 1993. In another embodiment, transgenic mammalian cells may be used to transport and/or express nucleic acids. Methods for producing nucleic acid constructs suitable for expressing a polypeptide in situ are well known to those skilled in the art.

In another embodiment of the invention the nucleic acid may be, for example, an antisense construct.

The nucleic acid may also be administered as naked nucleic acid. In such cases, suitable delivery systems for facilitating uptake of the nucleic acid may be used, for example, by coating the nucleic acid onto biodegradable beads which are efficiently delivered into the cell. Administration of naked nucleic acid may, for example, facilitate transient expression in the host or host cell.

Alternatively, the pharmaceutical composition may comprise one or more polypeptides. The polypeptide introduced into the pharmaceutical composition may be a human transketolase like-1 polypeptide in combination with one or more other known polypeptides, e.g., enzymes, antibodies, regulatory factors such as cyclins, cyclin-dependent kinases or CKIs or toxins.

The pharmaceutical compositions may be administered by any suitable method known to those skilled in the art. The administration may include, for example, injection, such as intradermal, intramuscular, intravenous or subcutaneous injection, intranasal administration, e.g., inhalation, or oral administration. The appropriate dosage should be selected to ensure the therapeutic effect of the drug according to parameters known to those skilled in the art, such as age, sex, body weight, etc. of the patient.

The type of carrier used in the pharmaceutical composition of the present invention varies depending on the mode of administration. For parenteral administration, e.g., subcutaneous injection, the carrier preferably comprises water, saline, alcohol, lipids, waxes and/or buffers. For oral administration, any of the above carriers or solid carriers, such as mannitol, lactose, starch, magnesium stearate, sodium sugar, talc, cellulose, glucose, sucrose, and/or magnesium carbonate, may be used. Biodegradable microspheres (e.g., polylactic glycolide) may also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are disclosed, for example, in: U.S. Pat. nos.4,897,268 and 5,075,109.

It is also possible for the compounds of the invention to be incorporated into immunogenic compositions.

The components of the immunogenic composition may comprise a vaccine, antigen, antigenic fragment or nucleic acid encoding an antigen or antigenic fragment expressed in situ. The compound may be in the form of a polypeptide or a nucleic acid which allows for expression of the polypeptide in situ. The immunogenic composition comprises a compound as described above and a further immunostimulant or immunogenic adjuvant.

The polypeptides of the invention, or fragments thereof, comprising an immunogenic portion of a human transketolase like-1 protein, are useful as immunogenic compositions, wherein the polypeptides are capable of stimulating an autoimmune response in a patient against tumor cells. The patient may or may not have a diagnosable disease. The compounds disclosed herein are therefore useful for treating or inhibiting the development of cancer. The compounds may be administered before or after conventional treatment of the tumor, such as surgical removal of the primary tumor, administration of radiation therapy, conventional chemotherapy, or any other form of treatment of the corresponding cancer or its precursor.

An immunogenic composition, such as a vaccine, 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 immunogenic antigen. Examples of non-specific immune response enhancers include adjuvants, biodegradable microspheres (such as polylactic acid), and liposomes, for example, into which polypeptides may be incorporated. The pharmaceutical compositions and vaccines may also comprise additional tumor antigen epitopes, either incorporated into the fusion protein described above (i.e., a single polypeptide comprising multiple epitopes) or present in separate polypeptides.

Any suitable immune response enhancer may be used in the vaccines of the present invention. For example: adjuvants may be included. Most adjuvants comprise materials that protect the antigen from rapid degradation, such as aluminum hydroxide or mineral oil, and non-specific immune response stimulators, such as lipid a, Bordetella pertussis (Bordetella pertussis), or mycobacterium tuberculosis (mycobacterium tuberculosis). Such adjuvants are commercially available, for example: freund's incomplete and complete Adjuvant (Difco laboratories, Detroit, Mich.) and Merck Adjuvant65(Merck and company, Inc., Rahway N.J.).

For therapeutic purposes, the polypeptide, polynucleotide or binding agent may be administered by various routes. Possible routes include, for example, intradermal, intramuscular, intravenous or subcutaneous injection, for example, intranasal administration by inhalation or oral administration.

It is another aspect of the present invention to provide a method of treatment and/or vaccination. According to the present invention, human transketolase like-1 polypeptides and/or polynucleotides may be used to treat cell proliferative disorders. The treatment may be, for example, immunotherapy or somatic gene therapy.

According to the present inventors transketolase like-1 polypeptides and/or polynucleotides may be used for vaccination against cell proliferative disorders. Vaccination according to the invention may comprise administering an immunogenic compound to an individual to stimulate an immune response against the immunogenic compound and thereby immunize the individual against the immunogenic compound. Stimulating an immune response may comprise inducing the production of antibodies to the above compounds and stimulating cytotoxic T-cells. For vaccination purposes, the polypeptides, nucleic acids and binding agents according to the invention may be administered in a physiologically acceptable form. The composition introduced into the subject may comprise one or more antigenic components, a physiologically acceptable carrier material or buffer solution, an immunostimulant and/or an adjuvant. The adjuvant may comprise, for example, Freund's incomplete adjuvant or Freund's complete adjuvant or other adjuvants well known in the art.

The compositions can be administered by any method that can be used, e.g., intravenously, subcutaneously, intramuscularly, and the like. The dosage of the composition depends on the particular case and the purpose of the vaccination. It may be desirable to adjust for parameters of the individual being treated, such as age, weight, sex, and the like. The type of immune response elicited is also considered. It is generally preferred that the individual receives 100ug-1g of a polypeptide according to the invention, 10⁶-10¹²The MOI contains a recombinant nucleic acid according to the invention in an in situ expressible form.

The vaccinated individual may be any organism that comprises a transketolase like-1 protein and that may be affected by a cell proliferative disorder.

For example, vaccination of individuals may be beneficial in the case of altered, non-wild-type sequences or structures of marker molecules associated with cell proliferative disorders.

The polypeptides disclosed herein may also be used in adoptive immunotherapy for the treatment of cancer. Adoptive immunotherapy can be broadly classified as active or passive immunotherapy. In active immunotherapy, treatment relies on stimulating the endogenous host immune system to fight the tumor by administering immune response modifiers (e.g., tumor vaccines, bacterial adjuvants, and/or cytokines).

In passive immunotherapy, treatment involves the delivery of biological agents (e.g., effector cells or antibodies) with defined tumor immunoreactivity, which may directly or indirectly mediate anti-tumor effects and do not necessarily rely on the entire host immune system. Examples of effector cells include T lymphocytes expressing the disclosed antigens (e.g., CD 8)⁺Cytotoxic T-lymphocytes, CD4⁺Helper T cells, tumor-infiltrating lymphocytes), killer cells (e.g., natural killer cells, lymphokine-activated killer cells), B cells, or antigen-presenting cells (e.g., dendritic cells and macrophages). The polypeptides disclosed herein may be used to generate antibodies for passive immunization or anti-idiotypic antibodies (see U.S. patent No.4,918,164).

The preferred method for producing sufficient adoptive immunotherapy T cells is the in vitro growth of immune T cells. Culture conditions that expand single antigen-specific T cells to billions and maintain their recognition of antigens in vivo are well known in the art. These in vitro culture conditions are typically stimulated intermittently with antigens, often in the presence of cytokines such as IL-2 and non-differentiated feeder cells. As described above, the immunoreactive polypeptides described herein can be used to rapidly culture expanded antigen-specific T cells to produce sufficient quantities of immunotherapeutic cells. In particular, antigen presenting cells, such as dendritic cell macrophages or B cells, may be pulsed with the immunoreactive polypeptide to stimulate or transfect the nucleic acid sequence using standard techniques well known in the art. For example: antigen-presenting cells can transfect nucleic acid sequences that contain promoter regions suitable for increased expression and can be expressed as part of a recombinant virus or other expression system. To culture T cells for effective therapy, the cultured T cells must be able to grow, be widely distributed, and survive for long periods in vivo. Studies have shown that by repeated stimulation With IL-2-supplemented antigens, Cultured T Cells can be induced to grow in vivo and survive for significant, long periods of time (see, e.g., Cheever M. et al, "Therapy With Cultured T Cells: Priniples Cultured" Immunological Reviews, 157: 177, 1997).

The polypeptides disclosed herein may also be used to generate and/or isolate tumor-reactive T cells for administration to a patient. In one approach, antigen-specific T cell lines can be generated by in vivo immunization with short peptides corresponding to immunogenic portions of the disclosed polypeptides. The resulting antigen-specific CD8+ CTL clone can be isolated from the patient, expanded using standard tissue culture techniques, and returned to the patient.

Alternatively, a peptide corresponding to an immunogenic portion of a polypeptide of the invention can be used to generate a subpopulation of tumor-reactive T cells, which are provided with antigen-specific T cells by selective in vitro stimulation and autologous T cell expansion, and subsequently transferred into a patient, for example as described by Chang et al (crit. Cells of the immune system, such as T cells, may be obtained using commercial cell separation systems, such as CEPRATE from CellPro (Bothell, Wash.).^TMSystems (see U.S. Pat. No.5,240,856; U.S. Pat. No.5,215,926; WO 89/06280; WO91/16116 and WO92/07243) are isolated from the peripheral blood of a patient. The isolated cells are stimulated with one or more immunoreactive polypeptides contained in a carrier, such as a microsphere, to provide antigen-specific T cells. The tumor antigen-specific T cell population is then expanded using standard techniques and the cells are returned to the patient.

In another embodiment, the polypeptide-specific T cell and/or antibody receptor can be cloned, expanded, and transferred into other vectors or effector cells for adoptive immunotherapy.

In another embodiment, homologous or autologous dendritic cells can be pulsed with a peptide corresponding to at least an immunogenic portion of a polypeptide disclosed herein. The generated antigen-specific dendritic cells can be transferred into a patient or used to stimulate T cells to provide antigen-specific T cells, which are then administered to the patient. Cheever et al describe the use of peptide-pulsed dendritic cells to generate antigen-specific T cells in a mouse model, followed by tumor elimination using such antigen-specific T cells (Cheever et al, Immunological Reviews, 157: 177, 1997).

Alternatively, vectors expressing the disclosed nucleic acids can be introduced into stem cells taken from a patient, clonally propagated in vitro, and returned to the same patient for autologous transplantation.

The monoclonal antibodies of the invention are also useful as therapeutic compounds for reducing or eliminating tumors. The antibody may be used alone (e.g., to inhibit metastatic disease) or in combination with one or more therapeutic agents. Suitable therapeutic agents include radionuclides, differentiation inducers, drugs, toxins, and derivatives thereof. Preferred radionuclides include 90Y, 123I, 125I, 131I, 186Re, 211At, and 212 Bi. Preferred drugs include methotrexate, pyrimidine and purine analogs. Preferred differentiation inducers include phorbol esters and butyric acid. Preferred toxins include ricin, abrin, diphtheria toxin (diphtheria toxin), cholera toxin, gelonin, pseudomonas exotoxin, shigella toxin, and pokeweed antiviral protein.

Further methods of treating a condition associated with a transketolase like-1 gene may comprise any method suitable for decreasing the activity of a transketolase like-1 polypeptide in a subject or in a cell of a subject. These methods may comprise decreasing the activity of a transketolase like-1 polypeptide by decreasing gene expression or decreasing enzyme activity. Examples may include administration of antisense constructs, ribozymes, enzyme inhibitors, administration of a transketolase like-1 polypeptide cofactor antagonist, e.g., an anti-thiamine compound, or administration of a cofactor (e.g., thiamine) necessary to reduce enzymatic activity.

Methods of administering ribozymes or antisense constructs are well known to those skilled in the art. Administration can be by administration of naked nucleic acid or by administration of nucleic acid suitable for in situ expression of the corresponding active product.

In a preferred embodiment, treating a condition associated with overexpression of a transketolase like-1 gene comprises administering an anti-thiamine compound, or reducing thiamine uptake in an individual characterized by a condition in which transketolase like-1 gene is overexpressed.

In another embodiment, the invention relates to a method of identifying and obtaining a drug candidate for the treatment of colon, gastric, pancreatic or lung cancer, comprising the steps of: contacting a TKT-L1 polypeptide or a cell expressing the polypeptide for use in the methods of the invention in the presence of a detectable signal component capable of providing an alteration in response to transketolase activity or regulation of cell proliferation; detecting the presence or absence or an increase in a signal generated by an alteration in the activity of a transketolase or the regulation of cell proliferation, wherein a lack or decrease in the signal is indicative of a putative agent.

The candidate agent may be a single compound or a plurality of compounds. The term "plurality of compounds" in the process of the invention is to be understood as a plurality of substances which may be identical or different.

The compound or compounds may be chemically synthesized or microbially produced and/or contained within cells extracted from a sample, such as a plant, animal or microorganism. Furthermore, the compounds may be known in the art, but are not known to date to inhibit or activate the TKT-L1 polypeptide. The reaction mixture may be a cell-free extract or comprise a cell or tissue culture. Suitable methods for the methods of the invention are well known in the art and are generally described in Albert et al, molecular Biology of the cell, third edition (1994) and the appendix examples. The plurality of compounds may be added, for example, to the reaction mixture, to the culture medium, injected into the cells or applied to the transgenic animal. The cells or tissues useful in the methods of the invention are preferably host cells, mammalian cells or non-human transgenic animals as described in embodiments of the invention.

If a sample comprising a compound or compounds is identified in the methods of the invention, it is possible to isolate the compound from an original sample determined to contain a compound capable of inhibiting or activating TKT-L1, or to re-isolate the original sample, for example if the sample consists of a plurality of different compounds, to reduce the number of different substances in each sample, and to repeat the method of isolating the original sample. Depending on the sample complexity, the above steps can be carried out several times, preferably until the sample identified according to the method of the invention contains only a defined number of or only substances. Preferably the samples comprise substances with similar chemical and/or physical properties, most preferably the substances are the same.

The compounds that can be tested and identified according to the methods of the invention can be peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs, and the like.

The compounds isolated by the above method can also be used as lead compounds to develop analogue compounds. The analogs have stable electronic configurations and molecular conformations, allowing key functional groups to be presented to TKT-L1 in substantially the same manner as the lead complex. In particular, the analogs have steric electronic properties comparable to the binding domain, but may be smaller molecules than the lead compound, generally having a molecular weight below about 2kD and preferably below about 1 kD. Analogs can be identified by using dynamic analytical techniques such as self-constant field (SCF) analysis, Conformational Interaction (CI) analysis, and normal modes. These analyses can be done with a computer program; for example, computer-aided modeling of receptor-ligand interactions of Rein (Alan Liss, New York, 1989). Methods for the preparation of chemical derivatives and analogues are well known in the art and are described, for example, in Beilstein, handbook of organic chemistry, Springer edition New York Inc., 175Fifth Avenue, New York, N.Y 10010 U.S.A. and in organic Synthesis, Wiley, New York, USA. Furthermore, the above derivatives and analogues can be tested for their effect according to methods known in the art (see also above). In addition, suitable derivatives and analogs of peptidomimetics and/or computer-aided design may be used, e.g., according to the methods described above.

Once identified and obtained, the compounds are preferably provided in a therapeutically useful form.

The present invention provides methods for diagnosing and treating disorders characterized by abnormal cell proliferation, such as cancer. One aspect of the present invention provides a method for detecting a disorder characterized by abnormal cell proliferation, such as cancer, based on the determination of the presence and/or expression level of a human transketolase like-1 gene in a biological sample. In a second aspect, the invention provides a method of treatment of a condition characterised by abnormal cell proliferation, such as cancer, using a human transketolase like-1 gene product as a therapeutically active agent. The invention also provides therapeutic methods based on reducing the activity of the transketolase like-1 gene polypeptide. One aspect of the invention is a method for providing rational tumor treatment and customizing a therapeutic regimen corresponding to the overexpression of a detected gene product based on the detection of a transketolase like-1 gene product in a patient sample. In addition, the present invention provides a kit for research or diagnosis for completing a reaction involved in the detection of the presence or absence and/or overexpression level of a human transketolase like-1 gene. Finally the invention relates to a pharmaceutical composition for the treatment of a condition according to the invention.

The following examples are intended to be illustrative only and not limiting to the scope of the invention disclosed herein.

Example 1: human transketolase like-1 mRNA levels in colon cancer tissues were determined.

The tumor biopsy dissects were semi-quantitatively analyzed for human transketolase like-1 gene mRNA levels by in situ staining reactions. The dyeing reaction was carried out as follows:

tissue dissectates were incubated in ethanol at increasing concentrations up to 100%. After ethanol evaporation the dissectates were boiled in 10mM citrate buffer (pH6,0) for tissue pretreatment. A hybridization mixture was prepared by mixing 50. mu.l of ready-to-use hybridization buffer (DAKO A/S, Glostrup, Danmark) with about 5 to 10pmol of the probe. The probe is a fluorescent-labeled oligonucleotide with the following sequence: TCTCATCACAAGCAGCACAGGAC

The hybridization mixture was heated to 95 ℃ and then equilibrated to 37 ℃. The dissected specimens were boiled and incubated with 50. mu.l of the hybridization mixture for 2 hours at 37 ℃. The dissectates were washed twice with excess wash buffer 2 XSSC solution at room temperature for 15 minutes each, and once again with 1 XSSC at 50 ℃ for 15 minutes. The dissectates were then rinsed twice with 2 x SSC at room temperature. After washing the dissectates were incubated with blocking buffer (NEN, blockangdrawer) for 30 min at room temperature. Then diluted with 1: 100Release (with blocking buffer, see above) of anti-fluorescein-AP (DAKO A/S) incubation for 1 h. The dissectates were washed twice with 1 XPBS/0.1% Triton X100 for 10 minutes at room temperature, followed by 1 XPBS, 50mM MgCl₂(pH 9.2) was washed once at room temperature for 10 minutes.

The staining reaction was performed with NBT/BCIP (Sigma) for about 30 minutes at room temperature. The staining reaction was stopped by a brief incubation with PBS containing 1mM EDTA. Finally, the dissectate is immersed in H₂Odest and fixation with Aquatex (Merck). The stained dissectates may be subjected to microscopic analysis.

The results showed that human transketolase like-1 gene was overexpressed in colon cancer tissues compared to normal colon tissues.

Example 2: the human transketolase like-1 gene and transketolase levels in cancer tissues and control tissues were determined by semi-quantitative RT-PCR.

The levels of human transketolase like-1 mRNA and human transketolase mRNA in colon cancer samples, lung adenocarcinoma samples and stomach cancer samples were determined by semi-quantitative RT-PCR. Tumor biopsy specimens were used in this study.

Tumors were harvested, snap frozen and stored at-80 ℃. They were confirmed by histopathological analysis to consist mainly of tumor cells. mRNA was isolated from patient tumors and corresponding normal tissues using Qiagen reagents (Qiagen, Hilden, Germany) and single stranded cDNA was synthesized using SuperScriptII (Life technologies, Inc). Quantitative PCR was performed using a 7700 sequencer (Taqman. TM.) and SYBR Green PCR Master-Mix according to the manufacturer's instruction manual (Applied Biosystems, Foster City, Calif.).

The PCR reaction was carried out in a volume of 25. mu.l, with a final concentration of 300nmol of each primer, 15 seconds at 95 ℃ and 60 seconds at 60 ℃ for 40 cycles. The following primers were used for quantitative PCR:

transketolase like-1: primer A: CACCTTGGGATTCTGTGTGC

And (3) primer B: TCTCATCACAAGCAGCACAG

Transketolase: primer A: TGTGTCCAGTGCAGTAGTGG

And (3) primer B: ACACTTCATACCCGCCCTAG are provided.

PCR product specificity was verified by gel electrophoresis (data not shown).

The results showed that human transketolase like-1 gene was highly overexpressed in 1/10 in colon cancer, 2/5 in lung adenocarcinoma and 3/5 in stomach cancer, compared to normal control tissue.

In particular, the degree of overexpression of the transketolase like-1 gene in the sample was significant. The total 6/20 cancers showed more than 8-fold overexpression of the TKT-L1 gene. In contrast, none of the cases significantly overexpressed the transketolase gene.

The results show that the transketolase like-1 gene is overexpressed in all cancer subtypes of different origins. There was no difference in expression of the phase-inverted ketolase genes in the tested tumor tissues.

Example 3: immunochemical detection of overexpression of tktl1 in cancer samples

Formalin-fixed paraffin-embedded sections of stomach tissue samples were immunocytochemically stained with antibodies against tktl 1.

Sections were rehydrated by incubation with xylene and fractionated ethanol and transferred to Aquabidest. Antigen retrieval was performed with 10mM citrate buffer (pH 6.0). Thus the sections were heated in a water bath at 95 ℃ for 40 minutes, cooled to room temperature for 20 minutes and transferred to washing buffer (PBS/0.1% Tween 20).

To inactivate endogenous peroxidase, the sample was incubated with 3% H₂O₂Incubated together at room temperature for 10 minutes and then washed with PBS/0.1% Tween20 for 10 minutes.

Sections were incubated with primary antibody, murine anti-tktl 1 antibody (1: 300), at room temperature for 1 hour, then rinsed with wash buffer and placed in a fresh buffer bath for 5 minutes. The antibody used was directed to the human tktl1 protein sequence shown in bold in figure 7.

The sections were then incubated with a second antibody, goat anti-mouse (1: 500) antibody, for 1 hour at room temperature. Wash 3 times for 5 minutes each. Sections were covered with 200. mu.l of chromogenic substrate solution (DAB) for 10 minutes. The sections were then rinsed as described above, counterstained in hematoxylin for 2 minutes, the remaining hematoxylin rinsed with distilled water, the specimens fixed and capped with a liquid sealant.

Microscopic examination of the sections revealed that tktl1 immunoreactive cells could be found in samples microscopically identified as gastric cancer. Tktl1 specific staining was seen in the cancer cell nucleus and cytoplasm. In addition, a granular staining pattern was observed in tumor cells.

The immunohistochemical staining method can also be used for tissues from breast cancer, lung cancer, neck Cancer (CINIII), gastric cancer, esophageal cancer, endometrial cancer, and ovarian cancer. In all of these examples, nuclear and cytoplasmic tktl1 staining in cancer cells was observed.

In addition, metastatic lesions of colorectal cancer into the liver were analyzed using the immunochemical method described above. The results showed strong overexpression of tktl1 protein.

Claims (33)

1. A method of detecting a condition characterized by abnormal cell proliferation in an individual comprising a detecting the presence and/or level of expression of a human transketolase like-1 gene in a biological sample obtained from said individual; b. performing a diagnostic assessment from the presence and/or level of expression, wherein overexpression is indicative of a condition characterised by abnormal cell proliferation.

2. The method according to claim 1, wherein the disorder characterized by abnormal cell proliferation is cancer.

3. The method of claim 2, wherein the cancer is colon cancer, lung cancer, stomach cancer or pancreatic cancer.

4. The method according to any one of claims 1 to 3, wherein the biological sample is a body fluid, a secretion, a smear, a biopsy, a liquid containing cells, lysed cells, cell debris, peptides or nucleic acids.

5. The method according to claim 4, wherein the sample is serum, urine, semen, stool, bile, biopsy or a cell sample or a tissue sample.

6. The method according to any one of claims 1 to 5, wherein the detection of the expression of the human transketolase like-1 gene is performed at the polypeptide level.

7. The method according to any one of claims 1 to 5, wherein the detection of the expression of the human transketolase like-1 gene is performed at the nucleic acid level.

8. The method according to claim 6, wherein the detection at the polypeptide level is performed using a binding agent directed against a human transketolase like-1 polypeptide.

9. The method according to claim 8, wherein the binding agent is an antibody, an antibody fragment, a peptidomimetic comprising an antigen binding epitope, or a minibody.

10. The method according to any one of claims 6, 8 or 9, wherein the detection is an immunocytochemical detection method.

11. The method according to claim 7, wherein at least one nucleic acid probe hybridizing to a human transketolase like-1 nucleic acid is used for the detection.

12. The method according to claim 11, wherein the probe is detectably labeled.

13. The method according to claim 12, wherein the label is selected from the group consisting of a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate or an enzyme.

14. The method according to any one of claims 7 or 11-13, wherein the detection reaction comprises a nucleic acid amplification reaction.

15. The method according to any one of claims 11-13, wherein the amplification reaction is PCR, LCR or NASBA.

16. A method according to any one of claims 7 or 11 to 13 for use in situ hybridization.

17. A method according to any preceding claim, for use during an in vivo or in vitro molecular imaging method.

18. A kit for carrying out the method of any one of claims 1 to 17, which is a research kit or a diagnostic kit.

19. The kit of claim 18, comprising a. at least one probe for detecting the expression product of a human transketolase like-1 gene in a biological sample; b. human transketolase like-1 gene product samples were used as positive control reactions.

20. The kit of claim 19, wherein the probe is a nucleic acid probe that specifically hybridizes to a human transketolase like-1 nucleic acid; or an antibody that specifically binds to a human transketolase like-1 protein.

21. A method of treating a condition characterized by abnormal cell proliferation, based on the administration of a pharmaceutical composition comprising a human transketolase like-1 gene or gene product in a pharmaceutically acceptable form.

22. The method according to claim 21, wherein the human transketolase like-1 gene or gene product is the sense strand or antisense strand of a nucleic acid or a polypeptide.

23. The method according to claim 22, wherein the pharmaceutical composition comprises a chimeric nucleic acid comprising a human transketolase like-1 nucleic acid or fragment thereof, or a fusion polypeptide comprising a human transketolase like-1 polypeptide or fragment thereof.

24. The method according to any one of claims 21-23, wherein the disorder characterized by abnormal cell proliferation is cancer.

25. The method of claim 24, wherein the cancer is colon, lung, stomach, or pancreatic cancer.

26. The method according to any one of claims 21-25, wherein the method of treatment is immunotherapy.

27. The method according to any one of claims 21-26, wherein the method of treatment is vaccination therapy.

28. Use of a human transketolase like-1 polypeptide or a human transketolase like-1 nucleic acid for the preparation of a pharmaceutical composition for the treatment of cancer.

29. A method of identifying and obtaining a drug candidate for treatment of colon, lung, pancreas or stomach tumors comprising the steps of: a. contacting a TKT-L1 polypeptide or a cell expressing such a polypeptide for use in the methods of the invention in the presence of a component that provides a detectable signal in response to an alteration in transketolase activity or regulation of cell proliferation; detecting the presence or absence or an increase in a signal generated by an alteration in the activity of a transketolase or the regulation of cell proliferation, wherein a lack or decrease in the signal is indicative of a putative agent.

30. A pharmaceutical composition for treating colon tumors, lung tumors, pancreatic tumors, or gastric tumors comprising a compound identifiable according to the method of claim 29, an anti-thiamine compound, an inhibitor of transketolase activity, an inhibitor of transketolase-like-1 activity, a transketolase-like-1 polypeptide, or a human transketolase-like-1 nucleic acid.

31. A rational method for tumor treatment comprising a. detecting the presence and/or overexpression of a transketolase like-1 gene in a biological sample, b. establishing a subpopulation based on the presence and/or level of a transketolase like-1 gene, c. tailoring a suitable therapy based on the subpopulation, comprising decreasing the activity of transketolase like-1 in an individual or in cells of an individual.

32. The method of claim 31, wherein the reduction of transketolase-like-1 activity is achieved by administering an anti-thiamine compound, a pharmaceutical composition of claim 31, an inhibitor of transketolase enzyme activity, a transketolase-like-1 antisense nucleic acid construct, a transketolase-like-1 specific ribozyme, or by reducing the administration of thiamine.

33. A pharmaceutical composition according to claim 30 for use in a method according to claim 31 or 32.