JP2005525789A - Cancer diagnosis method, cancer modulator screening composition and method - Google Patents

Abstract

Described herein are genes whose expression is up- or down-regulated in certain cancers or other diseases, or otherwise regulated in diseases. Related methods and compositions used for the diagnosis, prognosis, and treatment of these medical conditions are disclosed. Also described herein are methods that can be used to identify modulators of selected symptoms.

Description

Related Patents This application is filed in US Patent No. 60 / 340,376 filed on December 14, 2001; Attorney Docket No. 018501-006400US filed on February 8, 2002; US Patent No. 60 / filed on January 8, 2002. No. 347,211; US Patent No. 60 / 334,393 filed November 29, 2001; US Patent No. 60 / 355,394 filed November 15, 2001; US Patent No. 60 / 347,349 filed January 10, 2002 US Patent No. 60 / 368,809 filed March 29, 2002; US Patent No. 60 / 409,450 filed September 9, 2002; US Patent No. 60 / 359,077 filed February 20, 2002; U.S. Patent No. 60 / 386,614 filed on June 5, 2002; U.S. Patent No. 60 / 356,714 filed on Feb. 13, 2002; U.S. Patent No. 60 / 397,775 filed on July 22, 2002; U.S. Patent No. 60 / 332,464 filed on May 21, 2002; U.S. Patent No. 60 / 397,845 filed on July 22, 2002; U.S. Patent No. 60 / 370,110 filed on April 4, 2002; July 16, 2002 U.S. Patent No. 60 / 396,839, filed November 13, 2001; U.S. Patent No. 60 / 350,666, filed November 13, 2001; And claims priority to U.S. Pat. No. 60 / 372,246 of 12 filed, entirely incorporated by reference, supra herein for all purposes. This application also incorporates PCT / US02 / 29560; PCT / US02 / 02242; and PCT / US02 / 17594.

FIELD OF THE INVENTION The present invention relates to the expression characteristics of nucleic acids and proteins involved in cancer and other diseases, and the identification of nucleic acids, products, and antibodies thereto; and the expression characteristics in the diagnosis, prognosis, and treatment of those symptoms It relates to the use of the composition. Furthermore, the present invention relates to the identification and use of factors and / or targets that modulate those symptoms.

  Cancer is a major cause of mortality in the United States. For example, the American Cancer Society estimates that 1,359,150 people were diagnosed with malignant tumors and 554,740 people died from one of those diseases in 1996. Cancer accounts for 23.9% of all American deaths, with only heart disease being the leading cause of death (33%). Unfortunately, mortality from cancer is increasing and is expected to be the largest cause of death in the United States during the first half of this century, as it was already in Japan.

  Cancer has the property of disrupting control over normal cell division, growth and differentiation. Its initial clinical pathology is extremely miscellaneous, with more than 70 types of cancer occurring in virtually all organs and tissues of the body. In addition, similarly classified cancer types may represent multiple different molecular diseases. Unfortunately, some cancers are virtually disease-free until late, in which case treatment is more difficult and prognosis is severe.

  Cancer treatment usually includes surgery, chemotherapy, and / or radiation therapy. Although these therapies effectively treat about 50% of cancer patients, all current therapies induce serious side effects that reduce quality of life. Identification of new therapeutic targets and diagnostic markers is important for improved diagnosis and treatment of cancer patients.

Recent developments in molecular medicine have increased interest in tumor-specific antigens that can be targets for various immunotherapy or small molecule strategies. Antigens suitable for immunotherapeutic strategies should be highly expressed in cancer tissues, preferably obtained from the vascular and cell surfaces, and ideally not expressed in normal adult tissues. However, expression in tissues that are not present but are not life-threatening, such as genital organs, particularly those lacking one sex, may be tolerated. Examples of antigens currently available for specific cancer aspects and treatment include Her2 / neu and the B cell antigen CD20. Humanized monoclonal antibodies against Her2 / neu (Herceptin / trastuzumab) are currently used to treat metastatic breast cancer. See Ross and Fletcher (1998) Stem Cells 16: 413-428. Similarly, anti-CD20 monoclonal antibodies (Rituxin / rituximab) have been used for effective treatment of non-Hodgkin lymphoma. See Maloney, et al. (1997) Blood 90: 2188-2195; Leget and Czuczman (1998) Curr. Opin. Oncol. 10: 548-551.

  Elucidating the role of novel proteins and compounds in disease states to identify therapeutic targets and diagnostic markers is beneficial to improve current treatments for cancer patients. Accordingly, provided herein are molecular targets for therapeutic intervention in a variety of predetermined cancers. In addition, methods are provided herein that can be used for cancer diagnosis and prognosis. Furthermore, a method used for screening bioactive substance candidates for the ability to modulate cancer is also provided.

SUMMARY OF THE INVENTION The present invention is a method for determining whether a patient has pathological cells, and in a biological sample from said patient, at least 80 sequences and the sequences set forth in Table 2A-80. A method comprising detecting a nucleic acid consisting of a sequence that is% identical, thereby determining the presence or absence of a pathological cell. In a particular embodiment of the method, the disease comprising cancer is described in Table 1; the biological sample comprises isolated nucleic acid; the nucleic acid is mRNA; the biological sample comprises cancer. A tissue from an organ affected by the disease described in 1); prior to the step of detecting the nucleic acid, a step of amplifying the nucleic acid is further taken; detection is detection of a protein encoded by the nucleic acid; Comprises a sequence described in Table 2A-80; the detection step uses a labeled nucleic acid probe utilizing a biochip consisting of a sequence that is at least 80% identical to the sequence described in Table 2A-80 Or by detecting the polypeptide encoded by the nucleic acid; or the patient is suspected of receiving a treatment plan corresponding to the disease in Table 1 or having the disease or cancer.

  The following compositions are also provided: for example, labeled, isolated nucleic acid molecules consisting of the sequences set forth in Table 2A-80 as examples; an expression vector comprising the nucleic acid; a host cell comprising the expression vector; An isolated polypeptide encoded by the nucleic acid molecule consisting of the sequences set forth in Table 2A-80; or in particular an antibody that binds to the polypeptide. In certain embodiments, the antibody: binds to an effector component, binds to a detectable label (eg, including a fluorescent label, radioisotope, or cytotoxic chemical), is an antibody fragment, or humanized It is an antibody.

  Other methods have been provided that specifically target components against the patient's pathological cells, which include providing the target by administering an antibody to the patient, as described above. Other methods include, for example, a method for determining whether a patient has pathological cells, including a method of contacting a biological sample with an antibody as described above. In a more particular method, the antibody is conjugated to: an effector component or a fluorescent label; and the biological sample is a blood, serum, urine or stool sample.

  Further included is a method of identifying a component that modulates a polypeptide associated with a disease, the method comprising the following steps: a polypeptide associated with the component and the disease, and at least 80 of the sequences set forth in Table 2A-80. Contacting with a polypeptide encoded by a polynucleotide that selectively hybridizes to the identical sequence; and measuring the functional effect of the component on the polypeptide. Other drug screening assays involve the following steps: administering test components to mammals suffering from the diseases of Table 1 or cells isolated therefrom; and Table 2A in treated cells or mammals. The gene expression level of a polynucleotide that selectively hybridizes with a sequence that is at least 80% identical to the sequence described in -80 is compared to the gene expression level of said polynucleotide in a control cell or mammal Including that. At this time, the test component for adjusting the expression level of the polynucleotide is a therapeutic drug candidate for the above-mentioned disease.

DETAILED DESCRIPTION OF THE INVENTION Based on the objectives outlined above, the present invention is a novel method for the diagnosis and prognostic evaluation of various diseases, such as various predetermined forms of cancer including, for example, angiogenesis, fibrosis, and metastatic cancer. As well as methods for screening for compositions that modulate the condition. Also provided are methods for treating the disease or cancer.

See also: American Society of Clinical Oncology (ed. 2001) ASCO Curriculum: Symptom Management Kendall / Hunt, ISBN: 0787277851 ( ASCO Curriculum : Disease Management); Bonadonna, et al. (2001) Textbook of Breast Cancer (2d ed.) Dunitz Martin, ISBN: 1853178241; Devita and Hellman (eds. 2001) Cancer Principles and Practice of Oncology (2 Vols.), Lippincott Williams, ISBN: 0781723876; Howell, et al. (2001) Breast Cancer Isis Medical Media, ISBN: 1901865584; Kaye and Laws (2001) Brain Tumours: An Encyclopedic Approach Encyclopedia approach ”) (2d ed.) Churchill Livingstone, ISBN: 0443064261; Mihm, et al. (2001) The Melanocytic Proliferation: A Comprehensive Textbook of Pigmented Lesions Wiley-Liss, ISBN: 0471252719; Montgomery and Aaron (2001) Clinical Pathology of Soft-Tissue Tumors Marcel Dekker, ISBN: 0824702905; Petrovich, et al. (eds. 2001) Combined Modality of Central Nervous System Tumors Combined physical therapy (medical radiology) for tumors of the system ”(Medical Radiology) Springer Verlag, ISBN: 3540660534; Rosen (2001) Rosen's Breast Pathology (Rosen's breast pathology) Lippincott Williams and Wilkins, ISBN: 0781723795;

Shah, et al. (2001) Oral Cancer (oral cancer) Isis Medical Media, ISBN: 189906687X; Weiss and Goldblum (2001) Enzinger and Weiss's Soft Tissue Tumors (4th ed. ) Mosby, ISBN: 0323012000; Abeloff, et al. (Eds. 2000) Clinical Oncology (2d ed.) Churchill Livingstone, ISBN: 044307545X, American Society of Clinical Oncology (ed. 2000) Cancer Genetics and Cancer Predisposition Testing Kendall / Hunt, ISBN: 0787276154 (American Clinical Oncology Society, Cancer Genetics and Cancer Predisposition Test); Fletcher (2000) Diagnostic Histopathology of Tumors (Tumor Diagnostic Histopathology) ( 2 vols. 2d ed.) Churchill Livingstone, ISBN: 0443079927; Vogelzang (ed. 2000) Comprehensive Textbook of Genitourinary Oncology (2d ed.) Lippincott Williams and Wilkins, ISBN: 0683306456 ;

Holland, et al. (Eds. 2000) Holland-Frei Cancer Medicine (Book with CD-ROM 5th ed.) Turrisi, et al. (2000) Lung Cancer Isis Medical Media, ISBN: 1901865428; Bartolozzi and Lencioni (eds. 1999) Liver Malignancies: Diagnostic and Interventional Radiology (Medical Radiology) Diagnosis and interventional radiology (medical radiology)) Springer Verlag, ISBN: 3540647562; Gasparini (ed. 1999) Prognostic Variables in Node-Negative and Node-Positive Breast Cancer Prognostic variables for positive breast cancer ”) Kluwer, ISBN: 0792384474;

Hansen (ed. 1999) The LASLC Textbook of Lung Cancer: International Association for the Study of Lung Cancer Dunitz Martin, ISBN: 1853177083; Raghavan, et al. (Eds. 1999 ) Textbook of Uncommon Cancer (2nd ed.) Wiley, ISBN: 0471929212; Thawley, et al. (Eds. 1999) Comprehensive Management of Head and Neck Tumors Comprehensive management of cervical cancer ”(2 vols.) Saunders, ISBN: 0721655823; Whittaker and Holmes (eds. 1999) Leukemia and Related Disorders (3d ed.) Blackwell Science, ISBN : 0865426074; Aapro (ed. 1998) OncoMedia: Medical Oncology ("OncoMedia") (CD-ROM) Elsevier Science, ISBN: 0080427480;

Abeloff (1998) Clinical Oncology (Library Version 2 CD-ROM Individual Version 2.0 Windows and Macintosh) Harcourt Brace, ISBN: 0443075557 ("Clinical Oncology" (Library version 2CD-ROM Individual Version 2.0 Windows and Macintosh); Benson (ed. 1998) gastrointestinal oncology (cancer treatment and research, CTAR 98) ( "gastrointestinal oncology" (cancer treatment and research, CTAR 98) Kluwer, ISBN: 0792382056; Brambilla and Brambilla (. eds 1998) Lung tumors: Fundamental Biology and Clinical Management (Lung Tumors: Basic Biology and Clinical Management) (Vol 124) Marcel Dekker, ISBN: 0824701607; Canellos, et al. (Eds. 1998) The Lymphomas ( Lymphoma ) Saunders, ISBN: 0721650309 Greenspan and Remagen (1998) Differential Diagnosis of Tumors and Tumor-Like Lesions of Bones and Joints Lippincott Williams and Wilkins Publishers, ISBN: 0397517106; Hiddemann ( ed. 1998) Acute Leukemias VII: Experimental Approa ches and Novel Therapies (Acute Leukemia VII: Experimental Approach and Novel Treatment) (Haematologie Und Bluttransfusion, Vol 39), Springer Verlag, ISBN: 3540635041;

Husband and Reznek (1998) Imaging in Oncology (2 vols.) Mosby, ISBN: 1899066489; Leibel and Phillips (eds. 1998) Textbook of Radiation Oncology Saunders, ISBN: 0721653367; Maloney and Miller (eds. 1998) Cutaneous Oncology: Pathophysiology, Diagnosis, and Management (Skin Oncology: Pathophysiology, Diagnosis and Management ) Blackwell Science, ISBN: 0865425175; 1998) Advances in Radiation Therapy Kluwe, ISBN: 0792399811; Oldham (ed. 1998) Principles of Cancer Biotherapy (3d ed.) Kluwer , ISBN: 0792335074; Ozols (ed. 1998) Gynecologic Oncology (" Gynecological Oncology ") Kluwer, ISBN: 0792380703; Parkin, et al. (Eds. 1998) Cancer Incidence in Five Continents Incidence ”) (Iarc Scientific Publications, No 143) Oxford University Press, ISBN: 9 283221435 (Oxford University Press); Perez and Brady (eds. 1998) Principles and Practice of Radiation Oncology Lippincott Williams and Wilkins, ISBN: 0397584164; Black, et al. (Eds. 1997 ) Cancer of the Nervous System Blackwell Science, ISBN: 0865423849; Bonadonna, et al. (1997) Textbook of Breast Cancer: A Clinical Guide to Therapy ] Blackwell Science, ISBN: 1853173487;

Pollock (ed. 1997) Surgical Oncology ("surgical oncology") Kluwer, ISBN: 0792399005; Sheaves, et al. (Eds. 1997) Clinical Endocrine Oncology ("clinical endocrine oncology") Blackwell Science, ISBN: 086542862X; Vahrson (1997) Radiation Oncology of Gynecological Cancers Springer Verlag, ISBN: 0387567682; Walterhouse and Cohn (eds. 1997) Diagnostic and Therapeutic Advances in Pediatric Oncology Development ”) Kluwer, ISBN: 0792399781; Aisner (ed. 1996) Comprehensive Textbook of Thoracic Oncology (Lippincott, Williams and Wilkins, ISBN: 0683000624; Bertino, et al. (Eds. 1996) ) Encyclopedia of Cancer (3 vols.) Academic, ISBN: 012093230X;

Cavalli, et al. (1996) Textbook of Medical Oncology Dunitz Martin, ISBN: 1853172901; Peckham, et al. (Eds. 1995) Oxford Textbook of Oncology (Oxonology Oxford Textbook (2-Vols.) Oxford University Press, ISBN: 0192616854 (Oxford University Press); and Freireich and Kantarjian (eds. 1996) Molecular Genetics and Therapy of Leukemia (Cancer Treatment and Research, V. 84) Genetics and Leukemia Treatment ”(Cancer Treatment and Research, V. 84)) Kluwer, ISBN: 0792339126.

  In particular, the identification of a marker that is selectively expressed in a given cancer allows its expression to be used in diagnostic, prognostic or therapeutic methods. Thus, the present invention demonstrates various compositions, such as nucleic acids, polypeptides, antibodies, and small molecule agonists / antagonists, that help to selectively identify those markers. For example, treatment methods use selective localization and modulation of function (for markers with causative disease effects), expression of markers for vaccines, identification of binding partners, or using, for example, antisense or RNA interference (RNAi) May take the form of protein therapy using the antagonists.

  This marker is useful, for example, for molecular characterization of the subsets of diseases provided in Table 1, and these subsets may actually require completely different treatments. In addition, the markers are important in certain diseases and diseases associated with cancer, for example, non-malignant diseases that affect similar tissues or have similar induction / management mechanisms. The process or characteristics of the transition may also be targeted. Diagnosis and prognostic use are possible, for example, for determining related but separate diseases or treatment plans. This detection method is based on nucleic acids such as polymerase chain reaction, or hybridization techniques, or proteins such as ELISA, diagnostic imaging, immunohistochemistry and the like. Diagnosis is qualitative or quantitative and detects an increase or decrease in expression level.

  Table 2B-76B shows increased or decreased expression in affected samples (see Table 1-3), especially angiogenesis, arthritis, prostate cancer, breast cancer, colorectal cancer, cervical cancer, bladder cancer, head and neck cancer Gene showing a sequence including esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, kidney cancer, stomach cancer, skin cancer, testicular cancer, uterine cancer, glioblastoma, Ewing sarcoma, soft tissue sarcoma, and lung fibrosis Provide a UniGene cluster identification number for the nucleotide sequence. Tables 2A-80 also provide representative registration numbers that provide nucleotide sequences that are part of the UniGene cluster.

Definitions The term “oncoprotein”, “cancer polynucleotide”, or “cancer-associated transcription” refers to polymorphic variants, alleles, mutants, and interspecies homologs of nucleic acids and polypeptides, which are: (1) About 60% or more, 65%, 70%, 75%, 80%, 85%, 90% nucleotide sequence identity to the nucleotide sequence of the gene of Table 1-80 or related thereto, preferably About 92%, 94%, 96%, 97%, 98%, or 99% or more nucleotide sequence identity, preferably at least 25, 50, 100, 200, 500, 1000, or more Having a nucleotide sequence spanning a range of nucleotides; (2) an antibody, such as a polyclonal antibody, generated against an immunogen comprising an amino acid sequence encoded by the nucleotide sequence of Table 1-80 or a gene related thereto; And its conservatively modified mutations (3) hybridize specifically under stringent hybridization conditions with a nucleic acid sequence, or the complement of Table 1-80, and conservatively modified variants thereof; or (4) Table About 60% or more, 65%, 70%, 75%, 80%, 85% amino acid sequence identity to the amino acid sequence encoded by the nucleotide sequence of 1-80 or related genes, preferably about 90% 91%, 93%, 95%, 97%, 98%, or 99% or more amino acid sequence identity, preferably at least 25, 50, 100, 200, 500, 1000, or more It has an amino acid sequence that spans a range of amino acids. A polynucleotide or polypeptide sequence generally includes a primate, eg, a human; a rodent, eg, a rat, mouse, hamster; a cow, pig, horse, sheep, or other mammal; From animals. “Cancer polypeptides” and “cancer polynucleotides” include both natural and recombinant forms.

  A “full-length” oncoprotein or nucleic acid refers to a cancer polypeptide or polynucleotide sequence, or a variant thereof, usually contained in one or more natural wild-type cancer polynucleotide or polypeptide sequences. Contains ingredients. This “full length” may be prior to or after various stages of post-translational processing or splicing, including alternative splicing.

  As used herein, a “biological sample” is a sample of biological tissue or fluid that contains nucleic acids and polypeptides such as, for example, cancer proteins, polynucleotides, or transcripts. These samples include, but are not limited to, tissues isolated from primates such as humans or rodents such as mice and rats. Biological samples include biopsy or autopsy samples, frozen sections taken for histological use, pre-collected samples, blood, plasma, serum, saliva, stool, tears, mucus, hair, skin, etc. Also includes tissue fragments. Biological samples also include explants and primary and / or transformed cells from patient tissue. The biological sample is usually from a eukaryote, most preferably a mammal, eg a primate such as a chimpanzee or human; a cow; a dog; a cat; a rodent such as a guinea pig, rat, mouse; , Birds; reptiles; Livestock and livestock are also covered.

  “Providing a biological sample” means obtaining a biological sample for use in the methods described in the present invention. In most cases this is done by taking a cell sample from the animal, but using previously isolated cells (eg isolated from others, at other times and / or for other uses) It can also be achieved by performing the method of the present invention in vivo. Already harvested tissues or materials with treatment and effect records are particularly useful.

  In sequences of two or more nucleic acids or polypeptides, the term “identical” or percent “identity” is measured by default parameters described below, for example using the BLAST or BLAST 2.0 sequence comparison algorithm. Or when manual alignment and visual inspection (see eg NCBI website http://www.ncbi.nlm.nih.gov/BLAST/ etc.), two or more sequences or subsequences are identical A certain percentage of amino acid residues or nucleotides (e.g., when compared and aligned to correspond to the maximum in a comparison frame or specified region, about 70% are identical, preferably 75%, over a certain range, 80%, 85%, 90%, 91%, 93%, 95%, 97%, 98%, 99%, or more). These sequences are then said to be “substantially identical”. This definition also represents or applies to the complement of a test sequence. This definition further includes deleted and / or inserted, substituted sequences, and natural or synthetic variants, such as polymorphic or allelic variants. As described below, desirable algorithms can take into account gaps and the like. Preferably, the length ranges of at least 25 amino acids or nucleotides are the same, and more preferably the length ranges of about 50-100 amino acids or nucleotides are the same.

  In sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm parameters are designated. Preferably, default parameters can be used and alternative parameters are specified. A sequence comparison algorithm herein calculates the ratio of identical sequences of a test sequence to a reference sequence, based on program parameters.

As used herein, a “comparative frame” generally refers to a series of sites selected from the group comprising about 20 to 600, usually about 50 to about 200, usually about 100 to about 150 (sequences thereof). A reference to a portion is included, where after the two sequences are optimally aligned, a sequence is compared to the same number of reference sequences in consecutive positions. Methods for aligning sequences for comparison are known. Optimal alignment for comparison is performed using the following method: the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482-489; the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453; the search for similarity method of Pearson and Lipman (searching for similarity between Pearson and Lipman) (1988) Proc. Nat'l. Acad. Sci. USA 85: 2444-2448; Computerized implementation of these algorithms (Genetics Computer Group, 575 Science Dr., Madison, WI, Wisconsin Genetics Software Package) GAP, BESTFIT, FASTA and TFASTA); or manual alignment and visual inspection (eg Ausubel, et al. (Eds. 1995 and supplements) Current Protocols in Molecular Biology Le ”) See Wiley).

Preferred examples of suitable algorithms for measuring sequence identity and sequence similarity ratios are Altschul, et al. (1977) Nuc. Acids Res. 25: 3389-3402 and Altschul, et al. (1990 ) Including the BLAST and BLAST 2.0 algorithms described in J. Mol. Biol. 215: 403-410. BLAST and BLAST 2.0 are used to determine the percent identity of the nucleic acid and protein sequences of the invention using the parameters described herein. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The algorithm first identifies a short word of length W in the query sequence that, when aligned with a word of the same length in the sequence of the database, matches a positive threshold score T or meets the score T. Identifying high-scoring sequence pairs (HSPs). T is called neighborhood word score threshold (Altschul, et al., Supra). These initial neighborhood word hits initiate a search to find longer HSPs containing them.

Word hits are stretched in both directions along each sequence as long as the cumulative alignment score can be increased. The cumulative score is calculated using, for example, the nucleotide sequence, the parameter M (reward score of matched residue pair; always> 0) and N (penalty score of mismatched residue; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of word hits in each direction stops when: The cumulative alignment score decreases by X minutes from the maximum achieved; the cumulative score is due to the collection of one or more negative score residue alignments When is 0 or less; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X measure the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses word length (W) 11, expectation (E) 10, M = 5, N = -4, and comparison of both strands as defaults. For amino acid sequences, the BLASTP program has a word length of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-919), Alignment (B) 50, M = 5, N = -4, and comparison of both strands are used as defaults.

The BLAST algorithm also performs a statistical analysis of the similarity of two sequences. See, for example, Karlin and Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the minimum total probability (P (N)), which provides an indication of the probability of a chance match occurring between two nucleotides or amino acids. For example, a nucleic acid is similar to a reference sequence when the minimum total probability of comparing the test nucleic acid and the reference nucleic acid is about 0.2 or less, more preferably about 0.01 or less, and most preferably about 0.001 or less. Is considered. The logarithmic value is a negative large number such as 5, 10, 20, 30, 40, 40, 70, 90, 110, 150, 170, for example.

  An indication that two nucleic acid sequences are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically related to the antibody produced against the polypeptide encoded by the second nucleic acid. It is to cross-react with. Thus, for example, if two peptides differ only by conservative substitutions, one polypeptide will generally be substantially identical to the second polypeptide. Another indication that two nucleic acid sequences are substantially identical is that the two molecules and their complements hybridize to each other under stringent conditions. Yet another indication that two nucleic acid sequences are substantially identical is that the same primer can be used to amplify the sequence.

  A “host cell” is a natural cell or transformed cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be cultured cells, explants, in vivo cells, and the like. Host cells include prokaryotic cells such as E. coli, eukaryotic cells such as yeast, insects, and amphibians, or mammals such as CHO (established cell lines derived from Chinese hamster ovary) and HeLa (cervical cancer) cells. It may also be an animal cell (see, for example, the American Type Culture Collection catalog or website www.atcc.org).

  The terms "isolated", "purified" or "biologically pure" refer to substances that are substantially or essentially free from the components they normally add, as found in nature. Refers to that. Purity and homogeneity are usually measured using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Proteins or nucleic acids that are the predominant species present in the tissue specimen are substantially purified. In particular, an isolated nucleic acid is separated from several open reading frames that naturally flank the gene and encode proteins other than the protein encoded by the gene. The term “purified” in some embodiments means that the nucleic acid or protein produces essentially one band in the electrophoresis gel. This preferably means that the nucleic acid or protein is at least about 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure. In another aspect, “purify” or “purification” means removing at least one contaminant or component from the composition to be purified. In this sense, purification does not require the purified compound to be homogeneous, for example 100% pure.

  The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein and refer to a polymer of amino acid residues. These terms are not only natural amino acid polymers containing modified residues or non-natural amino acid polymers, but also amino acid polymers in which one or more amino acid residues are synthetic chemical mimics of the corresponding natural amino acids Is true.

  The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Natural amino acids are amino acids encoded by genetic information, and are amino acids that have been modified later, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs have the same basic chemical structure as natural amino acids, such as α-carbons that bind to hydrogen, carboxyl groups, amino groups, and R groups such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Refers to a compound. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but maintain some basic chemical structure of natural amino acids. An amino acid mimetic refers to a compound that has a structure different from the chemical structure of a normal amino acid but functions in the same manner as other amino acids.

  In the present specification, amino acids are represented by the commonly known three letter code or the one letter code recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides are represented by the commonly accepted single letter symbols.

  “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and if the nucleic acid does not encode an amino acid sequence, for example, naturally Refers to adjacent, essentially identical or related sequences. Because of the degeneracy of the genetic code, most proteins are encoded by many functionally identical nucleic acids. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a certain codon, the codon can be replaced with another corresponding codon described without altering the encoded polypeptide. Such nucleic acid changes are “silent (no amino acid substitution) mutations” and are a type of conservatively modified mutation. Any nucleic acid sequence herein that encodes a polypeptide represents a silent variation of the nucleic acid. In certain situations, each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to create functionally similar molecules . Thus, silent mutations of nucleic acids encoding polypeptides are potential in the described sequence for the expression product, but not necessarily for the actual probe sequence.

With respect to amino acid sequences, one of ordinary skill in the art will recognize each substitution, deletion, addition to a nucleic acid, peptide, polypeptide, or protein sequence that changes, adds, or deletes a single nucleic acid or a small percentage of nucleic acids in the encoded sequence. Will be recognized as “conservatively modified variants” and that the change results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables displaying functionally similar amino acids are known. Such conservatively modified variants are in addition to, but not exclusive of, polymorphic variants, interspecies homologues, and alleles of the present invention. Common conservative substitutions include the following combinations: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4 ) Arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, eg, Creighton (1984) Proteins: Structure and Molecular Properties Freeman).

Macromolecular structures, such as the structure of a polypeptide, can be described in terms of various levels of organization. For a general discussion of this structure see, for example, Alberts, et al. (Eds. 2001) Molecular Biology of the Cell (4th ed.) Garland; and Cantor and Schimmel (1980 ) Biophysical Chemistry Part I: The Conformation of Biological Macromolecules See Freeman. “Primary structure” refers to the amino acid sequence of a specific peptide. “Secondary structure” refers to a locally ordered three-dimensional structure within a polypeptide. These structures are usually known as domains. A domain is a portion of a polypeptide, often forming a small unit of the polypeptide, usually 25 to about 500 amino acids in length. Normal domains are made up of smaller components such as β-sheet and α-helix stretches. “Tertiary structure” refers to the complete three-dimensional structure of a polypeptide monomer. “Quaternary structure” refers to a three-dimensional structure usually formed by non-covalent connections of individual tertiary units. Anisotropic terms are also known as energy terms.

The terms “nucleic acid”, “oligonucleotide”, “polynucleotide”, and the like used herein grammatically, mean at least two or more nucleotides covalently linked. Oligonucleotides typically range in length from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides to about 100 nucleotides in length. Nucleic acids and polynucleotides are polymers of any length, including longer ones such as, for example, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000. The nucleic acids of the invention usually contain a phosphodiester bond, but in some cases, for example, phosphoramidates, phosphorothioates, phosphorodithioates, or O-methyl phosphoramidite bonds (Eckstein (1992) Oligonucleotides and Analogues: A Practical Nucleic acid analogs having at least one different bond, such as Approach Oxford Univ. Press (Oligonucleotides and Analogues: Practical Approach, Oxford University Press); and peptide nucleic acid backbones and bonds. Other similar nucleic acids are described in U.S. Patent Nos. 5,235,033 and 5,034,506, and Sanghvi and Cook (eds. 1994) Carbohydrate Modifications in Antisense Research, ACS Symposium Series 580, Chapters 6 and 7. Including analogs having a positive skeleton such as a nonionic skeleton and a non-ribose skeleton, including those described. Nucleic acids containing one or more carbocyclic carbohydrates are also included in one definition of nucleic acids. Modification of the ribose phosphate backbone can be done for a variety of reasons, for example, to increase the stability and half-life of the molecule in a physiological environment or as a probe for a biochip. Natural nucleic acid and analog mixtures can be made; while other nucleic acid analog mixtures and natural nucleic acid and analog mixtures can be made.

A number of references, such as the following, disclose nucleic acid analogs: Phosphoramidate (Beaucage, et al. (1993) Tetrahedron (“tetrahedron”) 49: 1925-1963, and its References; Letsinger (1970) J. Org. Chem. 35: 3800-3803; Sprinzl, et al. (1977) Eur. J. Biochem. 81: 579-589; Letsinger, et al. (1986) Nucl. Acids Res 14:.... 3487-499 ; Sawai, et al (1984) Chem Lett 805; Letsinger, et al (1988) J. Am Chem Soc 110:.... 4470-4471; and Pauwels, et al. (1986) Chemica Scripta 26: 141-149), phosphorothioate (Mag, et al. (1991) Nucleic Acids Res. 19: 1437-441; and US Pat. No. 5,644,048), phosphorodithioate (Brill, et al. ( 1989) J. Am. Chem. Soc. 111: 2321-2322), O-methyl phosphoramidite linkage (Eckstein (1992) Oligonucleotides and Analogues: A Practical Approach , Oxford Univ. Press Approach ”Oxf - see de University Press)), and combined with the peptide nucleic acid backbone (see below: Egholm (1992) J. Am Chem Soc 114:...... 1895-1897; Meier, et al (1992) Chem Int Ed. Engl. 31: 1008-1010; Nielsen (1993) Nature 365: 566-568; Carlsson, et al. (1996) Nature 380: 207, the entirety of which is incorporated herein by reference).

Other nucleic acid analogs include nucleic acid analogs having a positive backbone (Denpcy, et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6097-101; nonionic backbone (US Pat. No. 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863; Kiedrowski, et al. (1991) Angew. Chem. Intl. Ed. English 30: 423-426; Letsinger, et al. (1988) J. Am. Chem. Soc. 110: Letsinger, et al. (1994) Nucleoside and Nucleotide 13: 1597; Sanghvi and Cook (eds. 1994) Carbohydrate Modifications in Antisense Research ACS Symposium Series 580, Chapters 2 and 3; Mesmaeker, et al. 1994) Bioorganic and Medicinal Chem. Lett. 4: 395-398; Jeffs, et al. (1994) J. Biomolecular NMR 34:17; Horn, et al. (1996) Tetrahedron Lett. 37: 743), and the United States Patent Nos. 5, 235,033 and 5,034,506, and Sanghvi and Cook (eds. 1994) Carbohydrate Modifications in Antisense Research, ACS Symposium Series 580, Chapters 6 and 7 Non-ribose skeletons, including ones that include one or more carbocyclic sugars are also included in one of the definitions of nucleic acids (Jenkins, et al. (1995) Chem. Soc. Rev. pp 169- 176) Some nucleic acid analogues are described in Rawls (page 35, 1997, June 2) C & E News .

  Peptide nucleic acids (PNA) including peptide nucleic acid analogs are particularly preferred. In contrast to the highly charged phosphodiester backbones of natural nucleic acids, these backbones are substantially nonionic under neutral conditions. This provides at least two advantages. The PNA backbone exhibits improved hybridization mechanics. PNA represents a greater melting temperature (Tm) change for mismatched base pairs compared to perfect match. DNA and RNA usually show a Tm drop of 2-4 ° C versus internal incompatibility, but for nonionic PNA backbones, the temperature drop is around 7-9 ° C. Similarly, due to its non-ionic character, the hybridization of bases bound to these backbones is relatively insensitive with respect to salt concentration. In addition, PNA is not degraded by cellular enzymes and is therefore more stable.

  As clearly stated, nucleic acids may be single-stranded or double-stranded, and may contain portions that contain both single-stranded and double-stranded sequences. Single strand delineation also determines the sequence of complementary strands; therefore, the sequences described herein also provide complementary sequences. When the nucleic acid includes a combination of deoxyribonucleotide and ribonucleotide and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, etc., the nucleic acid is genomic DNA and cDNA Both (complementary DNA) can be DNA, RNA, or hybrid. “Transcription” is usually used for natural RNA, such as pre-mRNA (mRNA precursor), hnRNA (heteronuclear RNA), or mRNA (messenger RNA). As used herein, the term “nucleoside” includes modified nucleosides such as nucleotides, nucleosides, nucleotide analogs, and amino-modified nucleosides. In addition, “nucleoside” includes non-natural analog structures. Thus, individual units of peptide nucleic acids that each contain a base are considered herein as nucleosides.

A “label” or “detectable moiety” refers to a composition that can be detected by spectroscopic, photochemical, biochemical, immunochemical, physiological, chemical, or other physical methods. Common labels are classified into three categories: a) isotope labels that are radioactive or heavy isotopes; b) immunolabels such as antibodies, antigens, epitope tags; and c) colored or fluorescent dyes. These labels are incorporated into cancer nucleic acids, proteins, and antibodies. For example, the label should be capable of producing a detectable signal, either directly or indirectly. The detectable moieties are fluorescent or chemical, such as radioisotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I, high electron density reagents, fluorescein isothiocyanate, rhodamine, or luciferin. Luminescent compounds, or enzymes (such as commonly used in ELISA, for example), biotin, digoxigenin, or haptens or proteins, or alkaline phosphatase, beta galactosidase, or horseradish peroxidase. Methods for conjugating antibodies to labels are known. For example, Hunter, et al. (1962) Nature 144: 945; David, et al. (1974) Biochemistry 13: 1014-1021; Pain, et al. (1981) J. Immunol. Meth. 40: 219-230; And Nygren (1982) J. Histochem. And Cytochem. 30: 407-412.

  “Effector”, “moiety” or “effector component” is covalently linked by a linker or chemical bond, or non-covalently by an ionic or van der Waals force, electrostatic or hydrogen bond A molecule that is bound (or linked or bound) to an antibody. An “effector” is, for example, a radioactive moiety, an oral compound, an enzyme or substrate, a tag such as an epitope tag, a toxin or the like; a moiety; a chemotherapeutic agent; a lipase; an antibiotic; It can be a variety of molecules, such as an immunomodulating component (micA / B) and also a radioisotope that emits “hard” beta rays, for example.

  "Labeled nucleic acid probe or oligonucleotide" is, for example, covalently bound to a label by a linker or chemical bond, or non-covalently by ionic, van der Waals force, static electricity, or hydrogen bond, By detecting the presence of the label bound to the probe, the presence of the probe is detected. Alternatively, methods using high affinity interactions can produce similar results where one pair of binding partners binds to another partner, such as biotin, streptavidin, and the like.

  As used herein, a “nucleic acid probe or oligonucleotide” refers to a complementary sequence target, usually by one or more types of chemical bonds, eg, through complementary base pairing by formation of hydrogen bonds. A nucleic acid that can bind to a nucleic acid. As used herein, a probe includes a natural base (eg A, G, C or T) or a modified base (7-deazaguanosine, inosine, etc.). In addition, the base of the probe is linked in a way other than phosphodiester bonds, preferably functionally that does not interfere with hybridization. Thus, for example, a probe may be a peptide nucleic acid in which the constituent bases are linked by peptide bonds rather than phosphodiester bonds. The probe binds a target sequence lacking perfect complementarity to a probe sequence that is dependent on stringent hybridization conditions. The probe is preferably labeled directly, for example with isotopes, chromophores, luminophores, chromophores, etc., or indirectly with, for example, biotin, to which a streptavidin complex later binds. By analyzing the presence or absence of the probe, the presence or absence of the selected sequence or partial sequence can be detected. Diagnosis and prognosis is based on the genomic level, or the RNA level, or the level of protein expression.

  The term “recombinant” when used with respect to, for example, a cell, nucleic acid, protein, or vector, by incorporating a heterologous nucleic acid, protein, or modified natural nucleic acid or protein, such cell, nucleic acid, protein, or vector Indicates that the cell has been modified, or that the cell is obtained from a modified cell. Thus, for example, a recombinant cell expresses a gene that is not found in the native (non-recombinant) form of the cell, or is inherently abnormally expressed, underexpressed, or not expressed at all. Express the gene. As used herein, the term “recombinant nucleic acid” originally originated from the body by manipulating the nucleic acid in a form not normally found in nature, usually using, for example, polymerases or endonucleases. It means the nucleic acid formed in In this way, operational linkage of different sequences is possible. Thus, expression of vectors formed by joining linearly isolated nucleic acids or DNA molecules that do not normally bind in vitro is considered to be recombinant for the purposes of the present invention. Once a recombinant gene is created and reintroduced into a host cell or organism, the gene is known to replicate without recombination, for example, using cellular mechanisms within the host cell rather than in vitro manipulation. . However, once the recombination has occurred, the nucleic acid is still considered recombined for purposes of the present invention, even if it is subsequently replicated without recombination.

  Similarly, a “recombinant protein” is a protein made using, for example, recombinant techniques by expression of a recombinant nucleic acid as depicted above. A recombinant protein is distinguished from natural protein by at least one or more properties. The protein is separated or purified from a portion or most of the protein and compound normally associated with the wild-type host and is thus substantially pure. The isolated protein preferably comprises at least about 0.5%, more preferably at least about 5% by weight of the protein in a given sample normally associated with its native state Without the substance. A substantially pure protein comprises at least about 75% by weight of the total protein, preferably at least about 80%, and particularly preferably at least about 90%. This definition includes the production of oncoproteins from one organism in different organisms or host cells. Alternatively, the protein is produced at a significantly higher concentration than would normally be seen by using an inducible or high expression promoter to produce increased concentration levels of the protein. Alternatively, the protein may be in a form that is not normally found in nature due to the addition of an epitope tag, amino acid substitution, insertion, and deletion described below.

  The term “heterologous” when used in reference to a portion of a nucleic acid refers to a nucleic acid comprising two or more subsequences that are not normally found in the same relationship to each other in nature. For example, a nucleic acid usually has two or more sequences that make up a new functional nucleic acid by arranging unrelated genes, such as a promoter from one source and a coding part from another source, and is produced while recombining Is done. Similarly, a heterologous protein often refers to two or more subsequences that are not naturally found in the same relationship to each other (eg, a fusion protein).

  A “promoter” is usually a sequence of nucleic acid control sequences that directs transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as a TATA element in the case of polymerase II type promoters. A promoter also includes distal enhancers or repressor elements that, depending on the situation, may be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to between a nucleic acid expression control sequence (such as a promoter or transcription factor binding site) and a second nucleic acid sequence, for example, where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence. Functional combination of

  An “expression vector” is a nucleic acid construct that is produced recombinantly and synthetically by a series of specific nucleic acid components that allow transcription of a particular nucleic acid in a host cell. The expression vector can be part of a plasmid, virus or nucleic acid fragment. Expression vectors usually contain nucleic acid that is transcribed in operative linkage with a promoter.

  The phrase “selectively (or specifically) hybridizes” is under stringent hybridization conditions when the sequence is present in a complex mixture (eg, total cells or collected DNA or RNA). Refers to the selective binding, duplication or hybridization of molecules to specific nucleotide sequences.

The phrase “stringent hybridization conditions” refers to the condition in which a probe hybridizes to its target subsequence, usually in a complex mixture of nucleic acids, but not to any other sequence. Stringent conditions are sequence-dependent and are different under different circumstances. Long sequences hybridize at particularly high temperatures. For extensive guidance on nucleic acid hybridization, see Tijssen (1993) Hybridization with Nucleic Probes (Laboratory Techniques in Biochemistry and Molecular Biology). vol. 24) As seen in Elsevier's "Overview of principles of hybridization and the strategy of nucleic acid assays".

Generally, stringent conditions are selected to be about 5-10 ° C. lower than the melting temperature (T _m ) for the specific sequence at a defined ionic strength, pH. T _m is the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (the target sequence is excessively present at T _m and 50% of the probe is blocked at equilibrium) (determined) Measured ionic strength, pH, and nuclear concentration). Stringent conditions include a salt concentration of about 1.0 M or less, usually pH 7.0 to 8.3, a sodium ion concentration (or other salt) of about 0.01 to 1.0 M, and a short probe (eg, about 10 to about nucleotides). The temperature of 50) is at least about 30 ° C, and the temperature of long probes (eg, about 50 or more nucleotides) is at least about 60 ° C.

Stringent conditions may be achieved by the addition of destabilizing substances such as formamide. For selective or specific hybridization, a positive signal is usually at least twice background, preferably 10 times background hybridization. Examples of stringent hybridization conditions are as follows: Wash with 0.2x SSC and 0.1% SDS at 65 ° C, formamide 50%, 5xSSC, 1% SDS (sodium dodecyl sulfate), culture at 42 ° C, or 5xSSC Incubate at 1 ℃ SDS, 65 ℃. For PCR (Polymerase Chain Reaction), temperatures of about 36 ° C. are common for low stringency amplification, but annealing temperatures vary between about 32-48 ° C. depending on the length of the primer. For high stringency PCR amplification, the temperature is typically about 62 ° C, but high annealing temperatures range from about 50-65 ° C based on primer length and selectivity. Normal cycling conditions for both high and low stringency amplification are: a denaturation step at 90-95 ° C for 30-120 seconds, an annealing step that lasts 30-120 seconds, and an extension of 1-2 minutes at about 72 ° C Including stages. Protocols and guidelines for low or high stringency amplification reactions can be found in, for example, Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications , Academic Press, NY Is provided to.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is made using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acid typically hybridizes under moderately stringent hybridization conditions. Typical “moderately stringent hybridization conditions” are: hybridization at 37 ° C in 40% formamide buffer, 1 M NaCl, 1% SDS and washing with 1X SSC at 45 ° C. Including. Positive hybridization is usually twice the lowest background. Selective hybridization and wash conditions can be used to provide similar stringency conditions. Additional guidelines for determining hybridization parameters are available in numerous references, such as Ausubel, et al. (Eds. 1991 and supplements) Current Protocols in Molecular Biology Wiley. Provided by the literature.

  The phrase “functional effect” in the context of assays that test compounds that modulate the activity of oncoproteins is indirectly or directly, eg, physiological, functional, physical, or the ability to reduce cancer. Includes determination of parameters under the influence of cancer proteins or nucleic acids, such as chemical effects. Mediators include: ligand binding activity; cell survival; cell growth on soft agar; anchorage dependence; contact inhibition and density limitation of growth; cell proliferation; cell transformation; growth factor or serum dependence; tumor specificity Includes marker levels; invasiveness to Matrigel; tumor growth and metastasis in the body; mRNA and protein expression of metastatic cells; and other properties of cancer proteins. “Functional effects” include in vitro, in vivo, and ex vivo activities.

  “Measurement of functional effect” refers to a compound in which physiological, functional, enzymatic, physical, or chemical effects, for example, indirectly or directly increase or decrease a mediator variable under the effect of an oncoprotein. It means to assay. Functional effects such as: for example, spectroscopic properties (eg fluorescence, absorbance, refractive index), hydrodynamics (eg shape), protein chromatographic or dissolution properties, measurement of inducible markers or cancer protein Activation of transcription, measurement of binding activity or binding assay, eg binding to antibodies or other ligands, measurement of proliferation, cell proliferation, cell survival, cell transformation, growth factor or serum dependence, tumor-specific marker level , Matrigel invasiveness, tumor growth and metastasis in vivo, mRNA and protein expression, and other changes in cancer cell properties can be measured. Functional effects can be in many ways: for example, quantitative or qualitative measurement of changes in morphological features by microscopy, measurement of RNA or protein level changes in cancer-related sequences, measurement of RNA stability, eg chemistry Evaluated by identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP, etc.), such as by luminescent materials, fluorescent materials, colorimetric assay reactions, antibody binding, inducible markers, and ligand binding assays.

  “Inhibitors,” “activators,” and “modulators” of cancer polynucleotides and polypeptide sequences refer to molecules and compounds identified using in vivo and ex vivo assays of cancer polynucleotides and polypeptide sequences. Refers to activation, inhibition, or regulation. An inhibitory factor is, for example, an inhibitory factor that partially or completely interferes with activity, for example by binding, reduces, prevents, delays activation, inactivates, desensitizes or desensitizes oncoprotein activity or expression. A compound that down-regulates. Antisense or inhibitory nucleic acids appear to suppress protein expression and subsequent functions. An “activator” is a compound that increases, opens, triggers, promotes, enhances activation, sensitizes, activates, or upregulates the activity of oncoproteins. Inhibitors, activators or modulators can be genetically modified variants of a cancer protein, such as variants with altered activity, in addition to natural or synthetic ligands, antagonists, agonists, antibodies, chemical small molecules and the like. Including. Such assays of inhibitors and activators include, for example, in vitro, expression of cancer proteins in cells or cell membranes, application of putative modulator compounds, and measurement of functional effects on activities as described above. The cancer activators and inhibitors further comprise culturing the cancer cells with a test compound and one or more oncoproteins, such as oncoproteins encoded by the sequences specified in Table 1-80, eg, 1, 2, Identified by measuring the increase or decrease in the expression of 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more oncoproteins.

  Samples or assays containing oncoproteins treated with potential activators, inhibitors, or modulators are compared to control sample groups without inhibitors, activators, modulators to examine the extent of inhibition. The control sample group (not treated with the inhibitor) is given a relative protein activity value of 100%. Polypeptide inhibition occurs when the activity value relative to the control group is 80%, preferably 50%, more preferably 25-0%. The activity of the polypeptide of cancer is about 110%, more preferably 150%, and even more preferably 200-500% (eg, relative to the control group) relative to the control group (not treated with the activator). 2-5 times higher), and more preferably 1000-3000% higher.

The term "change in cell proliferation" refers to cell survival, lesion formation, anchorage independence, semi-solid or soft agar growth, growth contact inhibition and changes in density restriction, loss of growth factors or serum demand, cell morphology Or in immortalization, acquisition or loss of immortalization, acquisition or loss of tumor-specific markers, ability to form or suppress tumors when injected into a suitable animal host, and / or immortalization of cells, etc. Any change in the growth and growth characteristics of cells in the body. See, for example, Freshney (1994) Culture of Animal Cells a Manual of Basic Technique (2d ed.) Wiley-Liss, pages 231-241. This refers to precancerous cells, cancer cells, and normal cells.

“Cancer cells”, “transformed” cells or “transformation” in tissue culture refers to spontaneous or induced phenotypic changes that are not necessarily accompanied by the uptake of new genetic material. Transformation may occur from viral transformation and infection with new genomic DNA uptake or exogenous DNA uptake, but mutates endogenous genes either spontaneously or after contact with carcinogens It may occur by doing. Transformation involves phenotypic changes such as cell immortalization, control of overgrowth, non-morphological changes, and / or malignancy. See Freshney (2000) Culture of Animal Cells: A Manual of Basic Technique (4th ed.) Wiley-Liss.
“Antibody” refers to a polypeptide comprising a constituent part of an immunoglobulin gene, or a fragment thereof, which specifically recognizes and binds to an antigen. The recognized immunoglobulin genes include the constant region κ, λ, α, γ, δ, ε, and μ genes in addition to the myriad immunoglobulin genes in the variable region. Light chains are classified as kappa or lambda. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin class, IgG, IgM, IgA, IgD, and IgE. Usually, the binding region of an antibody to an antigen or a functionally equivalent region is most important in binding selectivity and affinity. Paul (ed. 1999) Fundamental Immunology (4th ed.) See Raven.

A typical immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each pair having a “light” chain (about 25 kD) and a “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of amino acids of approximately 100-110 or more, mainly responsible for antigen recognition. The terms variable light chain (V _L ) and variable heavy chain (V _H ) refer to the light or heavy chain, respectively.

Antibodies exist, for example, as complete immunoglobulins or as many characteristic fragments produced by digestion with various peptidases. Thus, for example, pepsin produces F (ab) ' ₂ , a dimer of Fab, which is itself a light chain linked to V _H -C _H 1 by a disulfide bond under a disulfide bond in the hinge region. Therefore, it absorbs antibodies. F (ab) ′ ₂ is cleaved under mild conditions to break the disulfide bond in the hinge region, and the F (ab) ′ ₂ dimer is converted to a Fab ′ monomer. The Fab ′ monomer is basically a Fab having a hinge region (see Paul (ed. 1999) Fundamental Immunology (“Basic Immunology”) (4th ed.) Raven). If the various antibody fragments are defined in terms of complete antibody digestion, those skilled in the art will recognize that these fragments have been synthesized de novo chemically or by recombinant DNA methodology. Thus, the term antibody as used herein refers to an antibody produced by modification of the whole antibody, or synthesized de novo using recombinant DNA methodology (eg, single chain Fv), or a phage display library. (See, eg, McCafferty, et al. (1990) Nature (“Nature”) 348: 552-554)) antibody fragments.

Many techniques are known for preparing antibodies, such as recombinant, monoclonal, or polyclonal antibodies. For example, Kohler and Milstein (1975) Nature 256: 495-497; Kozbor, et al. (1983) Immunology Today 4:72; Cole, et al. (1985) pp. 77-96 in Reisfeld and Sell (1985) Monoclonal Antibodies and Cancer Therapy Liss; Coligan (1991) Current Protocols in Immunology Lippincott; Harlow and Lane (1988) Antibodies : A Laboratory Manual (see “Antibodies: Experimental Guide”) CSH Press; and Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) See Academic Press. Single chain antibody production techniques (US Pat. No. 4,946,778) can be adapted to produce antibodies to the polypeptides of the invention. Other organisms such as transgenic mice and other mammals can also be used to express humanized antibodies. On the other hand, phage display technology can be used to identify antibodies, and in particular heteromeric Fab fragments that bind to selected antigens. See, for example, McCafferty, et al. (1990) Nature 348: 552-554; Marks, et al. (1992) Biotechnology 10: 779-783.

  “Chimeric antibody” refers to an antibody molecule as follows: (a) a constant region, or a portion thereof, a constant region of a class having a different or altered binding portion (variable region) to an antigen; And / or species, or modified to link to entirely different molecules that confer new properties to the chimeric antibody, such as enzymes, toxins, hormones, growth factors, drugs, effector functions, chemoattractants, immune modulators, etc. Or (b) a variable region, or a portion thereof, is changed, replaced, or replaced with a variable region having different or altered antigenic properties.

Identification of cancer-related sequences
In one form, the expression level of the gene is measured in a separate patient sample in need of diagnostic information to provide an expression profile. The expression profile of a particular sample is essentially a “fingerprint” of the state of that sample; even if two states have similar expression of a particular gene, by evaluating multiple genes, At the same time, it is possible to create a gene expression profile that is characteristic of the state of the cell. That is, normal cells can be distinguished from cancer or metastatic cancer cells, or cancer tissue or metastatic cancer tissue can be compared to the tissue of a surviving cancer patient. By comparing tissue expression profiles in various known cancer conditions, information is gained regarding which genes are important for each of these conditions, including both up- and down-regulated genes. Molecular profiling may identify currently sub-types of diseases that are currently collectively designated, such as different forms of cancer.

  Identification of sequences that are differentially expressed in cancer versus non-cancer tissues allows the information obtained thereby to be used in a number of ways. For example, a particular treatment plan is evaluated for cancer effects of down-regulation of chemotherapeutic drugs in a particular patient and the resulting tumor growth or recurrence. On the other hand, certain treatment steps may trigger other markers that are used as targets to destroy tumor cells. Similarly, the results of diagnosis and treatment can be confirmed by comparing patient samples with known expression profiles. Malignant diseases can also be compared to non-malignant conditions. Metastatic tissue can also be assayed to determine the stage of cancer in the tissue, or the origin of the primary tumor, eg, metastasis from a remote primary site. In addition, these gene expression profiles (or individual genes) allow screening of candidate substances for the purpose of mimicking or altering specific expression profiles; for example, screening for drugs that suppress cancer expression profiles Can do. This is done by making a biochip containing the important set of oncogenes used in these screens. These methods can also be achieved on a protein basis; that is, protein expression levels of oncoproteins can be assessed for diagnosis or for screening of candidate substances. In addition, cancer nucleic acid sequences can be administered for gene therapy purposes, including administration of antisense nucleic acids, or administration of cancer proteins (antibodies and other modulators) as therapeutic agents.

  Thus, the present invention provides nucleic acid and protein sequences that are differentially expressed in normal tissue and / or cancer against non-malignant diseases, or other types of related diseases referred to herein as “cancer sequences”. As summarized below, cancer sequences include cancer up-regulated (eg, expressed at high levels) sequences in addition to down-regulated (eg, expressed at low levels) sequences. In a preferred embodiment, the cancer sequence is human; however, cancer sequences from other organisms are useful in animal model disease and drug evaluation; where mammals, rodents (rats, mice) Other cancer sequences are provided from vertebrates, including primates, hamsters, guinea pigs, etc.), primates, livestock (including sheep, goats, pigs, cows, horses, etc.), and pets (eg, dogs, cats, etc.). Cancer sequences from other organisms are obtained by the methods outlined below.

  Cancer sequences include both nucleic acid and amino acid sequences. In a preferred embodiment, the skin cancer sequence is a recombinant nucleic acid. These nucleic acid sequences can be used in a variety of applications, including screening applications as well as diagnostic applications to detect natural nucleic acids; for example, PCR microtiter plates with selected probes for nucleic acid probes or cancer sequences. Useful with biochips.

  Cancer sequences can be initially identified by substantial nucleic acid and / or amino acid sequences that are homologous to the cancer sequences summarized herein. Such homology is based on the overall nucleic acid or amino acid sequence and is generally measured using, for example, homology programs or hybridization conditions, as summarized below.

  For the identification of sequences associated with cancer, cancer screens can include, for example, normal and cancerous tissues, cancer and non-malignant conditions, non-malignant and normal tissues, or tumor tissues from patients with metastatic disease Includes comparison of genes identified in non-metastatic tissues, usually different tissues, to the sample. Other suitable tissue comparisons include comparing cancer samples with metastatic cancer samples of other cancers such as lung, stomach, gastrointestinal cancer. Samples at different stages of cancer, such as survivor tissue, drug resistance status and tissue experiencing metastasis, are applied to a biochip containing nucleic acid probes. Samples are first microdissected if appropriate and processed for mRNA preparation. Suitable biochips are commercially available from, for example, Affymetrix of Santa Clara, California. The gene expression profile described herein is generated and the data is assayed.

  In one embodiment, the gene showing different expression in a normal state and a disease state includes lung, heart, brain, liver, stomach, kidney, muscle, colon, small intestine, large intestine, pancreas, bone, and / or placenta. Compared to genes expressed in other normal tissues, including but not limited to. In a preferred embodiment, genes identified in cancer screens that are expressed in significant amounts in other tissues (eg vital organs) are deleted from the profile, although in other embodiments this is not necessary (eg , Where the organs specific to women or men are not important). That is, when screening for drugs, it is usually desirable that the target expression be disease specific in order to minimize side effects that may occur in other organs.

  In a preferred embodiment, the cancer sequence is an up-regulated sequence of cancer; that is, the expression of these genes is higher in cancerous tissue than in non-cancerous or non-malignant tissue. “Up-regulation” as often used herein means at least a 2-fold change, preferably about a 3-fold change, and at least about 5-fold or more is desired. Other embodiments show sequences that are up-regulated in a non-malignant state relative to a normal state. It is also desired that the sample group is uniform.

The Unigene cluster identification number and registration number in this specification are for the GenBank sequence database, and the sequence of this registration number is specifically incorporated herein. See, for example, Benson, et al. (1998) Nuc. Acids Res. 26: 1-7; and http://www.ncbi.nlm.nih.gov/. Sequences can also be obtained from other databases, such as the European Institute for Molecular Biology (EMBL) or the Japan DNA Data Bank (DDBJ). In some situations, sequences are obtained from a collection of available sequences or predicted from genomic DNA using an exon prediction algorithm such as FGENESH. See Salamov and Solovyev (2000) Genome Res. 10: 516-522. In other situations, the sequence is obtained by cloning and sequencing the isolated nucleic acid.

  In other preferred embodiments, the sequence of the cancer is down-regulated in cancer; that is, the expression of these genes is lower in cancerous tissue than in non-cancerous tissue. “Down-regulation” as often used herein means a change of at least about 2-fold, preferably about 3-fold change, and at least about 5-fold or more is desired.

Informatics The ability to identify genes that are over- or under-expressed in cancers can be used in diagnostics, therapeutics, drug development, pharmacogenetics, protein structure, biosensor development, and other related fields. A database with high resolution and high sensitivity can be further provided. For example, the expression profile can be used for diagnosis or prognostic assessment of patients with cancer or related diseases. See Tables 1 and 3. Or, as another example, intracellular toxicological information provides a more direct correlation between drug composition and activity (an article published at the IBC Proteomics Conference in Coronado, California, Anderson (June 11- 12, 1998) Pharmaceutical Proteomics: Targets, Mechanism, and Function (see “ Pharmaceutical Proteomics: Targets, Mechanisms and Functions ”). Use intracellular toxicological information in biological sensor devices to predict potential toxicological effects of chemical exposures and exposure thresholds that are likely to be acceptable (see US Pat. No. 5,811,231) You can also Similar benefits can also be obtained from data sets associated with other biomolecules and bioactive agents (eg, nucleic acids, sugars, lipids, drugs, and the like).

  Thus, in another aspect, the present invention provides a database comprising at least one set of assay data. The data contained in the database was obtained, for example, using sequence assays alone or in library format. The database may be in any form that can maintain and transmit data, but is preferably an electronic database. The electronic database of the present invention can be maintained by any electronic device that can store and access the database, such as a personal computer, but is preferably distributed over a wide area network such as the World Wide Web.

  The focus of the current part of the database, including peptide sequence data, is for clarity of illustration only. A similar database of assay data obtained using the assay of the present invention can be constructed.

  Identify and / or survey the relative and / or absolute presence of various molecular species and macromolecular species from biological samples indicative of cancer, such as, for example, identification of sequences associated with cancer as described herein. Compositions and methods to do include correlation with pathological conditions, disease predisposition, drug testing, therapeutic monitoring, binding causing genetic disease, and identification of those that correlate with immune and physiological conditions, among others Provide a wealth of information. Data obtained from the assay of the present invention is amenable to manual testing and manual assays, but in a preferred embodiment, data processing using a high speed computer is used.

  A series of methods for indexing and searching biomolecule information can be used. For example, U.S. Pat.Nos. 6,023,659 and 5,966,712 provide a correlated database system that stores biomolecular sequence information so that sequences can be cataloged and searched based on the functional hierarchy of one or more proteins. Disclosure. US Pat. No. 5,953,727 catalogs and searches a collection of incomplete length DNA sequences based on association with one or more sequence plans to obtain a full length sequence from a collection of incomplete length sequences. Disclosed is a correlated database of sequence records containing information in a format that allows it. US Pat. No. 5,706,498 discloses a gene database search system that searches for gene sequences that are similar to sequence data items in the gene database based on the degree of similarity between the main sequence and the target sequence. US Pat. No. 5,538,897 is a fragment of a peptide mass spectrum for identifying amino acid sequences in a computer database by comparing the mass spectrum predicted using approximate fitting to the experimentally obtained mass spectrum. A method using a pattern is disclosed. U.S. Pat.No. 5,926,818 includes the functionality of a multidimensional data assay described as online assay processing (OLAP), with multiple mergers of forecast data and actual data based on one or more merge methods or scales. A dimensional database is disclosed. US Pat. No. 5,295,261 discloses a hybrid database structure in which the recording area of each database is divided into two classes, navigation data and information data, where the navigation area is a tree structure or more than two. Stored in a hierarchical topological map, which can be viewed as a merge of the tree structures.

In addition: Baxevanis, et al. (2001) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins. Wiley; Mount (2001) Bioinformatics: Sequence and Genome Analysis ( Bioinformatics : Sequences and Genome Assays) CSH Press, NY; Durbin, et al. (Eds. 1999) Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids And Probabilistic Models of Nucleic Acids ”) Cambridge University Press; Baxevanis and Oeullette (eds. 1998) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins (2d. Ed.) Wiley-Liss;

Rashidi and Buehler (1999) Bioinformatics: Basic Applications in Biological Science and Medicine CRC Press; Setubal, et al. (Eds. 1997) Introduction to Computational Molecular Biology ( “Introduction to Molecular Biology Using Computers”) Brooks / Cole; Misener and Krawetz (eds. 2000) Bioinformatics: Methods and Protocols (Humana Press; Higgins and Taylor (eds. 2000) Bioinformatics: Sequence, Structure, and Databanks: A Practical Approach (Oxford University Press); Brown (2001) Bioinformatics: A Biologist's Guide to Biocomputing and the Internet ( Bioinformatics : A guide to biocomputing and the Internet for biologists) Eaton Pub .; Han and Kamber (2000 ) Data Mining: Concepts and Techniques Kaufmann Pub .; and Waterman (1995) Introduction to Computational Biology: Maps, Sequences, and Genomes ("Introduction to Molecular Biology Using Computers: Maps, See Sequence and Genome)) Chap and Hall.

  The present invention includes computers and software for storing cross tabulated assay data records in a computer searchable form, such as data identifying samples containing targets from which each sequence specificity record is obtained, for example. Provide a computer database.

  In an exemplary embodiment, the sample containing at least one target is from a control tissue sample known to be free of pathological disease. In some variations, the at least one source is a known pathological tissue specimen, such as, for example, other tissue specimens that have been assayed for neoplastic lesions or cancer. In other variations, the assay record includes the following parameters for each target species in the sample: (1) Unique identification code, eg, target molecular structure and / or characteristic separation coordinates (eg, electrophoretic coordinates); (2) Sample source; and (3) Cross tabulate one or more of the absolute and / or relative amounts of the target species in the sample.

  The present invention also includes magnetic disks, optical disks, magneto-optical disks, DRAM, SRAM, SGRAM, SDRAM, RDRAM, DDR RAM, magnetic bubble storage devices, and other data storage devices including CPU registers, CPU data storage arrays. Providing storage and retrieval of target data accumulated in a computer data storage device; Typically, the target data record is charged as a bit pattern in a magnetic domain array on a magnetizable medium or as an array of cells in a DRAM device (eg, each cell includes a transistor or charge storage area on the transistor). Stored as an array of states or transistor gate states. In one embodiment, the present invention provides a bit encoding a protein expression fingerprint record, including a unique identifier of such a storage device and at least 10 target data records created therein, cross-tabulated into a target source. A computer system including a pattern is provided.

  When the target is a peptide or nucleic acid, the present invention is preferably computerized between an assay record of the peptide or nucleic acid sequence stored in or retrieved from a computer storage device or database and at least one other sequence. A method of identifying a sequence of related peptides or nucleic acids, comprising performing a given comparison. The comparison includes sequence assays, comparison algorithms, or computer programmatic aspects thereof (eg, FASTA, TFASTA, GAP, BESTFIT) and / or the comparison is in a sequence pool measured from a sample polypeptide or nucleic acid sample. Comparison of relative amounts of peptide or nucleic acid sequences.

  The present invention further preferably includes an IBM-ready (DOS) that includes bit pattern encoding data from the assay of the present invention in a file format suitable for search and processing in computerized sequence assays, comparisons, or relative metrics. , Windows, Windows95 / 98/2000, Windows NT, OS / 2) magnetic disk, floppy disk in other formats (eg Linux, SunOS, Solaris, AIX, SCO Unix, VMS, MV, Macintosh, etc.) or hard ( Provides fixed, Winchester disk drives.

  The present invention further provides a network including a plurality of computing devices linked by a data link such as an Ethernet cable (coax or 10BaseT), telephone line, ISDN line, wireless network, optical fiber, or other suitable signal transmission medium. Wherein at least one network device (eg, computer, disk array, etc.) includes a pattern of magnetic domains (eg, magnetic disks) and / or charge domains (eg, arrays of DRAM cells) are assayed according to the present invention; The bit pattern encoding data obtained from

  The present invention further provides a method for transmitting assay data; this includes generation of electronic signals of electronic communication devices such as modems, ISDN terminal adapters, DSL, cable modems, ATM switches, etc. Or in an encrypted format) including bit pattern encoding data from an assessment or database that includes multiple assay results obtained by the method of the invention.

  In a preferred embodiment, the present invention includes a query target and an array of data structures, such as the ranking of database targets based on the assay results obtained by the method of the present invention, the degree of identity to target data and gap weights. A computer system that compares databases. The central processing unit is preferably initialized to capture and execute a computer program for sequencing and / or comparing assay results. Query target data is input to the central processing unit by the I / O device. Running the computer program allows the central processing unit to retrieve assay data from a data file that contains a binary description of the assay results.

  Target data or records and computer programs are transferred to auxiliary memory, which is usually random access memory (eg, DRAM, SRAM, SGRAM, or SDRAM). Targets are ranked based on the degree of correspondence between the characteristics of the selected assay (eg, binding to the selected affinity moiety) and the same characteristics of the query target, and the results are determined by the I / O device. Is output. For example, the central processing unit is a conventional computer (eg, Intel Pentium, PowerPC, Alpha, PA-8000, SPARC, MIPS 4400, MIPS 10000, VAX, etc.); the program is a molecular biology software package in a commercial or public domain ( For example, UWGCG Sequence Analysis Software, Darwin); data files are optical disks, magnetic disks, data servers, storage devices (eg; DRAM, SRAM, SGRAM, SDRAM, EPROM, bubble memory, flash memory, etc.); I / The O device can be a terminal consisting of a video display, keyboard, modem, ISDN terminal adapter, Ethernet port, punch card reader, magnetic strip reader, or other suitable I / O device.

The present invention also preferably includes: (1) a computer; (2) a stored bit pattern stored in a computer that encodes a collection of records of peptide sequence specificity obtained by the method of the present invention; A comparison target, such as a query target; and (4) the use of a computer system as described above comprising a sequence and a comparison program, with ranking of comparison results based on normal computer similarity values. For example, see Ewens and Grant (2001) Statistical Methods in Bioinformatics: An Introduction (Springer-Verlag). A mathematical approach can also be used to conclude whether the similarity or difference in gene expression exhibited by the various samples is significant. For example, Golub, et al. (1999) Science 286: 531-537; Duda, et al. (2001) Pattern Classification Wiley; and Hastie, et al. (2001) See The Elements of Statistical Learning: Data Mining, Inference, and Prediction (Springer-Verlag). To determine whether a sample is more similar or has the greatest similarity to a given state between the sample and one or more pools representing different states for comparison One approach; and the pool with the smallest vector angle is chosen as the most similar to the biological sample between the compared pools.

Characteristics of cancer-related proteins The cancer proteins of the present invention are classified as secreted proteins, transmembrane proteins, or intracellular proteins. In one embodiment, the oncoprotein is an intracellular protein. Intracellular proteins are found in the cytoplasm and / or nucleus. Intracellular proteins are involved in all aspects of cell function and replication, including signal pathways; abnormal expression of these proteins often results in disordered or unrestricted cellular processing (Alberts, et al. (Eds. 1994) Molecular Biology of the Cell (see 3d ed.) Garland). For example, many intracellular proteins have enzyme activities such as protein kinase activity, protein phosphotase activity, protease activity, nucleotide cyclase activity, and polymerase activity. Intracellular proteins also function as docking proteins that are involved in organizing protein complexes, or targeting proteins to various intracellular localizations, and in maintaining the structural integrity of organelles.

An increasingly appreciated concept in protein characterization is the presence in the protein of one or more structural motifs that are attributed to a given function. In addition to being found in protein enzyme domains, highly conserved sequences have been identified in proteins involved in protein-protein interactions. For example, the Src-homology 2 region (SH2) domain binds to a phosphorylated tyrosine target in a sequence-dependent manner. SH2 domains and other PTB domains also bind to phosphorylated tyrosine targets. The SH3 domain binds proline-rich targets. In addition, to name a few, it has been found that PH domains, tetratricopeptide repeat domains, WD domains, etc. mediate protein-protein interactions. Some of these may also bind to phospholipids and other second messengers. These motifs are identified based on the amino acid sequence; therefore, assaying the sequences of these proteins provides insights into both the enzyme potential of the molecule and / or the molecule to which the protein binds. Pfam, a large collection of hidden Markov models that cover multiple sequence alignments and many common protein domains, is one useful database. Translations are available on the Internet from Washington University in St. Louis, the Sanger Center and the Karolinska Institute in Sweden. For example, Bateman, et al. (2000) Nuc. Acids Res. 28: 263-266, Sonnhammer, et al. (1997) Proteins 28: 405-420; Bateman, et al. (1999) Nuc Acids Res. 27: 260-262, and Sonnhammer, et al. (1998) Nuc. Acids Res. 26: 320-322.

  In other embodiments, the cancer sequence is a transmembrane protein. A transmembrane protein is a molecule that complements the phospholipid bilayer of a cell and has an intramolecular domain, an extramolecular domain, or both. Such intramolecular domains of proteins have many functions, including those already described for intramolecular proteins. For example, the intramolecular domain has enzymatic activity and / or functions as a binding site for additional proteins. Often the intracellular domain of a transmembrane protein plays both roles. For example, certain receptors, tyrosine kinases, have both protein kinase activity and SH2 domains. In addition, tyrosine autophosphorylation on the receptor molecule itself creates additional SH2 domain binding sites, including proteins.

  A transmembrane protein contains one to many transmembrane domains. For example, receptor tyrosine kinases, certain cytokine receptors, receptor guanylyl cyclase, and receptor serine-threonine protein kinases contain a single transmembrane domain. However, many other proteins, including channels and adenylyl cyclase, contain numerous transmembrane domains. Many cell surface critical receptors, such as G protein-coupled receptors (GPCRs), are referred to as “seven transmembrane domains” proteins because they contain seven transmembrane domains. The properties of the transmembrane domain include about 17 consecutive hydrophobic amino acids followed by charged amino acids. Thus, when assaying the amino acid sequence of a particular protein, the localization and number of transmembrane domains within the protein can be predicted (see, for example, the PSORT website http://psort.nibb.ac.jp/) . Important transmembrane protein receptors are insulin receptor, insulin-like growth factor receptor, human growth hormone receptor, glucose transporter, transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor, epidermal growth factor Receptors, leptin receptors, including but not limited to interleukin receptors such as IL-1 receptor, IL-2 receptor.

  The extracellular domains of transmembrane proteins are diverse; however, conserved motifs are repeatedly found between various extracellular domains. Conserved structures and / or functions are considered to be different extracellular motifs. Many extracellular domains are involved in binding to other molecules. In one form, the extracellular domain is found on the receptor. Factors that bind the receptor domain include circulating ligands, which are small molecules such as peptides, proteins or adenosine. For example, growth factors such as EGF, FGF, and PDGF are circulating growth factors that bind to cognate receptors and trigger various cellular responses. In addition, cytokines, mitogenic factors, neurotrophic factors and the like are included. The extracellular domain also binds to intracellular molecules. Thus, the extracellular domain mediates cell-cell interactions. The cell-related ligand is linked to the cell by a glycosylphosphatidylinositol (GPI) anchor, or the ligand itself is a transmembrane protein. The extracellular domain is also associated with the extracellular matrix and contributes to the maintenance of the cell structure.

  Transmembrane oncoproteins are particularly preferred in the present invention because they are readily available for immunotherapy as described herein. In addition, as summarized below, transmembrane proteins are also useful in diagnostic imaging methods. Antibodies are used to label these readily available proteins. On the other hand, antibodies can also label intracellular proteins. In this case, the sample is usually permeable to provide access to the protein in the cell. In addition, some membrane proteins are processed to release soluble proteins or to expose residual fragments. The released soluble protein is a useful diagnostic marker and the processed residual protein fragment is a useful lung disease marker.

  For example, it has also been evaluated that transmembrane protein solubility can be created by removing transmembrane sequences by recombinant methods. Furthermore, by adding an appropriate signal sequence by a recombinant method, a soluble transmembrane protein can be secreted.

  In other embodiments, the oncoprotein is a secreted protein; its secretion is constitutive or regulated. These proteins have a signal peptide or signal sequence that directs the molecule into the secretory pathway. Secreted proteins are involved in many physiological events; for example when circulated, they often serve to signal various other types of cells. Secreted proteins can be autocrine (acts on cells that secrete the factor), paracrine (acts on cells in close proximity to the cell that secreted the factor), endocrine (eg, secretion into the bloodstream) Act on cells at the time of disease) or in an exocrine manner (eg secretion through the duct or adjacent epithelial surface, such as sweat glands, sebaceous glands, pancreatic ducts, lacrimal glands, mammary glands, ear glands, etc.). Thus, secreted molecules are often used to regulate or alter many aspects of physiology. Oncoproteins that are secreted proteins are particularly preferred in the present invention because they function as good targets for diagnostic markers, for example in blood, plasma, serum or stool tests. What is an enzyme (oncoprotein) is a target for antibodies or small molecules. Others are useful as vaccine targets, for example by the CTL mechanism.

Use of Cancer Nucleic Acids As noted above, cancer sequences are initially identified by the amino acid sequences that are homologous or related to the essential nucleic acids and / or the cancer formulations outlined herein. Such homology is based on the overall nucleic acid or amino acid sequence and is usually measured using homology programs or hybridization conditions, as summarized below. Usually, the combination bound on the mRNA is found on the same molecule.

As detailed elsewhere, the identity ratio is measured using an algorithm such as BLAST. The preferred method uses the WU-BLAST-2 BLASTN module set to default parameters with overlap ranges and overlap ratios of 1 and 0.125 respectively. Alignment involves the introduction of aligned sequence gaps. Further, for sequences containing more or fewer nucleotides than the nucleic acid described, the percentage homology is determined based on the number of homologous nucleotides relative to the total number of nucleotides. Thus, for example, the homology of a shorter sequence than the identified sequence is measured using the number of nucleotides in the shorter sequence.
In one embodiment, hybridization studies measure nucleic acid homologues. Thus, for example, a nucleic acid that is hybridized with the nucleic acid, or its complement, or even found on native mRNA under highly stringent conditions is considered a cancer sequence. In other embodiments, hybridization conditions are less stringent, eg, moderate to low severity conditions are used; see Ausubel, supra, and Tijssen, supra.
The sequence of the cancer nucleic acid of the invention, such as the sequence of Table 68, can be a fragment of a large gene, for example a portion of the nucleic acid. “Gene” in this context includes coding regions, non-coding regions, and mixtures thereof. Thus, using the sequences provided herein, the sequence of an oncogene can be extended in either direction by well-known techniques of cloning long or full-length sequences; Ausubel, et al. , supra. Information science makes much possible. And many sequences can be collected to include multiple sequences corresponding to a single gene, such as a system such as UniGene (see http://www.ncbi.nlm.nih.gov/UniGene/) .

  Once a cancer nucleic acid has been identified, it can be cloned and, if necessary, its constituent parts can be recombined to form the complete cancer nucleic acid coding region, or the complete RNA sequence. Once isolated from a natural source, eg, contained in a plasmid or other vector, or excised there as a linear nucleic acid fragment, the recombinant cancer nucleic acid may contain other coding regions, such as extended coding regions. It is further used as a probe to identify and isolate cancer nucleic acids. It can also be used as a “precursor” nucleic acid to make modified or different cancer nucleic acids and proteins.

  The cancer nucleic acids of the present invention are used in several ways. In one embodiment, nucleic acid probes for cancer nucleic acids are made and combined with biochips and used as screens and diagnostic methods summarized below, or for example, gene therapy, vaccines, RNA interference (RNAi) and / or anti-antibody Administered for use in sense. On the other hand, a cancer nucleic acid containing a cancer protein coding region can also be introduced into an expression vector for expression of the cancer protein for screening purposes or for administration to a patient.

  In preferred embodiments, nucleic acid probes of cancer nucleic acids (both nucleic acid sequences summarized in the figure and / or their complements) are made. The nucleic acid probe bound to the biochip is a substantial complement of a cancer nucleic acid, such as a target sequence (such as a target sequence of a sample or other in a sandwich assay), such that target sequence hybridization or probe hybridization in the present invention occurs. One of the probe sequences). As summarized below, this need not be a perfect complement and there may be mismatched base pairs that prevent hybridization of the target sequence to the single stranded nucleic acid of the invention. However, if the number of mutations is so great that hybridization does not occur even under the mildest conditions, the sequence will not be the complement of the target sequence. Thus, “substantially complement” herein refers to a probe that is substantially the complement of the target sequence, and under normal reaction conditions, particularly a string as summarized herein. It means to hybridize under a gentle condition.

  A nucleic acid probe is usually single-stranded, but may have a single-stranded portion and a double-stranded portion. Probe heavy chain properties are measured by composition, composition, and target sequence characteristics. Usually, nucleic acid probes range in length from about 8 to 100 bases, preferably from about 10 to 80 bases, particularly preferably from about 30 to 50 bases. That is, the entire gene is not normally used. In some embodiments, fairly long nucleic acids up to 100 bases may be used.

  In a preferred embodiment, one or more probes are used for one sequence, such as overlapping probes or probes of different parts of the target used. That is, two, three, four or more, preferably three probes are used to create a particular target iteration. Probes may overlap (eg have some common sequences) or be separated. In some cases, PCR primers are used to amplify a more sensitive signal.

  Nucleic acids are bound or immobilized on the immobilization support by various methods. As used herein, “fix” and its grammatical synonyms indicate that the connection or binding between the nucleic acid probe and the immobilized support is stable under conditions such as binding, washing, assaying and removal as summarized. Means enough to do. The bond is usually covalent or non-covalent. "Noncovalent bond" and its grammatical synonyms mean herein one or more electrostatic, hydrophilic, and hydrophobic interactions. Non-covalent bonds include, for example, a covalent bond between a molecule such as streptavidin and a support, a non-covalent bond between a biotinin probe and streptavidin, and the like. “Covalent bond” and its grammatical synonyms means herein that two parts of a solid support and a probe are bound by at least one bond, including a sigma bond, a pi bond and a coordination bond. To do. Covalent bonds can be formed directly between the probe and the solid support, or can be formed by cross-linking agents or by including specific reactive groups on either the solid support or the probe, or both molecules. Sometimes. Immobilization also includes shared or non-shared interactions.

  Usually, the probe binds to the biochip in various ways. As described herein, nucleic acids are first synthesized and then bound to the biochip or may be synthesized directly on the biochip.

  The biochip contains a suitable solid substrate. As used herein, “substrate”, “solid support” or their grammatical synonyms means a substance that can be modified such that a nucleic acid probe binds or is linked and is responsive to at least one detection method. . Often the substrate contains distinct individual sites suitable for individual separation and identification. The number of potential substrates is very large, including glass and modified glass or functional glass, plastics (including acrylic, copolymers of materials such as polystyrene and styrene, polypropylene, polyethylene, polybutylene, polyurethane, Teflon J, etc.), Examples include, but are not limited to, polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, and the like. Usually the substrate is optically detectable and does not emit enough fluorescence to be detected. See WO 0055627.

  Usually the substrate is planar, but other shapes of substrates may be used. For example, a probe may be placed on the inner surface of a tube through which the sample of the assay flows to minimize sample volume. Similarly, the substrate may be flexible, such as a flexible foam including closed cell foam made of a special plastic.

  In a preferred embodiment, the biochip and probe surfaces are coated with chemical functional groups for subsequent binding of both. Thus, for example, biochips are coated with chemical functional groups that include, but are not limited to, amino groups, carboxyl groups, oxo groups, thiol groups, although amino groups are particularly desirable. These functional groups can be used to bind the probe to the functional group on the probe. For example, an amino group-containing nucleic acid can be bound to an amino group-containing surface using, for example, a cross-linking agent; for example, the same or different bifunctional cross-linking agents are well known (1994 Pierce Chemical Company). Catalog, crosslinker technical section, pages 155-200). Furthermore, in some cases, additional cross-linking agents such as alkyl groups (including substituents and heteroalkyl groups) may be used.

  In this embodiment, oligonucleotides are synthesized and attached to the solid support surface. Binding occurs by binding to the solid support at the 5 'or 3' end, or by binding to an internal nucleotide. In other embodiments, immobilization to a solid support may be very strong while being non-covalent. For example, a biotinylated oligonucleotide is made that covalently binds to a surface coated with streptavidin and binding occurs.

  On the other hand, oligonucleotides may be synthesized on the surface. For example, photopolymerization compounds and photoactivation techniques using photopolymerization techniques are used. In preferred embodiments, nucleic acids can be synthesized intracellularly using well-known photolithography techniques. These photolithography techniques are described in WO 95/25116; WO 95/35505; US Pat. Nos. 5,700,637 and 5,445,934; and their references, all incorporated as is apparent. These coupling methods create a substrate for Affymetrix's GeneChip technology.

Often, amplification-based assays are performed to measure the expression level of cancer-related sequences. These assays are usually performed in conjunction with reverse transcription. In such assays, cancer-associated nucleic acid sequences serve as templates for amplification reactions (eg, polymerase chain reaction or PCR). In quantitative amplification, the amount of amplification product is proportional to the amount of template in the original sample. Comparison with an appropriate control group provides a measure of the amount of cancer-related RNA. Methods for quantitative amplification are well known. Detailed protocols for quantitative PCR are provided, for example, in Academic Press, Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications .

  In some embodiments, TaqMan-based assays are used to measure expression. TaqMan-based assays use a fluorogenic oligonucleotide probe containing a 5 ′ fluorescent dye and a 3 ′ quenching agent. The probe hybridizes with the PCR product but cannot extend itself due to the blocking agent at the 3 'end. When the PCR product is amplified in subsequent cycles, the TaqMan probe is disrupted by the nuclease activity of a 5 'polymerase, such as AmpliTaq. This disruption separates the 5 'fluorescent dye and the 3' quenching agent, thereby increasing the fluorescence emission as a function of amplification (see eg literature provided by Perkin-Elmer, eg www2.perkin-elmer.com) ).

Other suitable amplification methods include the following: ligase chain reaction (LCR) (Wu and Wallace (1989) Genomics ) 4: 560-569, Landegren, et al. (1988) Science 241: 1077-1080, and Barringer, et al. (1990) Gene 89: 117-122), transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86 : 1173-1177), self-sustaining sequence replication (Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874-1878), dot PCR, crosslinker adapter PCR, etc. It is not limited to.

Expression of oncoproteins from nucleic acids In a preferred embodiment, for example, cancer nucleic acids encoding oncoproteins are used to make various expression vectors that express oncoproteins for later use in screening assays, as described below. Is done. Expression vectors and recombinant DNA techniques for expressing proteins are well known (see Ausubel, supra, and Fernandez and Hoeffler (eds. 1999) Gene Expression Systems (Academic Press)). The expression vector is either a self-regenerating extrachromosomal vector or a vector that binds to the host genome. These expression vectors usually contain transcriptional and translational regulatory nucleic acids that are operably linked to the nucleic acid encoding the oncoprotein. The term “regulatory sequence” refers to a DNA sequence that is used to express a functionally linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter gene and optionally an operator sequence and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

  Nucleic acids are “operably linked” when they are placed into a functional relationship with another nucleic acid sequence. For example, a pre-linked or secretory-directed DNA is operably linked to the polypeptide's DNA if it is expressed as a preprotein that is involved in the secretion of the polypeptide; a promoter or enhancer is effective for transcription of the sequence. A ribosome binding site is operably linked to the coding sequence if it is in a position that facilitates translation. Usually, “functional linkage” means that DNA sequences are continuously linked, and in the case of secretion-induction, it is continuous and in the reading stage. However, enhancers do not have to be continuous. Ligation is usually accomplished by ligation at appropriate restriction sites. If such sites do not exist, synthesis of oligonucleotides or crosslinkers is used based on conventional methods. Transcriptional and translational regulatory nucleic acids are suitable for host cells normally used to express oncoproteins. Numerous suitable expression vectors and suitable regulatory sequences are known for a variety of host cells.

  In general, transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activation sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.

  A promoter sequence is either a constitutive or an inducible promoter. The promoter is also either a natural promoter or a hybrid promoter. Hybrid promoters that combine one or more promoter elements are also known and useful in the present invention.

The expression vector may contain other elements. For example, an expression vector may comprise two replication systems so that expression is maintained in two organisms, for example, in mammalian or insect cells, clones and amplification in prokaryotic hosts, etc. It becomes possible. Further, to integrate the expression vector, the expression vector often contains sequences homologous to the genome of at least one host cell, preferably two homologous sequences that are flanked by the expression construct. An integrated vector is directed to a specific location in the host cell by selecting appropriate homologous sequences for inclusion in the vector. The construction theory of integrated vectors is available. See Fernandez and Hoeffler, supra; and Kitamura, et al. (1995) Proc. Nat'l Acad. Sci. USA 92: 9146-9150.

  Further, in a preferred embodiment, the expression vector contains a selectable marker gene that allows for selection of transformed host cells. Selection genes are well known and will vary with the host cell used.

  The oncoprotein of the present invention is usually produced by culturing host cells transformed with an expression vector containing a nucleic acid encoding the oncoprotein under conditions suitable for inducing or inducing oncoprotein expression. Conditions suitable for the expression of the oncoprotein vary depending on the choice of expression vector and host and can be readily determined by routine experimentation or optimization. For example, using an inducible promoter requires growth conditions suitable for induction, but using a constitutive promoter in an expression vector requires optimization of host cell growth and proliferation. In addition, the timing of harvesting is important in some embodiments. For example, the baculovirus tissue used for expression in insect cells is a cytolytic virus, so selection of harvest time is important for product yield.

  Suitable cells for host cells include yeast, bacteria, protozoa, fungi, and animal cells including insect and mammalian cells. Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, HUVEC (umbilical vein endothelial cells), THP1 cells (macrophage nuclei), and other diverse human

  In a preferred embodiment, the oncoprotein is expressed in mammalian cells. Mammalian expression systems are used and include retroviral and adenoviral systems. One expression vector system is a retroviral vector system such as is usually described in PCT / US97 / 01019 and PCT / US97 / 01048. Because viral genes are often highly expressed and have a wide host range, promoters from mammalian gene cells are particularly used as mammalian promoters. Examples include SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and CMV promoter (see Fernandez and Hoeffler, supra). Usually, transcription termination and polyadenylation sequences found in mammalian cells are in the regulatory region located 3 'from the translation stop codon and thus flanked by the coding sequence together with the promoter element. Examples of transcription terminators and polyadenylation signals include those derived from SV40.

  There are methods for incorporating exogenous nucleic acid into mammalian hosts and other hosts, and the methods vary depending on the host cell used. Technologies such as dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of polynucleotides into liposomes, and direct microinjection of DNA into the nucleus included.

  In a preferred embodiment, the oncoprotein is expressed in a bacterial system. Promoters from bacteriophages are also used. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of trp and lac promoter sequences. In addition, bacterial promoters may include natural promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. In addition to a functioning promoter sequence, an effective ribosome binding site is desired. The expression vector also includes a signal peptide sequence that provides for secretion of the oncoprotein into the bacteria. Proteins are secreted into the growth medium (Gram positive bacteria) or within the periplasmic region (Gram negative bacteria) located between the inner and outer membranes of the cell. Bacterial expression vectors also contain a selectable marker gene that allows for selection of transformed bacterial strains. Suitable selection genes include genes that confer bacterial resistance to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes present in the histidine, tryptophan, and leucine biosynthetic pathways. An expression vector incorporating these components can be produced. Bacterial expression vectors are well known and include, among others, Bacillus subtilis, Escherichia coli, Streptococcus cremoris, and Streptomyces lividans (see Fernandez and Hoeffler, supra). Bacterial expression vectors are transformed into bacterial host cells using calcium chloride treatment, electroporation, or other techniques.

  In one embodiment, oncoproteins are produced in insect cells using, for example, expression vectors for transformation of insect cells, particularly baculovirus-based expression vectors.

  In a preferred embodiment, the oncoprotein is produced in yeast cells. Yeast expression systems are well known and include Saccharomyces cerevisiae, Candida albicans, C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis Expression of K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, Yarrowia lipolytica, etc. Is included.

  Cancer proteins can also be made as fusion proteins using existing technology. Thus, for example, for the production of monoclonal antibodies, when the desired epitope is small, the oncoprotein is fused with a carrier protein to form an immunogen. On the other hand, oncoproteins increase expression or are made as fusion proteins for other reasons. For example, when the oncoprotein is a cancer peptide, the nucleic acid encoding the peptide binds to other nucleic acids for expression. The fusion with the detection epitope tag can be made with, for example, FLAG, His6, myc, HA and the like.

In a preferred embodiment, the oncoprotein is purified or isolated after expression. Oncoproteins are separated or purified in a variety of ways depending on what other components are present in the sample and the requirements for the product to be purified, eg, the natural structure or denaturation. Standard purification methods include ammonium sulfate precipitation, electrophoretic, molecular, immunological, and ion exchange, hydrophobicity, affinity, reverse phase HPLC chromatography, chromatographic methods, and chromatofocusing. For example, a cancer protein can be purified using a standard anti-cancer protein antibody column. Combinations of ultrafiltration and filtration dialysis with protein concentration are also useful. For example, Walsh (2002) Proteins: Biochemistry and Biotechnology (Wiley; Hardin, et al. (Eds. 2001) Cloning, Gene Expression and Protein Purification . "Oxford Univ. Press (Oxford University Press); Wilson, et al. (Eds. 2000) Encyclopedia of Separation Science (" Encyclopedia of Separation Science ") Academic Press; and Scopes (1993) Protein Purification ]) See Springer-Verlag. The degree of purification required will depend on the use of the oncoprotein. In some embodiments, no purification is required.

Once expressed and purified as needed, oncoproteins and nucleic acids are useful in many applications. They are used as immunoselective agents, vaccine agents, screening agents, for therapeutic entities, for the production of antibodies, and as transcription or translation inhibitors.
Oncoprotein variants One embodiment of an oncoprotein also includes amino acid variants of naturally occurring sequences, as measured herein. Preferably, the variant is about 75% or more homologous to the wild type sequence, more preferably about 80%, more preferably about 85% or more, and even more preferably 90% or more. In some embodiments, homology is as high as about 93-95% or 98%. With respect to nucleic acids, homology in this context is similarity or identity, preferably meaning sequence identity. Similar to that described above for nucleic acid homology, this homology can also be measured by standard techniques.

  The oncoprotein of the present invention may be shorter or longer than the wild-type amino acid sequence. Thus, in a preferred embodiment, parts or fragments of the wild type sequence are also included within the definition of oncoprotein. In addition, as summarized above, the cancer nucleic acids of the invention may be used to obtain additional coding regions and thus additional protein sequences.

  In one embodiment, the oncoprotein is a derivative or variant of the oncoprotein as compared to the wild type sequence. That is, as outlined in more detail below, cancer peptide variants often contain at least one amino acid substitution, deletion, or insertion for a particularly desired amino acid substitution. Amino acid substitutions, insertions, or deletions occur at many residue positions within the cancer peptide.

  Furthermore, one embodiment of the oncoprotein of the present invention includes amino acid sequence variants. These variants usually fall into one or more of the following three categories: substitution, insertion, or deletion variants. These variants are usually made by site-directed mutagenesis of nucleotides in the DNA encoding the oncoprotein and using the cassette method or PCR mutagenesis or other methods to make the DNA encoding the variant Thereafter, the DNA is expressed in recombinant cell culture as described above. However, mutant oncoprotein fragments having about 100-150 residues may be produced by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by a predetermined nature of the variant, the property of distinguishing them from natural alleles, or an interspecies variant of an oncoprotein amino acid sequence. Variants usually show qualitative biological activity similar to natural analogues, but variants with altered properties may be chosen.

  Although the position or range for introducing amino acid sequence variants is often predetermined, the substantial variation need not be predetermined. For example, to optimize the performance of the transformation at a given site, any mutagenesis performed and expressed in the target codon or region is screened for the optimal combination of desired activities. The Techniques for causing substitution mutations at a predetermined site of DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Variant screening is often performed using an oncoprotein activity assay.

  Amino acid substitutions are usually 1 residue, and insertions are usually about 1 to 20 amino acids, although fairly large ones are acceptable. Deletions are usually about 1-20 residues, and in some cases can be quite large.

  Substitutions, deletions, insertions or combinations are used to arrive at the final derivative. These changes usually occur in a small number of amino acids to minimize molecular changes. However, large changes are allowed under certain circumstances. If small changes in oncoprotein characteristics are desired, substitutions are made based on the amino acid substitution relationships described.

  Variants usually show essentially the same qualitative biological activity and stimulate the same immune response as natural analogues, but variants are also selected to modify oncoprotein properties as needed Is done. On the other hand, the variant may be designed to alter the biological activity of the oncoprotein. For example, sugar chain formation sites may be added, changed, or removed.

  Selecting substitutions that are less conservative than those described above can cause significant changes in function and immunological identification. For example, substitutions may be made that have a more pronounced effect on: the structure of the polypeptide backbone at the altered site, eg, the alpha helix or beta sheet structure; the charge and hydrophobicity of the molecule at the target site; Or most of the side chain. For substitutions that are thought to have the greatest effect on the properties of the polypeptide, (a) a hydrophilic residue such as serine or threonine is replaced with a hydrophobic residue such as leucine, isoleucine, phenylalanine, valine, alanine (by (B) a cysteine or proline is replaced with (by) other residues; (c) a residue having an electropositive side chain such as, for example, lysine, arginine, or histidine is, for example, glutamic acid Or substituted with an electronegative residue such as aspartic acid; (d) a residue with a large side chain, such as phenylalanine, is replaced with (by) a non-side chain such as glycine, for example. (E) a proline residue is incorporated or substituted to change the rotational freedom of the peptidyl bond.

  Variants usually show similar qualitative biological activity and promote the same immune response as natural analogues, but variants are also selected to modify the properties of skin cancer proteins as needed. On the other hand, the variant may be designed to alter the biological activity of the oncoprotein. For example, the sugar chain formation site may be changed or removed.

  Covalent modifications of cancer polypeptides are included within the scope of the present invention. One type of covalent modification is an organic inducer capable of reacting with a target amino acid residue of a cancer polypeptide and a selected side chain or N- or C-terminal residue of the cancer polypeptide. Reaction with. Derivatization with a bivalent drug is, for example, as described in more detail below, a cancer polypeptide and a water-insoluble supporting substance or surface used in purification methods, screening assays, etc. of anti-cancer polypeptide antibodies. Useful in cross-linking. Commonly used crosslinking agents are, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, 3,3′-dithiobis Identical divalent imide esters, including disuccinimidyl esters such as (succinimidyl propionate), divalent maleimides such as N-maleimide-1,8-octane and methyl-3-((p-azido Includes drugs such as phenyl) dithio) propioimidate.

Other modifications include deamination of glutaminyl and asparaginyl residues to the corresponding glutamyl and asparatyl residues, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, threonyl, tyrosyl residues, lysine, Methylation of amino groups on arginine and histidine side chains (eg, pages 79-86, Creighton (1992) Proteins: Structure and Molecular Properties Freeman), acetylation of N-terminal amines And amidation of the C-terminal carboxyl group.

  Covalent modifications of other types of cancer polypeptides within the scope of the claimed invention include alteration of the native glycosylation pattern of the polypeptide. As used herein, “changing the native glycosylation pattern” refers to removing one or more carbohydrate moieties found in the native sequence of the cancer polypeptide and / or in the native sequence of the cancer polypeptide. It is intended to mean adding one or more non-existing sugar chain formers. Glycosylation patterns can be altered in many ways. Different cell types that express cancer-related sequences result in different glycosylation patterns.

  The addition of a glycosylation site to a cancer polypeptide is also made by changing its amino acid sequence. This change is made, for example, by addition or deletion of one or more serine or threonine residues to the native cancer polypeptide sequence (due to O-linked glycosylation). The cancer amino acid sequence is optionally altered by changes at the DNA level, particularly variations in preselected bases in which codons have been created to translate the DNA encoding the cancer polypeptide into the desired amino acid.

Another way to increase the number of carbohydrate moieties on the cancer polypeptide is to attach glycosides chemically or enzymatically to the polypeptide. For example, WO 87/05330; pages 259-306, Aplin and Wriston (1981) CRC Crit. Rev. Biochem .

Removal of the carbohydrate moiety present on the cancer peptide is accomplished by chemical, enzymatic, or mutational substitution of codons encoding amino acid residues that function as glycosylation targets. Chemical deglycosylation methods can be applied. See, for example, Sojar and Bahl (1987) Arch. Biochem. Biophys. 259: 52-57 and Edge, et al. (1981) Anal. Biochem. 118: 131-137. The carbohydrate portion of the polypeptide can be enzymatically cleaved through the use of various endo-type or exo-type glycosidases. See, for example, Thotakura, et al. (1987) Meth. Enzymol. 138: 350-359.

  Other types of covalent modification of cancer are described in US Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. It includes a linkage between the polypeptide and one of a variety of non-proteinaceous polymers such as, for example, polyethylene glycol, polypropylene glycol, or polyoxyalkylene.

  The cancer polypeptides of the present invention can also be modified in a method to form a chimeric molecule comprising a cancer polypeptide fused to another heterologous polypeptide or amino acid sequence. In one embodiment, the chimeric molecule comprises a fusion of a cancer polypeptide and a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is usually located at the amino terminus or carboxyl terminus of the cancer polypeptide. The presence of such epitope-tagged forms of cancer polypeptides can be detected using an antibody against the tag polypeptide. In addition, the supply of the epitope tag allows easy purification of the cancer polypeptide by affinity purification using an anti-tag antibody or other type of affinity substrate that binds to the epitope tag. In other embodiments, the chimeric molecule comprises a fusion of a cancer polypeptide with an immunoglobulin or a specific region of an immunoglobulin. For a bivalent chimeric molecule, this fusion is to the Fc region of an IgG molecule.

A variety of tag polypeptides and their other antibodies are available. Examples include the following: poly-his or poly-his-gly tags; HIS6 and metal chelation tags, influenza HA tag polypeptides and their antibodies 12CA5 (Field, et al. (1988) Mol. Cell. Biol. 8: 2159-2165); c-myc tag and its 8F9, 3C7, 6E10, G4, B7, and 9E10 antibodies (Evan, et al. (1985) Molecular and Cellular Biology Cell biology ”) 5: 3610-3616); Herpes simplex virus glycoprotein D (gD) tag and its antibody (Paborsky, et al. (1990) Protein Engineering 3 (6): 547-553 ). Other tag polypeptides include: Flag peptide (Hopp, et al. (1988) BioTechnology 6: 1204-1210); KT3 epitope peptide (Martin, et al. (1992) Science ( Science) 255: 192-194); tubulin epitope peptide (Skinner, et al. (1991) J. Biol. Chem. 266: 15163-15166); and T7 gene 10 protein peptide tag (Lutz-Freyermuth, et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6393-6397).

  Also included are other cancer proteins of the cancer group, and cancer proteins from other organisms expressed in clones as outlined below. Thus, probes or denatured polymerase chain reaction (PCR) primer sequences are used to find other oncoproteins of interest from humans or other organisms. Particularly useful probe and / or PCR primer sequences include unique regions of cancer nucleic acid sequences. Preferred PCR primers are about 15-35, preferably about 20-30 nucleotides in length, optionally including inosine. The conditions for PCR reactions are well described (eg, Innis, PCR Protocols, supra).

  In addition, oncoproteins are made longer than those encoded by the nucleic acids in the table, eg, by elucidating extended sequences, adding epitopes or purification tags, adding other fusion sequences, and the like.

  Oncoproteins are also identified as encoded by cancer nucleic acids. Thus, oncoproteins are encoded by nucleic acids that hybridize to sequences in the sequence list or their complements, as outlined herein.

Antibodies against oncoproteins In a preferred embodiment, oncoproteins must share at least one epitope or determinant with the full-length protein when the oncoprotein is used to produce antibodies for immunotherapy or immunological diagnosis. . As used herein, “epitope” or “determinant” usually refers to a portion of a protein that makes and / or binds to an antibody or T cell receptor in the environment of a major histocompatibility complex (MHC). means. Thus, in most cases, antibodies made for small oncoproteins can bind to full-length proteins, particularly linear epitopes. In preferred embodiments, the epitope is unique; that is, antibodies made for a specific epitope show little cross-reactivity. In preferred embodiments, the epitope is selected from the protein sequences shown in the table.

  There are methods to prepare polyclonal antibodies (eg, Coligan, supra; and Harlow and Lane, supra). Polyclonal antibodies can be raised in mammals by one or more injections of an immunizing agent and optionally an adjuvant. Usually, the immunizing agent and / or adjuvant is injected into the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent includes proteins encoded by the nucleic acids of Tables 1-80, or fragments thereof, or fusion proteins thereof. It is effective to bind the immunizing agent to a protein known to be immunogenic in the mammal to be immunized. Examples of such immunogenic proteins include, but are not limited to, clam hemocyanin, serum albumin, bovine thyroglobulin, soybean trypsin inhibitor. Examples of adjuvants that may be used include Freund's complete adjuvant, MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). Various immunization protocols are used.

The antibody, on the other hand, may be a monoclonal antibody. Monoclonal antibodies are prepared by the hybridoma method, as described in Kohler and Milstein (1975) Nature 256: 495. In the hybridoma method, a mouse, hamster or other suitable host animal is usually immunized with an immunizing agent that expresses lymphocytes that are or are capable of producing antibodies that specifically bind the immunizing agent. On the other hand, lymphocytes may be immunized in vitro. The immunizing agent usually comprises a polypeptide encoded by the nucleic acids in the table, or fragments thereof, or fusion proteins thereof. Usually, peripheral blood lymphocytes ("PBLs") are used when cells of human origin are desired, and spleen cells or lymph node cells are used when non-human mammalian sources are desired. The lymphocytes are then fused with an immortal cell line using an appropriate fluxing agent such as polyethylene glycol to form hybridoma cells (eg, pages 59-103, Goding (1986) Monoclonal Antibodies: Principles and Practice (“monoclonal”). Antibody: Principles and Practices)) See Academic Press). Immortal cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, or human origin. Usually, rat or mouse myeloma cell lines are used. Hybridoma cells are cultured in a suitable culture medium containing one or more substances that prevent the growth or survival of unfused, immortalized cells. For example, if the parent cell lacks the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium of the hybridoma usually contains hypoxanthine, aminopterin, and thymidine (“ HAT media ”).

  In one embodiment, the antibody is a bispecific antibody. Bispecific antibodies are preferably human or humanized monoclonal antibodies that have binding specificities for two or more different antigens or that have binding specificities for two epitopes of the same antigen. In some embodiments, one of the binding specificities is for a protein encoded by a nucleic acid in the table, or a fragment thereof, and the other is that of another antigen, preferably a cell surface protein, or receptor, or receptor sub Unit, preferably tumor-specific. On the other hand, tetramer-type technology produces multivalent reagents.

  In a preferred embodiment, the oncoprotein antibody can reduce or eliminate the biological function of the oncoprotein in a naked form or in a form associated with an effector moiety as described below. That is, when an anti-cancer protein antibody (either polyclonal or preferably monoclonal) is added to cancer cells (or cells containing cancer), the cancer can be reduced or eliminated. Usually, a reduction of at least 25%, particularly preferably at least about 50%, more particularly about 95-100% in activity, growth, size, etc. is desired.

  In a preferred embodiment, the antibody of the oncoprotein is a humanized antibody (eg, Xenerex Biosciences, Medarex, Inc., Abgenix, Inc., Protein Design Labs, Inc.). Humanized forms of non-human (eg, murine) antibodies are immunoglobulins, immunoglobulin chains, or fragments thereof (Fv, Fab, Fab ′, F () that minimally contain sequences from immunoglobulins that are not of human origin. ab ′) 2 or other antibody antigen-binding substance).

  Humanized antibodies include human immunoglobulins (receptor antibodies), wherein the residues from the complementarity measurement region (CDR) of the receptor have the desired properties, affinity, and ability, mouse, rat, or Replaced with residues from CDRs of a non-human origin (donor antibody) such as rabbits. In some cases, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human origin residues. Humanized antibodies may also contain residues not found in the CDRs or framework sequences incorporated in the receptor antibody. Usually, a humanized antibody comprises substantially all of at least one, generally two variable regions, wherein all or substantially all of the CDR regions are defined as immunoglobulin regions that are not of human origin. Correspondingly, all or substantially all framework (FR) regions are regions of the human immunoglobulin consensus sequence.

Humanized antibodies typically comprise at least a portion of an immunoglobulin constant region (Fc), usually human immunoglobulins (Jones, et al. (1986) Nature (321)) 321: 522- 525; Riechmann, et al. (1988) Nature (332) 323-329; and Presta (1992) Curr. Op. Struct. Biol. 2: 593-596). Humanization basically follows the method of Winter and co-workers (Jones, et al. (1986) Nature (321) Nature) 321: 522-525; Riechmann, et al. (1988) Nature ("Nature") 332: 323-327; Verhoeyen, et al. (1988) Science ("Science") 239: 1534-1536), replacing rodent CDRs or CDR sequences with corresponding sequences of human antibodies. Is done by. Accordingly, these humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567), wherein substantially less than the human variable region is replaced with the corresponding sequence of a species not of human origin.

Human antibodies can also be obtained using a phage display library (Hoogenboom and Winter (1992) J. Mol. Biol. 227: 381-388; Marks, et al. (1991) J. Mol ). Biol. (Journal of Molecular Biology) 222: 581-597) or using human monoclonal antibodies (eg, page 77, Cole, et al. Reisfeld and Sell (1985) Monoclonal Antibodies and Cancer Therapy (“monoclonal antibodies”). And Cancer Treatment ”) Liss; and Boerner, et al. (1991) J. Immunol. (Immunology Journal) 147: 86-95).

Similarly, human antibodies can be made by incorporating human immunoglobulin sites into transformed animals, eg, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Due to suspected legitimacy, the production of human antibodies was observed that closely resembled that found in almost every situation in humans, including gene rearrangement, assembly, and antibody coverage. This approach is described, for example, in US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016 and the following scientific publications: Marks, et al. (1992) Bio / Technology 10: 779-783; Lonberg, et al. (1994) Nature 368: 856-859; Morrison (1994) Nature (Nature) ) 368: 812-13; Fishwild, et al. (1996) Nature Biotechnology ( Nature Biotechnology ) 14: 845-851; Neuberger (1996) Nature Biotechnology ( Nature Biotechnology ) 14: 826; Lonberg and Huszar (1995) Intern. Rev. Immunol. 13: 65-93).

  Immunotherapy refers to the treatment of cancer with antibodies raised against cancer proteins. As used herein, immunotherapy can be passive or active. As defined herein, passive immunotherapy is the passive transfer of antibodies to a recipient (patient). Active immunization is the induction of antibody and / or T cell responses in a recipient (patient). Induction of the immune response occurs as a result of providing the recipient with a source of antigen against which the antibody has been raised. Antigens, on the other hand, can be expressed by injecting into a recipient a polypeptide in which it is desired that antibodies be raised, or they can express an antigen and cause an immune response under the conditions of expression of the antigen Provided by contacting the recipient.

  In a preferred embodiment, the oncoprotein against which the antibody was raised is a secreted protein, as described above. Without being bound by theory, antibodies used in therapy bind to the secreted protein and prevent it from binding to the receptor, thereby inactivating the secreted oncoprotein, for example, in an autocrine signal Disturb.

  In another preferred embodiment, the oncoprotein against which the antibody was raised is a transmembrane protein. Without being bound by theory, antibodies used for therapy bind to the extracellular region of the oncoprotein and prevent binding to other proteins such as circulating ligands and cell-related molecules. Antibodies cause down-regulation of transmembrane oncoproteins. An antibody can be a competitive, non-competitive or non-competitive inhibitor of a protein bound to the extracellular region of an oncoprotein. The antibody may also be an oncoprotein antagonist. In addition, antibodies prevent activation of transmembrane oncoproteins or induce or suppress specific cellular pathways. In one form, an antibody prevents cell growth when the antibody prevents binding of other molecules to the oncoprotein. Antibodies also include cytotoxic drugs including but not limited to TNF-α, TNF-β, IL-1, INF-γ, and IL-2, or 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, etc. For chemotherapeutic drugs containing, it is used to target cells or to make cells sensitive to them. In some instances, the antibody belongs to a subtype that activates serum complement titers when complexed with a transmembrane protein, thereby modulating cytotoxicity or antibody-dependent cytotoxicity (ADCC). Thus, cancer is treated by administering to the patient an antibody directed to a transmembrane oncoprotein. Antibody labeling provides a way to activate toxin complement, localize the toxin payload, target toxic-containing liposomes, or partially delete cells.

  In other preferred embodiments, the antibody binds to an effector moiety. The effector moiety can be a variety of molecules including a labeling moiety, such as a radioactive label or a fluorescent label, or can be a therapeutic moiety. In one form, the therapeutic moiety is a small molecule that mediates oncoprotein activity. In other forms, the therapeutic moiety mediates the activity of molecules associated with or adjacent to the oncoprotein. The therapeutic moiety suppresses enzyme or signal activity, such as the activity of a protease or collagenase or protein kinase associated with cancer, or becomes an inducer of other cells such as NK cells. See, for example, US Patent No. 09 / 544,494.

  In preferred embodiments, the therapeutic moiety is a cytotoxic agent. In this method, targeting a cytotoxic agent to cancer tissue or cells reduces the number of cells affected by the cancer, thereby reducing the pathology associated with the cancer. Cytotoxic drugs are diverse and include, but are not limited to, cytotoxic drugs, toxins, or active fragments of those toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, cursin, crotan, phenomycin, enomycin, saporin, auristatin and the like. Cytotoxic drugs also include radiochemicals that can be produced by binding radioisotopes to antibodies raised against oncoproteins and by binding radionuclides. Targeting a therapeutic moiety to a transmembrane protein not only functions to increase the local concentration of the therapeutic moiety in the affected area, but also reduces harmful side effects associated with the untargeted therapeutic moiety. There is a function. Antibody fragments are used to target toxic-containing liposomes.

  In another preferred embodiment, the oncoprotein against which the antibody is produced is an intracellular protein. In this case, the antibody is bound to a protein that promotes invasion into the cell. In one case, the antibody invades into the cell by endocytosis. In other embodiments, nucleic acid encoding the antibody is administered to the individual or cell. In addition, oncoproteins are targeted in cells, such as the nucleus, whereas antibodies to it contain signals that localize the target, such as, for example, nuclear localization signals.

The cancer antibody of the present invention specifically binds to a cancer protein. As used herein, “specifically binds” means that the antibody has a K _{d of} at least about 0.1 mM, more usually at least 1 μM, preferably 0.1 μM, and more preferably 0.01 μM or more. It means binding to a protein having _Kd . The selectivity of binding to a particular target rather than a related sequence is often important.

Detection of cancer sequences for diagnostic and therapeutic applications In one aspect, gene expression levels of RNA are measured by different cellular conditions in the cancer phenotype. Genes in normal tissue (eg, not affected by cancer), cancer tissue (and in some cases, for various cancer severity associated with prognosis, as outlined below), or non-malignant disease Expression levels are evaluated to provide expression characteristics. A particular cell state, or gene expression profile at the time of development, is basically a “fingerprint” of the cell state. Even if the two states express a specific gene in the same manner, gene expression characteristics that reflect the state of the cell can be created by simultaneously evaluating a large number of genes. Comparing the expression characteristics of cells in different states gives information about which genes are important for each of those states (including both up-regulated and down-regulated genes). Therefore, a diagnosis is made or confirmed, and it is determined whether the gene expression characteristics of the tissue sample are those of normal tissue or cancerous tissue. This provides a molecular diagnosis of the relevant condition.

  “Differential expression” or its grammatical synonym as used herein is a qualitative or quantitative expression of temporal and / or cellular gene expression patterns in or between cells and tissues. It makes a difference. Thus, differentially expressed genes can be qualitatively altered in their expression, including activation or inactivation, for example in normal versus cancerous tissues. A gene is turned on or off in one state in response to another, thereby allowing comparison of two or more states. A qualitatively regulated gene displays an expression pattern in a condition or cell type that can be detected by standard techniques. Some genes are expressed in one of the states or cell types and not in both.

On the other hand, the difference in expression may be quantitative, for example, the expression increases or decreases; for example, gene expression may be up-regulated and the amount of transcription may be increased, or down-regulated to reduce the amount of transcription. May decrease. The degree of expression difference need only be large enough for measurement by standard characterization techniques, such as the use of the Affymetrix GeneChip expression sequence outlined below. See Lockhart (1996) Nature Biotechnology 14: 1675-1680. Other techniques include, but are not limited to, quantitative reverse transcriptase (reverse transcriptase) PCR, Northern assay, and ribonuclease (RNase) protection. As outlined above, preferably the change in expression (eg, up-regulation or down-regulation) is at least about 50%, more preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%. Particularly preferred is 300 to at least 1000%.

  Evaluation is performed at the gene transcription or protein level. The amount of gene expression is monitored using nucleic acid probes to the RNA or DNA equivalent of the gene transcript and by measuring the level of gene expression. Optionally, the final gene product (protein) itself is monitored by other methods including, for example, antibodies to cancer proteins, standard immunoassays (such as ELISA) or mass assays, 2D gel electrophoresis assays, etc. . For example, a protein corresponding to an oncogene that has been identified as important in a cancer or disease phenotype is evaluated by a cancer diagnostic test. In a preferred embodiment, gene expression monitoring is performed on many genes simultaneously. Multiple protein expression monitoring is also performed.

  In this aspect, a cancer nucleic acid probe is attached to a biochip as outlined herein for detection and surveying of cancer sequences in specific cells. The assay is described in more detail below by way of example. PCR methods are used to provide better sensitivity.

  In a preferred embodiment, a nucleic acid encoding an oncoprotein is detected. While DNA or RNA encoding oncoproteins are detected, methods of detecting mRNA encoding oncoproteins are of particular interest. Probes that detect mRNA are nucleotide deoxynucleotide probes that are complementary to, and hybridize to, and include, but are not limited to, oligonucleotides, cDNA, or RNA. The probe should also include a detectable label as defined herein. In one method, mRNA is detected after the nucleic acid to be tested is immobilized on a solid support such as a nylon membrane and the sample and probe are hybridized. After washing to remove unspecifically bound probes, the label is detected. In other methods, mRNA detection is performed as is. In this manner, a permeabilized cell or tissue sample is contacted with a detectably labeled nucleic acid probe for a sufficient amount of time to hybridize the probe and target mRNA. After washing to remove unspecifically bound probes, the label is detected. For example, a digoxigenin-labeled riboprobe (RNA probe), which is the complement of mRNA encoding oncoprotein, is detected by binding digoxigenin and an anti-digoxigenin secondary antibody, and nitroblue tetrazolium and 5-bromo-4-chromo Developed with indoyl phosphate.

  In preferred embodiments, a variety of proteins from the three classes of proteins described herein (secreted, transmembrane, or intracellular proteins) are used in diagnostic assays. Cells containing cancer proteins, antibodies, nucleic acids, modified proteins, and cancer sequences are used in diagnostic assays. This is done at the individual gene or corresponding polypeptide level. In preferred embodiments, expression characteristics are used, preferably in conjunction with high-throughput screening techniques, to allow monitoring of gene and / or corresponding polypeptide expression characteristics.

  As described and defined herein, oncoproteins, including intracellular, transmembrane, or secreted proteins, are found as markers of cancer, for example, for prognosis or diagnostic purposes. Detection of these proteins in putative cancer tissues allows detection of cancer or similar diseases, prognosis, or selection of diagnostic and therapeutic methods. In one embodiment, antibodies are used to detect oncoproteins. A preferred method is to separate the protein from the sample by gel electrophoresis (usually denaturing and reducing protein gels, but other types of gels including isoelectric focusing gels etc.). Following protein separation, the cancer protein is detected, for example, by immunoblotting with antibodies raised against the cancer protein.

In other preferred embodiments, antibodies against oncoproteins are used in in situ imaging techniques, eg, in histology. For example, Asai, et al. (Eds. 1993) Methods in Cell Biology: Antibodies in Cell Biology (see “ Cell Biology Techniques: Antibodies in Cell Biology ”) (vol. 37) Academic Press). In this way, the cell is contacted with one or more antibodies against the oncoprotein. After washing to remove unspecific antibody binding, the presence of antibodies or antibodies are detected. In one embodiment, the antibody is detected by incubation with a secondary antibody that contains a detectable label. In other methods, the primary antibody against the oncoprotein comprises a detectable label, such as an enzyme marker that acts on the substrate. In other preferred embodiments, each of the plurality of primary antibodies comprises a separate detectable label. This method is particularly used when simultaneously screening multiple oncoproteins. Many other histological imaging techniques are provided by the present invention.

In a preferred embodiment, the label is detected with a fluorimeter equipped with the ability to detect and discriminate between different wavelength emissions. In addition, a fluorescence activated cell sorter (FACS) is used in the method.
In other preferred embodiments, the antibodies are used in the diagnosis of cancer from blood, serum, plasma, stool, and other samples. These samples are therefore useful as well as samples that are investigated and tested for the presence of oncoproteins. Antibodies are used to detect oncoproteins by the immunological assay techniques described above, including ELISA, immunoblotting (Western blot), immunoprecipitation, BIACORE technology and others. On the contrary, the presence of the antibody shows an immune response against the endogenous oncoprotein.

  In preferred embodiments, in situ hybridization of the labeled cancer nucleic acid probe to the tissue sequence is performed. For example, a sample sequence containing cancer tissue and / or normal tissue is prepared and in situ hybridization is performed (see, eg, Ausubel, supra). Individuals and standard fingerprints are compared and diagnosis, prognosis, or prediction is based on findings. It is further understood that genes that indicate a diagnosis are different from those that indicate a prognosis, and that molecular characteristics of the cellular state indicate a response or identify a refractory state or predict outcome.

  In preferred embodiments, cells containing cancer proteins, antibodies, nucleic acids, modified proteins, and cancer sequences are used in prognostic assays. As described above, gene expression characteristics are generated in correlation with cancer, clinical, pathological, or other information regarding long-term prognosis. This is also done at the protein or gene level, but the use of genes is desired. Single or multiple genes are used in various combinations. As described above, cancer probes are attached to the biochip to detect and quantify the tissue or patient cancer sequence. The assay proceeds as outlined in the diagnosis above. PCR methods provide more sensitive and accurate quantification.

Therapeutic Compound Assays In a preferred embodiment, the proteins, nucleic acids, antibodies described herein are used in drug screening assays. Cells containing cancer proteins, antibodies, nucleic acids, modified proteins, and cancer sequences are used in drug screening assays or to evaluate the efficacy of candidate substances in “gene expression characteristics” or polypeptide expression characteristics. . In preferred embodiments, expression profiles are preferably used in conjunction with high-throughput screening techniques to allow monitoring of gene expression profiles after treatment with candidate agents (eg, Zlokarnik, et al. (1998) Science (“ Science) 279: 84-88; Heid (1996) Genome Res. (Genome Res.) 6: 986-994).

  In preferred embodiments, cancer proteins, antibodies, nucleic acids, modified proteins, and cells containing native or modified oncoproteins are used in screening assays. Thus, the present invention provides a novel method of screening for compositions that modulate the cancer phenotype or the identified physiological function of the cancer protein. As mentioned above, this can be done either at the individual gene level or by evaluating the effect of the candidate substance on the “gene expression characteristics”. In a preferred embodiment, the expression profile is preferably used in conjunction with high throughput screening techniques to allow monitoring of gene expression profile after treatment with a candidate substance. See Zlokarnik, supra.

  Differentially expressed genes are identified herein and various assays are performed. In preferred embodiments, the assay is performed at the individual gene or protein level. That is, a particular gene in cancer is identified as being up-regulated and the test compound is screened for the ability to modulate gene expression or binding to a cancer protein. “Modulation” thus includes both an increase and a decrease in gene expression. A preferred amount of modulation is based on the initial change in gene expression in normal tissue versus tissue affected by cancer, the change being at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% Or more. That is, if a gene shows a 4-fold increase in cancer tissue compared to normal tissue, a reduction of about 4-fold is often desired; similarly, a 10-fold decrease in cancer tissue compared to normal tissue is often Provides induction of an increase in expression by 10 times the target value by the test compound.

The amount of gene expression is monitored by quantification of gene expression levels using nucleic acid probes, or optionally the gene product itself is monitored, for example, by use of antibodies against cancer proteins and standard immunological tests. . Proteomics and separation techniques also allow quantification of expression.
In a preferred embodiment, the gene expression or entity that the number of proteins monitor, eg, expression characteristics, are monitored simultaneously. Such characteristics typically include a plurality of entities described herein.

  In this aspect, a cancer nucleic acid probe is attached to a biochip as outlined herein for detection and surveying of cancer sequences in specific cells. On the other hand, PCR is sometimes used. Thus, for example, a series of microtiter plates or the like are used with primers dispensed into the desired cells. A PCR reaction is then performed and each cell is assayed.

Cancer Modulators An expression monitor is performed to identify compounds that modify the expression of one or more cancer-related sequences, such as the polynucleotide sequences shown in the table. Usually, in a preferred embodiment, a test modulator is added to the cells prior to the assay. In addition, screens are also provided that identify substances that regulate cancer, regulate oncoproteins, bind to oncoproteins, or prevent oncoproteins from binding to antibodies and other binding partners.

  As used herein, the terms “test compound”, “candidate substance”, “modulator” or their grammatical synonyms are used to describe the expression of a cancer phenotype or cancer sequence, such as a nucleic acid or protein sequence, Describes molecules such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides that are tested for their ability to be directly or indirectly altered. In preferred embodiments, the modulator alters expression characteristics or the expression characteristics of the nucleic acids or proteins provided herein. In one embodiment, the modulator suppresses the cancer phenotype, eg, to a normal or non-malignant tissue fingerprint. In other embodiments, the modulator induces a cancer phenotype. Typically, multiple assay combinations are performed in parallel at different drug concentrations to obtain different concentrations of different responses. Usually, one of these concentrations functions as a negative control, eg, concentration 0 or below the detection level.

  Candidate substances include various chemical classifications, but are usually organic molecules, preferably small organic molecules having a molecular weight of 100 to 2500 daltons. The desired small molecule is 2000D or less, or 1500D or less, or 1000D or less, or 500D or less. Candidate substances contain functional groups necessary for structural interaction with proteins, in particular hydrogen bonds and usually contain at least two of amino groups, carbonyl groups, hydroxyl groups, carboxyl groups, preferably chemical functional groups. Candidate materials often contain cyclic carbon, heterocyclic structures and / or aromatic or heteroaromatic structures substituted with one or more of the above functional groups. Candidate substances are also found in biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural homologues, or combinations thereof. Peptides are particularly preferred.

In one situation, the modulator neutralizes the effect of the oncoprotein. “Neutralize” refers to the effect on a cell in which the activity of the protein is inhibited or prevented.
In certain embodiments, the ability of the combinatorial library of potential modulators to screen for the ability to bind to or modulate the activity of a cancer polypeptide. Conventionally, identifying chemical compounds (referred to as “lead compounds”) with desirable properties or activities, such as suppression of activity, creation of variants of lead compounds, and evaluation of properties or activities of those mutant compounds This creates new chemical entities with useful properties. Often, high throughput screening (HTS) methods are used for these assays. For example, Janzen (2002) High Throughput Screening: Methods and Protocols (Humana; Devlin (ed. 1997) High Throughput Screening: The Discovery of Bioactive Substances Active substance discovery ”) Dekker; and Mei and Czarnik (eds. 2002) Integrated Drug Discovery Techniques . See Dekker.

  In one embodiment, the high-throughput screening method involves providing a library containing a large number of potential therapeutic compounds (candidate compounds). These “combined chemical libraries” are then screened by one or more assays that identify those library components (especially chemical species or subclasses) that exhibit the desired characteristic activity. The compounds thus identified function as conventional “lead compounds” or themselves as potential or de facto therapeutic agents.

A combinatorial chemistry library is a collection of diverse chemical compounds created by either chemical synthesis or biological synthesis by combining a number of chemical “basic components” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (eg, mutein) library, is a chemical compound called an amino acid in as many ways as possible for a given compound length (eg, the number of amino acids in the polypeptide compound). Formed by combining a set of basic components. A huge number of chemical compounds are synthesized by combining and mixing such chemical basic components (Gallop, et al. (1994) J. Med. Chem. (Medical Chemistry Journal)) 37: 1233-1251 ).

The preparation and screening of combinatorial chemical libraries is well known. Such combinatorial chemical libraries include, but are not limited to: peptide libraries (eg, US Pat. No. 5,010,175, Furka (1991) Pept. Prot. Res. 37: 487-493, Houghton, et al. ( 1991) Nature ("Nature") 354: 84-88), peptoids (PCT publication number WO 91/19735), encoded peptides (PCT publication WO 93/20242), optional biooligomers (PCT publication WO 92/00091) ), Benzodiazepines (US Pat. No. 5,288,514), variants such as hydantoin, benzodiazepines and dipeptides (Hobbs, et al. (1993) Proc. Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides ( Hagihara, et al. (1992) J. Amer. Chem. Soc. (Journal of the American Chemical Society) 114: 6568-570), non-peptide peptidomimetics with beta-D-glucose conjugates (Hirschmann, et al (1992) J. Amer. Chem. Soc. Null ”) 114: 9217-9218), similar organic synthesis of small compound libraries (Chen, et al. (1994) J. Amer. Chem. Soc. (Journal of the American Chemical Society) 116: 2661-662), Oligocarbamic acid (Cho, et al. (1993) Science 261: 1303-1305) and / or peptidyl phosphonate (Campbell, et al. (1994) J. Org. Chem. Journal ”) 59: 658). For customary information, see Gordon, et al. (1994) J. Med. Chem. (Medical Chemistry Journal) 37: 1385-1401.

Nucleic acid libraries (see eg Stratagene, Corp.), peptide nucleic acid libraries (see eg US Pat. No. 5,539,083), antibody libraries (eg Vaughn, et al. (1996) Nature Biotechnology ) 14 (3 ): 309-314, and PCT / US96 / 10287), carbohydrate libraries (see, eg, Liang, et al. (1996) Science 274: 1520-1522, and US Pat. No. 5,593,853) and small organic molecules Libraries (eg, benzodiazepines, page 33, Baum (Jan 18, 1993) C &EN; isoprenoids, US Pat. No. 5,569,588; thiazolidinones and metathiazanones, US Pat. No. 5,549,974; pyrrolidines, US Pat. Nos. 5,525,735 and 5,519,134; No. 5,506,337; benzodiazepines, US Pat. No. 5,288,514; others).

  Equipment for preparing combinatorial libraries is commercially available (eg, MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Kentucky), Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA (California), 9050 Plus, Millipore, Bedford, MA (Massachusetts)).

  Many well-known robotic systems have also been developed for solution chemistry. These systems are automated workstations such as automated synthesizers developed by Takeda Chemical Industries, LTD (Osaka, Japan), and robotics that mimic manual synthesis operations performed by chemists. Includes many robotic systems utilizing arms (Zymate II, Zymark Corporation, Hopkinton, Mass .; Orca, Hewlett-Packard, Palo Alto, Calif.). The apparatus described above is suitable for use with the present invention. The nature and accomplishment of changes (if any) to these devices to operate as described herein will be apparent. In addition, many combinatorial libraries are commercially available per se (eg, ComGenex, Princeton, NJ (New Jersey), Asinex, Moscow, Ru (Moscow, Russia), Tripos, Inc., St. Louis, MO ( Missouri), ChemStar, Ltd, Moscow, RU (Moscow, Russia), 3D Pharmaceuticals, Exton, PA (Pennsylvania), Martek Biosciences, Columbia, MD (Maryland)).

  Modulator identification assays are amenable to high-throughput screening. A preferred assay thus detects an increase or suppression of oncogene transcription, suppression or enhancement of polypeptide expression, and suppression or increase of polypeptide activity.

  High-throughput assays with the present invention for the presence, absence, quantification, or other characteristics of a particular nucleic acid or protein product are well known. Similarly, binding assays and reporter gene assays are equally well known. For example, US Pat. No. 5,559,410 discloses a high-throughput screening method for proteins, while US Pat. No. 5,585,639 discloses a high-throughput screening method for nucleic acid binding (eg, sequence), while US Pat. No. 5,576,220. And 5,541,061 disclose high throughput screening methods for ligand / antibody binding.

In addition, high-throughput screening methods are commercially available (eg, Zymark Corp., Hopkinton, MA (Massachusetts); Air Technical Industries, Mentor, OH (Ohio); Beckman Instruments, Inc. Fullerton, CA (California) ); Precision Systems, Inc., Natick, MA (Massachusetts, et al.)). These systems typically automate the entire process, including sample and reagent dispensing, liquid dispensing, timed incubation, and final measurement of a detector microplate suitable for the assay. These configurable systems offer high throughput and fast startup as well as a high degree of flexibility and customization. The manufacturers of these systems provide detailed protocols for various high throughput systems. For example, Zymark provides a technical bulletin describing a screening system for detecting gene transcription, ligand binding, and other modulations.
In one embodiment, the modulator is a protein, often a natural protein or a fragment of a natural protein. Thus, for example, cell extracts containing proteins or any or directed digests of proteinaceous cell extracts are used. In this way, a library of proteins is created for screening by the method of the present invention. Particularly desirable in this aspect are bacterial, fungal, viral, and mammalian protein libraries, the latter being more desirable, especially human proteins. Particularly useful test compounds are directed to the protein class to which the target belongs, for example enzymes or ligands and receptor substrates.

  In a preferred embodiment, the modulator is a peptide of about 5-30 amino acids, preferably 5-20 amino acids, particularly preferably 7-15 amino acids. Peptides are natural proteins, random peptides, or digests of “biased” random peptides. “Randomized” or grammatical synonyms herein means that each nucleic acid and peptide consists essentially of random nucleotides and amino acids, respectively. Since these random peptides (or nucleic acids described below) are usually chemically synthesized, nucleotides or amino acids can be incorporated at any position. The synthesis process is set up to produce a randomized protein or nucleic acid, allowing the formation of all or most possible combinations over the length of the sequence, thereby allowing the randomized organism A library of active protein candidates is formed.

  In one embodiment, the library is fully randomized and there is no sequence selection or constant at any position. In a preferred embodiment, the library is deflected. That is, some of the positions in the sequence are kept constant or selected from limited possibilities. For example, in a preferred embodiment, the nucleotide or amino acid residue is a histidine or purine such as a nucleic acid binding region, cysteine, crosslink, SH-3 region proline, serine, threonine, tyrosine, or phosphorylation site. For example, hydrophobic amino acids, hydrophilic residues, sterically biased residues (either large or small) are randomized within a clear classification range.

The modulator of cancer may be a nucleic acid as described above.
As noted above, for proteins, usually the nucleic acid that modulates the drug is a natural nucleic acid, a random nucleic acid, or a “biased” random nucleic acid. For example, digests of prokaryotic or eukaryotic genomes are used as outlined for the above proteins.

In a preferred embodiment, the candidate compound is an organic chemical moiety, the diversity of which is available from the literature.
After the candidate substance is added and the cells are cultured for some time, a sample containing the target sequence to be assayed is added to the biochip. If necessary, the target sequence is prepared using known techniques. For example, the sample is treated to lyse cells using known lysis buffers, electroporation, etc., in conjunction with purification and / or amplification such as PCR performed as necessary. For example, a label is covalently attached to a nucleotide and in vitro transcription is performed. Usually, nucleic acids are labeled with biotin-FITC or PE, or cy3 or cy5.

  In a preferred embodiment, the target sequence is labeled with, for example, a fluorescent, chemiluminescent, chemical, or radioactive signal to provide a means for detecting special binding of the target sequence to the probe. The label may be an enzyme that produces a detectable product when provided with a suitable substrate, such as alkaline phosphatase or horseradish peroxidase. On the other hand, the label may be a labeled compound or small molecule, such as an enzyme inhibitor that binds but is not catalyzed or altered by the enzyme. The label may also be a moiety or compound that specifically binds to streptavidin, such as an epitope tag or biotin. In the biotin example, streptavidin is labeled as described above, thereby providing a detectable signal of the bound target sequence. Unbound labeled streptavidin is usually removed prior to the assay.

  These assays may be direct hybridization assays or may include “sandwich assays”. Sandwich assays involve the use of multiple probes, U.S. Patent Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670 No. 5,591,584, No. 5,624,802, No. 5,635,352, No. 5,594,118, No. 5,359,100, No. 5,124,246, and No. 5,681,697. Incorporate inside. In this embodiment, the target nucleic acid is typically prepared as outlined above and added to a biochip containing a plurality of nucleic acid probes under conditions that allow the formation of hybridization complexes.

  In the present invention, a variety of hybridization conditions are used, including the very stringent conditions outlined above, moderate conditions, and less stringent conditions. Assays are usually performed under stringent conditions that allow the formation of labeled probes of the hybridization complex only in the presence of the target. The degree of severity is controlled by changes in stage parameters, which are thermodynamic variables including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration, pH, organic solvent concentration, and the like.

  These parameters may be used for non-specific binding controls, generally outlined in US Pat. No. 5,681,697. Therefore, in order to reduce non-specific binding, it is desirable to carry out specific steps under stringent conditions.

  The reactions outlined herein can be accomplished in a variety of ways. The elements of the reaction are added simultaneously or sequentially in a separate order in the preferred embodiments described below. In addition, the reaction includes various other reagents. These include neutral proteins such as salts, buffers such as albumin, synthetic detergents that facilitate optimal hybridization and detection and / or suppress non-specific or background interactions. Other reagents that enhance the effectiveness of the assay, such as proteolytic enzyme inhibitors, nucleolytic enzyme inhibitors, antibacterial agents, etc. are also used based on sample preparation and target purity, as appropriate.

  The assay data is examined to measure expression levels and to measure changes in expression levels in creating gene expression characteristics between each gene state.

  Screening is performed to identify modulators of the cancer phenotype. In one embodiment, screening is performed to identify modulators that can promote or suppress specific expression characteristics, thereby preferably creating a related phenotype. In other embodiments, for example, diagnostic applications, screening is performed to identify differentially expressed genes that are important in a particular condition and to identify modulators that bias the expression of individual genes. In other embodiments, screening is performed to identify modulators that alter the biological function of the expression product of a differentially expressed gene. Again, screening is performed to identify the importance of the gene in a particular state and to identify agents that bind to the gene product and / or modulate the biological activity of the gene product.

  In addition, screening for genes induced in response to candidate substances or treatment processes may be performed. After identifying a modulator based on the ability to modulate the expression characteristics of one oncogene to suppress the cancer expression pattern (or vice versa) leading to a normal expression pattern, or to mimic gene expression in normal tissue, The above screening is performed to identify genes that are specifically regulated corresponding to the drug. By comparing the expression characteristics of normal tissue and drug-treated cancer tissue, genes that are not expressed in normal tissue or cancer tissue but are expressed in the tissue subjected to drug treatment become clear. These drug specific sequences are identified and used in the oncogene or protein methods described herein. In particular, the sequences and proteins encoded by them are useful for marking or identifying cells that have undergone drug treatment. In addition, antibodies against drug-derived proteins can be generated and used to target new therapies such as toxic-containing liposomes to treated cancer tissue samples.

  Thus, in one embodiment, the test compound is administered to a population of cancer cells having associated cancer expression characteristics. As used herein, “administration” or “contact” is a method that allows an agent to act on a cell, whether by uptake and intracellular activity, or by cell surface activity, where the candidate substance is a cell. Means to be administered. In some embodiments, a nucleic acid encoding a proteinaceous candidate substance (eg, peptide) is injected into a viral structure, such as an adenovirus or retroviral structure, and administered to a cell, eg, a peptide drug such as PCT US97 / 01019. Expression is achieved. Regulatable gene therapy systems are also used.

  Once the test compound is administered to the cells, the cells are washed as necessary and preferably incubated for some time under physiological conditions. The cells are then collected and new gene expression characteristics are created as outlined herein.

  Thus, for example, cancerous tissue or non-malignant tissue is screened for agents that modulate, eg, induce or suppress a cancer phenotype. Changes in the expression characteristics of at least one, and preferably many genes, suggest that the drug has an effect on cancer activity. By defining such phenotypic indicators of cancer, screening for new pharmaceutical agents that revise the phenotype is considered. This method does not require the drug target to be known or displayed on the initial expression screening platform, nor does it require changing the transcription level of the target protein.

  In a preferred embodiment, screening of individual genes and gene products (proteins) is performed as described above. That is, a specific differentially expressed gene is identified as important in a specific state, and screening for modulators of either gene expression or gene product itself is performed. The gene product of a differentially expressed gene is sometimes referred to herein as a “cancer protein” or “cancer modifier protein”. Cancer modification proteins are protein fragments or full length proteins of the fragments encoded by the nucleic acids in the table. Preferably the cancer modifying protein is a fragment. In a preferred embodiment, the cancer nucleic acids used to measure sequence identity or similarity are encoded by the nucleic acids in the table. In other embodiments, the sequence is a natural allelic variant of the protein encoded by the nucleic acids in the table. In other embodiments, the sequence is a sequence variant as further described herein.

  Preferably, the cancer modifying protein is a fragment of 14-24 amino acids in length. More preferably, the fragment is a soluble fragment. Preferably the fragment comprises a non-transmembrane region. In a preferred embodiment, the fragment has an N-terminal Cys that aids solubility. In one embodiment, the C-terminal of the fragment is kept as a free acid and the N-terminal is a free amine that serves for conjugation with eg cysteine.

In one embodiment, the oncoprotein binds to an immunogenic agent as described herein. In one embodiment, the oncoprotein binds to BSA.
Measurement of cancer polypeptide activity, cancer, or cancer phenotype can be performed using a variety of assays. For example, the effect of a test compound on the function of a cancer polypeptide can be measured by the test parameters described above. Appropriate physiological changes that affect activity are used to assay the effects of the polypeptide-like test compounds of the present invention. When the functional sequence is measured using intact cells or animals, it is also possible to measure a variety of effects such as: if the cancer is associated with a tumor, the growth of the tumor, Metastasis, neovascularization, hormone release, changes in transcription to known and uncharacterized genetic markers (eg, Northern blots), changes in cellular metabolism such as cell proliferation or pH, and intracellular such as cGMP Second messenger change. The assay of the present invention particularly uses mammalian cancer polypeptides, such as mice, and preferably humans.

  Assays identifying compounds with modulatory activity can be performed in vitro. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for an appropriate length of time, such as 0.5-48 hours. In one embodiment, cancer polypeptide levels are measured in vitro by measuring protein or mRNA levels. Protein levels are usually measured by Western blot, ELISA, and other immunological assays with antibodies that selectively bind to cancer polypeptides or fragments thereof. For the measurement of mRNA, for example, PCR, amplification using LCR, hybridization assay such as Northern hybridization, RNase protection, and dot blotting are desired. The level of protein or mRNA is usually as described herein, for example, directly or indirectly with a labeled detection agent such as a fluorescently labeled or radioactively labeled nucleic acid, a radioactively labeled or enzymatically labeled antibody. It is detected by using it.

  On the other hand, reporter gene systems are devised using oncoprotein promoters that are operably linked to reporter genes such as luciferase, green fluorescent protein, CAT, or β-gal. Reporter constructs are usually transfected into cells. After being processed by the potential modulator, the amount of transcription, translation or activity of the reporter gene is measured based on standard techniques.

  In a preferred embodiment, screening of individual genes and gene products (proteins) is performed as described above. That is, a specific differentially expressed gene is identified as important in a particular state and screened for modulators of gene expression or gene product expression. The gene product of a differentially expressed gene is sometimes referred to herein as “oncoprotein”. An oncoprotein is a fragment of a protein or a full-length protein of the fragment shown herein.

  In one embodiment, screening for modulators of specific gene expression is performed. Usually, the expression of only one or a few genes is evaluated. In other embodiments, the screening is first set up to find compounds that bind to differentially expressed proteins. These compounds are then evaluated for their ability to modulate differentially expressed activity. Furthermore, once the first candidate compound is identified, the variants are further screened for better structure-activity relationship assessment.

  In a preferred embodiment, a binding assay is performed. Typically, a purified or isolated gene product is used; that is, the gene product of one or more differentially expressed nucleic acids is made. For example, antibodies against protein gene products are made and standard immunological assays are performed to determine the amount of protein present. On the other hand, cells containing oncoproteins are used in the assay.

  Accordingly, in a preferred embodiment, a method for binding a cancer protein to a candidate compound and a method for measuring the binding of a compound to a cancer protein are included. Preferred embodiments use human cancer proteins, although other mammalian proteins may be used, for example, to develop animal models of human disease. In some embodiments, a mutant or derivative of an oncoprotein is used as described above.

  In general, preferred embodiments of this method preferably include an isolated sample-receiving region (eg, microtiter plate, sequence, etc.) that binds the oncoprotein or candidate substance so that it does not diffuse to the insoluble support. An insoluble support is made of a composition to which the composition binds, is easily separated from soluble material, or is compatible with the entire screening method. The surface of the support is solid or porous and has an appropriate shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are usually made of glass, plastic (eg polystyrene), polysaccharides, nylon or nitrocellulose, Teflon and the like. Microtiter plates and arrays are particularly convenient because a large number of assays can be performed simultaneously using small amounts of reagents and samples. As long as it is compatible with the reagents and overall methods of the present invention, maintains the activity of the composition and is non-diffusible, the particular method of binding of the composition is usually not critical. Preferred binding methods include the use of antibodies (which do not sterically block the ligand binding site or activation sequence when the protein is bound to the support), “sticky” or to an ionic support. Direct binding, chemical cross-linking, synthesis of proteins or drugs on the surface, etc. Following binding of the protein or drug, excess unbound material is removed by washing. The sample receiving area is then shielded by incubation with bovine serum albumin (BSA), casein, or other innocuous proteins or other parts.

  In a preferred embodiment, the oncoprotein binds to the support and a test compound is added to the assay. On the other hand, the candidate substance binds to the indicator and the oncoprotein is added. Novel binding agents include special antibodies, non-natural binding agents identified on chemical library screens, peptide analogs, and the like. Of particular interest are screening assays for drugs that are less toxic to human cells. A variety of assays are used for this purpose, including labeled in vitro protein / protein binding assays, electrophoretic mobility change assays, protein binding immunological assays, functional assays such as phosphorylation assays .

  Measurement of the binding of test modulatory compounds and oncoproteins is done in a number of ways. In a preferred embodiment, the compound is labeled, for example, binding all or part of the oncoprotein to a solid support, adding a labeling candidate substance (eg, a fluorescent label), washing away excess reagents, and on the solid support. Binding is measured directly by measuring whether the label is present. Various shielding and cleaning steps are used as needed.

In some embodiments, only one of the compounds is labeled, for example a protein (or proteinaceous candidate compound). On the other hand, one or more compounds are labeled with different labels, such as ¹²⁵ I for proteins and fluorescence for compounds. Proximity reagents such as quenching agents or energy transfer agents are also useful.

  In one embodiment, test compound binding is measured by a competitive binding assay. A competitor is a binding moiety that is known to bind to a target molecule (eg, a cancer protein), such as an antibody, peptide, binding partner, or ligand. Under certain circumstances, there is a competitive bond between the compound and the binding moiety, where the binding moiety is replaced with the compound. In one embodiment, the test compound is labeled. If present, either the compound or the competitor, or both, is first added to the protein for a time sufficient to allow binding. Culturing is carried out at a temperature that promotes optimal activity, usually between about 4-40 ° C. The incubation time is usually optimized, for example, to facilitate high speed high throughput screening. Usually 0.1 to 1 hour is sufficient. Excess reagent is usually removed or washed away. A second compound is then added, followed by the presence or absence of a labeled compound to indicate binding.

  In a preferred embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor indicates that the test compound binds to the oncoprotein and can therefore bind to the oncoprotein and modulate the activity of the oncoprotein. In this manner, any compound can be labeled. Thus, for example, if the competitor is labeled, the presence of the label in the wash solution indicates displacement by the drug. On the other hand, if the test compound is labeled, the presence of the label on the support indicates substitution.

  In an alternative embodiment, the test compound is added first by incubation, washing, followed by the competitor. The absence of binding by the competitor indicates that the test compound has bound the oncoprotein with higher affinity. Thus, if the test compound is labeled, the presence of the label on the support with the absence of competitor binding indicates that the test compound is capable of binding to the oncoprotein.

  In a preferred embodiment, the method comprises a differential screen that identifies agents capable of modulating oncoprotein activity. In one embodiment, the method includes binding the oncoprotein and the competitor in the first sample. The second sample contains test compounds, oncoproteins, and competitors. Competitor binding is measured for both samples, and changes in binding between the two samples, or differences, indicate the presence of an agent capable of binding to the cancer protein and modulating its activity. That is, if the binding of the competitor in the second sample is different compared to the first sample, the drug has the ability to bind to the oncoprotein.

  On the other hand, differential screening is used to identify candidate substances that bind to native oncoproteins but cannot bind to modified oncoproteins. A model of the oncoprotein structure was created and used to synthesize drugs that interact with the site in rational drug design. Candidate substances that affect the activity of cancer proteins are also identified by screening for drugs for either the ability to promote or suppress the activity of the protein.

  In the assay, positive and negative controls are used. Preferably the control group and the test sample are tested at least 3 times to obtain a statistically valid result. All samples are incubated for a time sufficient for the drug to bind to the protein. After incubation, the sample is washed away with non-specifically bound material and the amount bound, usually the amount of labeled drug, is measured. For example, if a radiolabel is used, the sample is measured with a scintillation counter to determine the amount of bound compound.

  A variety of other reagents are included in the screening assay. These include reagents that promote optimal protein / protein binding and / or suppress non-specific or background interactions, such as salts and neutral proteins such as albumin, synthetic detergents, and the like. In addition, reagents that enhance the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antibacterial agents, and the like are also used. The mixed composition is added in order to provide the necessary bonds.

  In a preferred embodiment, the present invention provides a method of screening for compounds that can modulate the activity of oncoproteins. The method includes adding a test compound to the cell containing the oncoprotein as described above. Preferred cell types are almost all cells. The cell contains a recombinant nucleic acid encoding a cancer protein. In a preferred embodiment, the library of candidate substances is tested on a plurality of cells.

  In one state, the assay is physiological, such as drugs including hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, chemotherapy, radiation, carcinogenicity, other cells (eg cell / cell contacts) Assessed for the presence, absence, or previous or continuous exposure of the signal. In other examples, measurements are taken at other stages of the cell cycle process.

  Compounds that modulate cancer drugs are thus identified. A compound having pharmacological activity can promote or suppress the activity of the oncoprotein. Similar structures, once identified, are evaluated to identify important structural features of the compound.

  In one embodiment, a method for inhibiting cancer cell division is provided. The method includes administration of an inhibitor of cancer. In another aspect, a method for inhibiting cancer is provided. The method includes administration of an inhibitor of cancer. In a further aspect, there is provided a method of treating a cell or individual associated with cancer, including, for example, administration of an inhibitor of cancer.

In one embodiment, the cancer inhibitor is an antibody as described above. In other embodiments, the inhibitor of cancer is an antisense molecule.
As described below, various cell growth, proliferation, viability, and metastasis assays are available.

Soft agar growth or colony formation in test solution Normal cells require a solid substrate to adhere and grow. When transformed, the cell loses its phenotype and grows away from the substrate. For example, transformed cells can be grown in a stirred test medium or suspended in a semi-solid medium such as semi-solid or soft agar. When transformed cells are transfected with a tumor suppressor gene, they regenerate a normal phenotype and require a solid substrate for adhesion and growth. Soft agar growth or colony formation in assay test solutions is used to identify modulators of cancer sequences that suppress abnormal cell growth and transformation when expressed in host cells. The therapeutic compound reduces or eliminates the ability of the host cells to grow in a stirred test medium and to float in a semi-solid medium such as semi-solid or soft agar.

For example, Freshney (1998) Culture of Animal Cells: A Manual of Basic Technique (3d ed.) Wiley-Liss Freshney (2000) Culture of Animal Cells: A Manual of Basic Technique (4th ed.) Wiley-Liss; and Garkavtsev, et al. (1996) Nature Genet. 14 : 415-20.

Contact inhibition and growth concentration limits Normal cells usually grow in a flat systematic pattern until they come into contact with other cells in a Petri dish. Cells are contact-inhibiting and growth stops when they come into contact with each other. However, once transformed, the cells are no longer contact-inhibiting and continue to grow in a dense, unorganized nest. Therefore, transformed cells grow at a higher saturation concentration than normal cells. This is detected morphologically by non-directional cell monolayer formation, or by round cells in the nest within a regular pattern of surrounding normal cells. On the other hand, the labeling rate with ( ³ H) -thymidine at a saturating concentration is used to measure the growth concentration limit. See Freshney (2000), supra. Transformed cells, when transfected with a tumor suppressor gene, regenerate normal phenotype, become contact-inhibiting and grow to a low concentration.

In this assay, the rate of labeling with ( ³ H) -thymidine at a saturating concentration is a preferred method for measuring the concentration limit of growth. Transformed host cells are transfected with cancer-related sequences and grown for 24 hours at saturating concentrations in unrestricted medium conditions. The labeling rate with ( ³ H) -thymidine is measured by autoradiography. See Freshney (1998), supra.

Growth factors or serum dependence Transformed cells are usually less serum dependent than their normal counterparts (eg Temin (1966) J. Natl. Cancer Insti. (National Cancer Institute Journal)) 37: 167-175; Eagle, et al. (1970) J. Exp. Med. ( Exp. Med . Journal) 131: 836-879); see Freshney, supra. This is in part due to the release of various growth factors by the transformed cells. The growth factor or serum dependence of the host cell can be compared to that of the control group.

Tumor Specific Marker Levels Tumor cells release a greater amount of specific factors (hereinafter “tumor specific markers”) than the corresponding normal cells. For example, plasminogen activator (PA) is released at higher levels from human gliomas compared to normal brain cells (eg Mihich (ed. 1985) Biological Responses in Cancer ). See pages 178-184 of Plenum, Gullino "Angiogenesis, tumor vascularization, and potential interference with tumor growth"). Similarly, tumor angiogenic factor (TAF) is released from tumor cells at higher levels than the corresponding normal cells. See Folkman (1992) Sem. Cancer Biol. 3: 89-96.

Various techniques for measuring the release of these factors are described in Freshney (1998), supra. Furthermore, Unkeless, et al. (1974) J. Biol. Chem. (Biochemistry Journal) 249: 4295-4305; Strickland and Beers (1976) J. Biol. Chem. (Biochemistry Journal) 251: 5694-5702; Whur, et al. (1980) Br. J. Cancer (Br. J. Cancer) 42: 305-312; Mihich (ed. 1985) Biological Responses in Cancer ) Pp. 178-184, Plenum, Gullino "Angiogenesis, tumor vascularization, and potential interference with tumor growth" 1985) Biological Responses in Cancer Plenum, 178-184; See Freshney (1985) Anticancer Res. 5: 111-130.

Invasion to Matrigel The degree of invasiveness to Matrigel or other extracellular matrix structures is used as an assay to identify compounds that modulate cancer-associated sequences. Tumor cells show a good correlation between cell malignancy and invasiveness to Matrigel or other extracellular matrix structures. In this assay, carcinogenic cells are usually used as host cells. Expression of tumor suppressor genes in these host cells reduces the invasiveness of the host cells.

The technique described in Freshney (1994), supra is used. Briefly, the level of host cell invasion is measured using a filter coated with Matrigel or other extracellular matrix structure. Invasion into the gel, or penetration into the distal side of the filter, is assessed as invasive, and the cells are pre-labeled with ¹²⁵ I by the number of cells and distance traveled, or by ¹²⁵ I, and the distal side of the filter or dish It is assessed histologically by measuring the radioactivity at the bottom. See, for example, Freshney (1984), supra.

In vivo tumor growth The effect of cancer-associated sequences on cell growth is tested in transgenic or weakly immunized mice. A knockout transgenic mouse is produced in which the oncogene is split or the oncogene is inserted. A knockout gene-introduced mouse is produced by inserting a marker gene or other heterologous gene into the endogenous oncogene site of the mouse genome via homologous recombination. These mice can also be made by substituting a mutant form of a cancer gene with an endogenous oncogene, or by mutating the endogenous oncogene, for example, by contact with a carcinogen.

The DNA construct is introduced into the embryonic stem cell nucleus. Cells containing the newly created genetic lesion are injected into the host mouse embryo and reimplanted into the female receptor. Some of these embryos develop to become chimeric mice with germ cells partially derived from the mutant cell line. Therefore, by breeding chimeric mice, new types of mice containing the introduced genetic damage can be obtained. (See, for example, Capecchi, et al. (1989) Science 244: 1288-1292). Chimeric target mice can be obtained based on the following: Hogan, et al. (1988) Manipulating the Mouse Embryo: A Laboratory Manual CSH Press; and Robertson (ed. 1987) Teratocarcinomas and Embryonic Stem Cells: A Practical Approach ( IRT Press, Washington, DC).

On the other hand, various immunity-deficient or immunodeficient host animals are used. For example, genetically athymic “nude” mice (see, eg, Giovanella, et al. (1974) J. Natl. Cancer Inst. (National Cancer Institute Journal) 52: 921-930), SCID (severe complex Type immunodeficiency) mice, thymectomized mice, or irradiated mice (eg Bradley, et al. (1978) Br. J. Cancer 38: 263-272; Selby , et al. (1980) Br. J. Cancer (see Br. J. Cancer) 41: 52-61) are used as hosts. Transplanted tumor cells injected into isogenic hosts (usually about 10 ⁶ cells) produce invasive tumors in very high proportions, but normal cells of similar origin do not. In the host that produced the invasive tumor, cells expressing cancer-related sequences are injected subcutaneously. After an appropriate period, preferably about 4-8 weeks, tumor growth is measured (by volume or by its two largest dimensions) and compared to a control group. Tumors with a statistically effective decrease (eg using Student's T test) are said to have growth inhibition.

Cancer Polynucleotide Modulators Antisense and RNAi (RNA Interference) Polynucleotides In certain embodiments, the activity of a cancer-associated protein is an inhibitor or a nucleic acid sequence encoding a nucleic acid complement such as a cancer protein mRNA or a continuation thereof. Preferably hybridize specifically with it, down-regulated by the use of antisense polynucleotides, or completely suppressed. By binding the antisense polynucleotide to the mRNA, the translation and / or stability of the mRNA is reduced.

  In the context of the present invention, antisense polynucleotides can include naturally occurring nucleotides, or synthetic species formed from naturally occurring subunits or congeners very close thereto. Antisense polynucleotides may also alter sugar moieties or intrasugar linkages. Examples of these are thiophosphate and other sulfur-containing species. Analogs are encompassed by the present invention as long as they function effectively to hybridize with oncoprotein mRNA. See, for example, Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.

  These antisense polynucleotides can be easily synthesized by recombinant methods or synthesized in vitro. The equipment needed for the synthesis is sold by several specialists including Applied Biosystems. The preparation of other oligonucleotides such as thiophosphates and alkylate derivatives is also well known.

Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can be used, for example, to bind to the antisense strand and block transcription. Antisense and sense oligonucleotides contain single-stranded nucleic acid sequences (either RNA or DNA) that allow cancer molecules to bind to target mRNA (sense) or DNA (antisense). Preferred antisense molecules are for the cancer sequences in the table, or ligands or activators thereof. According to the present invention, an antisense or sense oligonucleotide usually comprises a fragment of at least about 14 nucleotides, preferably about 14-30 nucleotides. For the ability to extract antisense or sense oligonucleotides based on a cDNA sequence encoding a given protein, see, eg, Stein and Cohen (1988) Cancer Res. (“Cancer Res.”) 48: 2659-2668; And van der Krol, et al. (1988) BioTechniques (Biotechnique) 6: 958-976.

RNA interference is a mechanism that suppresses gene expression in a sequence-specific manner. For example, Brumelkamp, et al. (2002) Sciencexpress (21March2002); Sharp (1999) Genes Dev. (Genetic Dev.) 13: 139-141; and Catew (2001) Curr. Op. Cell Biol. (Curr. Op. Cell Biology) 13: 244-248. In mammalian cells, for example, 21 nt, double-stranded short RNA interference (siRNA) has been shown to be effective in inducing RNA interference responses. See, for example, Elbashir, et al. (2001) Nature (411) 411: 494-498. This mechanism is used to down-regulate the expression level of identified genes, for example, treatment of disease or confirmation of association.

Ribozymes In addition to antisense nucleotides, ribozymes are used to target cancer-associated nucleotide sequences or to repress their transcription. Ribozymes are RNA molecules that catalytically disrupt other RNA molecules. Various types of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P (ribonuclease P), and axhead ribozymes (eg, synthesis of various ribozyme properties) For review, see Castanotto, et al. (1994) Adv. In Pharmacology 25: 289-317).

The overall properties of hairpin ribozymes are described, for example, in Hampel, et al. (1990) Nucl. Acids Res. (Nucleic Acid Res.) 18: 299-304; European Patent Publication No. 0 360 257; US Pat. Has been. For example, WO 94/26877; Ojwang, et al. (1993) Proc. Natl. Acad. Sci. USA ("Proc. Natl. Acad. Sci. USA") 90: 6340-6344; Yamada, et al (1994) Human Gene Therapy 1: 39-45; Leavitt, et al. (1995) Proc. Natl. Acad. Sci. USA (Proc. Natl. Acad. Sci. USA) 92: 699-703; Leavitt, et al. (1994) Human Gene Therapy 5: 1151-120; and Yamada, et al. (1994) Virology 205: 121- 126.

  As described in WO91 / 04753, a polynucleotide cancer modulator is introduced into a cell containing a target nucleotide sequence by forming a bond with a ligand binding molecule. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the binding of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor and prevents the sense or antisense oligonucleotide or its binding species from invading the cell. Absent. On the other hand, a polynucleotide cancer modulator is introduced into a cell containing a target nucleic acid sequence by forming a polynucleotide / lipid complex, for example, as described in WO 90/10448. Antisense molecules, knockout models, or knock-in models have been found to be used in screening assays as described above in addition to therapeutic methods.

  Accordingly, in one embodiment, a method for modulating cancer in a cell or organism is provided. In one embodiment, the method comprises administering to the cell an anti-cancer antibody that reduces or eliminates the biological activity of the endogenous cancer protein. On the other hand, the method involves the administration of a recombinant nucleic acid encoding an oncoprotein to a cell or organism. This can be achieved in a number of ways. In preferred embodiments, such as when the cancer sequence is down-regulated in cancer, such a situation can be reversed by increasing the amount of oncogene product in the cell. This is accomplished using known gene therapy techniques, for example by overexpressing an endogenous oncogene or administering a gene encoding a cancer sequence. In a preferred embodiment, the gene therapy technique involves merging exogenous genes by enhanced cognate recombination (HER), as described, for example, in PCT / US93 / 0386. On the other hand, when the cancer sequence is up-regulated in cancer, for example, by administering an inhibitor such as cancer antisense or RNA interference, the activity of the endogenous oncogene is reduced.

  In one embodiment, the oncoproteins of the invention are used to make oncoprotein polyclonal or monoclonal antibodies. Similarly, oncoproteins bind to affinity chromatography columns by use of standard techniques. These columns are then used to purify cancer antibodies that are useful for manufacturing, diagnostic or therapeutic purposes. In a preferred embodiment, the antibody is made at an epitope unique to an oncoprotein; that is, the antibody shows little cross-reactivity to other proteins. The cancer antibody is bound to a standard affinity chromatography column and used to purify the cancer protein. The antibody is also used to block the polypeptide, as described above, because it specifically binds to the cancer protein.

Methods for identifying mutant cancer-related sequences Without being bound by theory, the expression of mutant cancer sequences is associated with cancer. Therefore, diseases based on mutant or mutated oncogenes can be measured. In one embodiment, the present invention provides a method for identifying a cell containing a mutated oncogene, such as a method for measuring all or a portion of at least one endogenous oncogene sequence in a cell. This is usually done in at least one tissue of the individual and includes the assessment of many tissues or different samples of the same tissue. The method involves a sequence comparison between a sequenced oncogene and a known oncogene, such as a wild type gene.

  The sequence of all or part of the oncogene is then compared to a known oncogene to determine if there is a difference. This is done using a known homology program such as Bestfit. In preferred embodiments, as outlined herein, the presence of a sequence difference between a patient oncogene and a known oncogene is associated with a disease state or disease state trend.

In a preferred embodiment, the oncogene is used as a probe to measure the copy number of the oncogene in the genome.
In another preferred embodiment, the oncogene is used as a probe to measure its chromosomal locality. Information such as chromosomal locality is particularly used to provide diagnosis or prognosis when chromosomal abnormalities such as translocations are identified at the oncogene locus.

Administration of drugs and vaccine preparations
In one embodiment, a therapeutically effective dose of an oncoprotein or modulator thereof is administered to the patient. In the present specification, the “therapeutically effective dose” means a dose whose administration exhibits an effect. The exact dosage depends on the purpose of the treatment and can be ascertained using known techniques. For example, Ansel, et al. (1999) Pharmaceutical Dosage Forms and Drug Delivery Lippincott; Lieberman (1992) Pharmaceutical Dosage Forms (vols. 1-3) Dekker, ISBN 0824770846, 082476918X, 0824712692, 0824716981; Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding ; Amer. Pharmaceut. Assn .; and Pickar (1998) Dosage Calculations ( Dosage calculation '') See Thomson. Along with the worsening of cancer, systemic versus local delivery, and the ratio of new protease synthesis, there is a need to adjust for age, weight, general health, sex, diet, time of administration, drug interactions and disease severity. US patent application Ser. No. 09 / 687,576 further discloses the use of the composition and methods for diagnosing and treating cancer.

  “Patient” for the purposes of the present invention includes humans and other animals, particularly mammals. The method is therefore adaptable to both human therapy and veterinary applications. In a preferred embodiment, the patient is a mammal, preferably a primate, and in the most preferred embodiment, the patient is a human.

  Administration of oncoproteins and modulators thereof of the present invention includes, but is not limited to, oral, subcutaneous, intravenous injection, intranasal, transdermal, intraperitoneal, intramuscular injection, intrapulmonary, transvaginal, transanal, intraocular. It is done in various ways. For example, in the case of external phase and inflammation treatment, oncoproteins and modulators may be applied directly in liquids or sprays.

  The pharmaceutical composition of the present invention comprises a cancer protein in a form suitable for administration to a patient. In a preferred embodiment, the pharmaceutical composition is in a water-soluble form, such as a pharmaceutically acceptable salt, including a salt with both an acid and a base added. “Pharmaceutically acceptable acid addition salts” retain the biological effectiveness of the free base and are formed from the following biological and other undesirable salts: hydrochloric acid, hydrobromic acid, sulfuric acid , Nitric acid, phosphoric acid and other inorganic acids, and acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid , Methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and other organic acids. “Pharmaceutically acceptable base addition salts” include salts obtained from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include primary, secondary, and tertiary amines, substituted amines including natural substituted amines, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. And salts such as cyclic amines and basic ion exchange resins.

The pharmaceutical composition also includes one or more of the following: carrier proteins such as serum albumin; buffers; excipients such as microcrystalline cellulose, lactose, corn starch and other starches; binders; flavoring agents such as sweeteners A colorant; and polyethylene glycol.
The pharmaceutical composition can be administered in various unit dosage forms based on the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powders, tablets, pills, capsules and troches. It is recognized that oncoprotein modulators (eg, antibodies, antisense constructs, ribozymes, small organic molecules, etc.) must be protected from digestion when administered orally. This is usually accomplished by complexing the molecule with a composition to confer resistance to acidic or enzymatic hydrolysis, or loading the molecule into a suitable resistant carrier, such as a liposome or protective barrier. There are ways to protect the drug from digestion.

Dosage compositions usually comprise an oncoprotein modulator dissolved in a pharmaceutically acceptable carrier, preferably a water soluble carrier. Various water-soluble carriers are used, such as buffered saline. These solutions are sterilized and usually free of undesirable materials. These compositions are sterilized by conventional known disinfection techniques. The composition may be pharmaceutically acceptable, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, pH regulators and buffers, toxicity regulators, etc., as necessary to approach physiological conditions. Contains possible auxiliary substances. The concentration of activator in these formulations is wide-ranging, and is initially selected based on the volume, viscosity, weight, etc. of the liquid, tailored to the particular form of administration chosen and patient needs (eg (1980) Remington's Pharmaceutical Science (18th ed.) Mack and Hardman and Limbird (eds. 2001) Goodman and Gilman: The Pharmacologial Basis of Therapeutics ed.) McGraw-Hill).

  Therefore, the usual pharmaceutical composition for intravenous administration is 0.1-10 mg per patient per day. A dosage of 0.1 to about 100 mg per patient per day may be used, especially when the drug is administered to an isolated site, such as a body cavity or organ lumen, rather than into the blood. In topical administration, substantially higher doses are possible. The actual method for preparing a parenteral composition is known or apparent.

  A composition comprising a modulator of an oncoprotein is administered for therapeutic or prophylactic treatment. In therapeutic applications, the composition is administered to a patient suffering from a disease (eg, cancer) in an amount sufficient to treat or at least partially stop the disease and its complications. An amount adequate to accomplish this is defined as a “pharmaceutically effective dose”. The effective amount for this use depends on the severity of the disease and the overall health of the patient. Single or multiple administrations of the composition are administered at a dosage and frequency as required and tolerated by the patient. In any case, the composition should provide a sufficient amount of the agent of the present invention to effectively treat the patient. In a mammal, the amount of a modulator that can prevent or delay the progression of cancer is referred to as a “prophylactically effective dose”. The particular dosage required for prophylactic treatment is determined by the mammal's medical condition and history, the particular cancer to be prevented, as well as other factors such as age, weight, sex, route of administration, and effects. Such prophylactic treatment is, for example, to prevent recurrence of cancer in a mammal previously suffering from cancer, or a mammal suspected of having a significant potential for developing cancer, based at least in part on gene expression characteristics. Used in animals. The vaccine method uses either a DNA vaccine or a protein vaccine.

It is contemplated that current cancer protein modulating compounds can be administered alone or in combination with additional cancer modulating compounds or other therapeutic agents such as other anti-cancer antibodies and treatments.
In many embodiments, one or more nucleic acids, such as polynucleotides, including the nucleic acid sequences shown in the table, such as RNA interference, antisense polynucleotides, or ribozymes, are introduced into cells ex vivo or in vivo. . The present invention provides methods, reagents, vectors and cells useful for the expression of cancer-related polypeptides and nucleic acids using ex vivo (cell-free), ex vivo, or in vivo (cell or organism based) recombinant expression systems. .

Particular means of introducing nucleic acids into host cells for protein and nucleic acid expression are application specific. Many means are used to introduce foreign nucleotide sequences into the host cell. These include calcium phosphate transfection, spheroplasts, electroporation, liposomes, microinjection, plasma vectors, viral vectors, and other genomic DNA clones, cDNA, synthetic DNA, or other foreign genetic material. Including well-known methods to be introduced (eg, Berger and Kimmel (1987) Guide to Molecular Cloning Techniques from Methods in Enzymology ) (vol. 152) Academic Press; Ausubel, et al. (Eds. 1999 and supplements) Current Protocols Lippincott; and Sambrook, et al. (2001) Molecular Cloning: A Laboratory Manual (3d ed. , Vol. 1-3) See CSH Press).

  In a preferred embodiment, the oncoprotein and modulator are administered as a therapeutic agent and formed as described above. Similarly, oncogenes (full-length sequences, both partial sequences, or regulatory sequences of cancer coding regions) are administered for gene therapy. These oncogenes include, for example, suppressive applications such as suppressor RNA, gene therapy (eg, incorporation into the genome), or antisense constructs.

Cancer polypeptides and polynucleotides are also administered as vaccine compositions for stimulating HTL, CTL, and antibody responses. These compositions include: For example, lipidated peptides (see, eg, Vitiello, et al. (1995) J. Clin. Invest. 95: 341-349), poly (DL-Lacticoglycolide) (“PLG”) peptide compositions encapsulated in microspheres (eg Eldridge, et al. (1991) Molec. Immunol. 28: 287-294; Alonso, et al. (1994) Vaccine (“Vaccine”) 12: 299-306; Jones, et al. (1995) Vaccine (“Vaccine”) 13: 675-681), immunostimulatory complex (ISCOMS) Peptide compositions contained therein (eg, Takahashi, et al. (1990) Nature (“Nature”) 344: 873-875; Hu, et al. (1998) Clin Exp Immunol. (“Clinical Exp Immunology”)) 113: 235-243), multiple antigen peptide systems (MAPs) (eg, Tam (1988) Proc. Natl. Acad. Sci. USA 85: 5409-5413; Tam (1996) J. Immunol. Methods Method Journal ”) 196: 17-32 See), peptides formed as multivalent peptides;

Peptides used in impact delivery systems, usually crystallized peptides, virus delivery vectors (Perkus, et al., P. 379, in Kaufmann (ed. 1996) Concepts in Vaccine Development ) de Gruyter Chakrabarti, et al. (1986) Nature (“Nature”) 320: 535-537; Hu, et al. (1986) Nature (“Nature”) 320: 537-540; Kieny, et al. (1986) Bio / Technology 4: 790-795; Top, et al. (1971) J. Infect. Dis. 124: 148-154; Chanda, et al. (1990) Virology (see Virology ) 175: 535-547),

Particles of viral or synthetic origin (eg, Kofler, et al. (1996) J. Immunol. Methods 192: 25-35; Eldridge, et al. (1993) Sem. Hematol. 30:16 -24; Falo, et al. (1995) Nature Med. (See Natural Med.) 1: 649-653), adjuvant (Warren, et al. (1986) Annu. Rev. Immunol. 4: 369-388 Gupta, et al. (1993) Vaccine (Vaccine) 11: 293-306), liposomes (Reddy, et al. (1992) J. Immunol. ) 148: 1585-1589; Rock (1996) Immunol. Today 17: 131-137) or naked or particle-absorbed cDNA (Ulmer, et al. (1993) Science 259: 1745 Robinson, et al. (1993) Vaccine (Vaccine) 11: 957-960; Shiver, et al., P 423, in Kaufmann (ed. 1996) Concepts in Vaccine Development ) De Gruyter; Cease and Berzofsky (1994) Annu. Rev. Immunol. 12: 923-989; and Eldridge, et al. (1993) ) Sem. Hematol. 30: 16-24). Toxin target delivery techniques, also known as receptor mediation targets, such as those from Avant Immunotherapeutics (Needham, Mass.) Are also used.

  Vaccine compositions often include an adjuvant. Many adjuvants contain substances designed to protect antigens from rapid catabolism, such as aluminum hydroxide or mineral oil, and enhance immune responses, such as lipid A, Bortadella pertussis, or proteins from Mycobacterium tuberculosis. Including. Certain adjuvants are commercially available: eg Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ) AS-2 (SmithKline Beecham, Philadelphia, PA); Aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; calcium, iron, or zinc salts; insoluble suspension of acylated tyrosine; acyl Polysaccharides derived from negative ions or cationic substances; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quill A. Cytokines such as GM-CSF, interleukin-2, -7, -12 and other similar growth factors are also used as adjuvants.

A vaccine can be administered as a nucleic acid composition in which DNA or RNA encoding one or more polypeptides or fragments thereof is administered to a patient. This approach is described, for example, in Wolff et. Al. (1990) Science 247: 1465-1468 and US Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; Details are explained. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivacaine, polymer, peptide mediated) delivery, cationic lipid complex, and particle mediated (“gene gun”) or pressure regulated Includes delivery (see US Pat. No. 5,922,687).

For therapeutic and prophylactic immunization purposes, the peptides of the invention are expressed by viral or bacterial vectors. Examples of expression vectors include attenuated viral hosts such as vaccines or fowlpox. This approach involves the use of vaccine viruses, for example, as vectors that express nucleic acid sequences encoding cancer polypeptides or polypeptide fragments. Once introduced into the host, the recombinant vaccine virus expresses the immunogenic peptide and elicits an immune response. Useful methods for vaccine vectors and immunization protocols are described, for example, in US Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover, et al. (1991) Nature ("Nature") 351: 456-460. A wide range of vectors are available for therapeutic administration and immunization, such as adeno and adeno-associated virus vectors, retrovirus vectors, typhoid vectors, detoxified anthrax toxin vectors, and the like. For example, Shata, et al. (2000) Mol Med Today (Molecular Medicine Today) 6: 66-71; Shedlock, et al. (2000) J. Leukoc. Biol. (Leukoc. Biol. Journal) 68: 793-806; See Hipp, et al. (2000) In Vivo (In Vivo) 14: 571-85.

  Methods for using genes as DNA vaccines are well known and include positioning an oncogene or part of an oncogene under the control of a promoter whose expression is controllable or a tissue-specific promoter in cancer patients. Oncogenes used in DNA vaccines can encode full-length oncoproteins, but more preferably encode portions of oncoproteins, including peptides derived from oncoproteins. In one embodiment, the patient is vaccinated with a DNA vaccine comprising a plurality of nucleotide sequences derived from an oncogene. For example, a cancer associated gene or sequence encoding a selective fragment of an oncoprotein is introduced into an expression vector and tested for the ability to generate an immunogenic and cytotoxic T cell response for Class I MHC. This step provides for the generation of a cytotoxic T cell response against cells having an antigen containing an intracellular epitope.

  In a preferred embodiment, the DNA vaccine comprises a gene encoding an adjuvant molecule with a DNA vaccine. Such adjuvant molecules include cytokines that increase immunogenic response to cancer polypeptides encoded by DNA vaccines. Additional or selective adjuvants can also be used.

  In other preferred embodiments, oncogenes are used to create animal models of cancer. When the identified oncogene is suppressed or reduced in cancer tissue, gene therapy techniques such as suppressor RNA or antisense RNA directed at the oncogene also reduce or suppress gene expression. Animal models of cancer are used to screen for modulators of cancer-related sequences or modulators of cancer. Similarly, animal genetic recombination techniques, including gene knockout techniques, for example as a result of cognate recombination with an appropriate gene target vector, result in the absence or increase of oncoprotein expression. If desired, tissue specific expression or oncoprotein knockout is required.

  It is also possible for cancer proteins to be overexpressed in cancer. In such a case, a transformed animal that overexpresses the oncoprotein is produced. Depending on the desired expression level, various strength promoters can be used for transgene expression. It is also possible to measure the total transgene copy number and compare to determine the transgene expression level. An animal produced by such a method is used as an animal model for cancer, and is also useful in screening for modulators for cancer treatment.

Kits used for diagnostic and / or prognostic applications Kits for the diagnostic, research and therapeutic uses suggested above are also provided by the present invention. For diagnostic and research purposes, these kits include at least one of the following: assay reagents, buffers, cancer-specific nucleic acids or antibodies, hybridization probes and / or primers, antisense polynucleotides, ribozymes, dominant negative cancer polypeptides. Alternatively, polynucleotides, small molecule inhibitors of cancer-related sequences, and the like. The therapeutic product contains sterile saline or other pharmaceutically acceptable emulsion base and suspension base.

  In addition, the kit includes instructional materials including instructions (eg, a protocol) for performing the method of the present invention. Regular teaching materials include, but are not limited to, written or printed materials. A medium capable of storing such instructions and transmitting them to the end user is contemplated by the present invention. Such media include, but are not limited to, electronic storage media (eg, magnetic disks, tapes, cartridges, chips), optical media (eg, CDROM), and the like. The medium may include an address of an Internet site that provides the teaching material.

  The present invention also provides kits for screening for modulators of cancer-related sequences. These kits can be prepared from readily available materials and reagents. For example, the kit includes one or more of the following substances: a cancer-related polypeptide or polynucleotide, a reagent tube, and instructions for a cancer-related activity test. Optionally, the kit includes a biologically active oncoprotein. A wide range of kits and components are prepared according to the present invention based on the intended use of the kit and the special needs of the user. Diagnosis usually involves the evaluation of multiple genes or products. The gene is usually selected based on correlation with important parameters of the disease identified in historical or outcome data.

Example 1: Gene chip assay Using a gene chip, the molecular properties of various normal and cancerous tissues are measured and assayed. RNA is isolated and a gene chip assay is performed as described (Glynne, et al. (2000) Nature (“Nature”) 403: 672-676; Zhao, et al. (2000) Genes Dev. (“Gene Dev. ] 14: 981-993).

  It will be appreciated that the above examples are presented for illustrative purposes, not to limit the scope of the present invention. All publications, sequence registration numbers, and patent applications shall be considered as specific and individually indicated as individual publications, registration numbers, or patent applications are incorporated herein. It is incorporated in the description.

Claims (19)

  A method for determining the presence or absence of pathological cells in a patient comprising a nucleic acid comprising a sequence at least 80% identical to the sequences set forth in Tables 2A to 80 in a biological sample from said patient. Said method comprising detecting and thereby determining the presence or absence of said pathological cell. a) the condition comprises cancer and is listed in Table 1; and / or
b) the biological sample comprises isolated nucleic acid;
The method of claim 1.   2. The method of claim 1, wherein the biological sample is tissue from an organ containing cancer and affected by the pathology in Table 1.   3. The method according to claim 2, wherein the nucleic acid is mRNA. a) further comprising the step of amplifying the nucleic acid prior to the step of detecting the nucleic acid; or
b) the detection is detection of a protein encoded by the nucleic acid;
The method of claim 2.   The method of claim 1, wherein the nucleic acid comprises a sequence set forth in Tables 2A-80. a) The detection step is:
i) use labeled nucleic acid probes;
ii) utilizing a biochip comprising a sequence that is at least 80% identical to the sequences set forth in Tables 2A-80; or
iii) a method performed by detecting a polypeptide encoded by the nucleic acid; or
b) The patient:
i) is receiving a treatment plan to treat the conditions in Table 1; or
ii) suspected of having said medical condition or cancer,
The method of claim 2.   An isolated nucleic acid molecule comprising a sequence set forth in Table 2A-Table 80.   9. The nucleic acid molecule according to claim 8, which is labeled.   An expression vector comprising the nucleic acid according to claim 8.   A host cell comprising the expression vector according to claim 10.   An isolated polypeptide encoded by a nucleic acid molecule comprising the sequence set forth in Tables 2A-80.   An antibody that specifically binds to the polypeptide of claim 12. a) associated with an effector component;
b) conjugated with a detection label comprising a fluorescent label, radioisotope or cytotoxic chemical;
c) an antibody fragment; or
d) is a humanized antibody,
14. The antibody according to claim 13.   14. A method of specifically targeting a compound to a patient's pathological cells, comprising administering to the patient the antibody of claim 13 thereby providing the targeting.   14. A method for determining the presence or absence of pathological cells for a patient, the method comprising contacting a biological sample with the antibody of claim 13. a) said antibody is:
i) an effector component; or
ii) fluorescent labels;
Or
b) the biological sample is a blood, serum, urine or stool sample;
The method of claim 16. A method for identifying a compound that modulates a polypeptide associated with a medical condition, comprising the following steps:
a) contacting said compound with a polypeptide related to a disease state encoded by a polynucleotide that selectively hybridizes with a sequence that is at least 80% identical to a sequence set forth in Tables 2A-80; and
b) measuring the functional effect of the compound on the polypeptide,
Including the above method. The following steps:
a) administering a test compound to a mammal having the pathology of Table 1 or cells isolated from said mammal; and
b) the gene expression level of a polynucleotide that selectively hybridizes with a sequence that is at least 80% identical to the sequence set forth in Table 2A-Table 80 in the treated cell or mammal, and in the control cell or mammal Comparing the gene expression level of the polynucleotide,
A test compound that modulates the expression level of the polynucleotide, wherein the test compound is a candidate for the treatment of the disease state.