Definitions

• the invention relates to the identification of nucleic acid and protein expression profiles and nucleic acids, products, and antibodies thereto that are involved in cancer and other diseases; and to the use of such expression profiles and compositions in the diagnosis, prognosis, and therapy of these conditions.
• the invention further relates to methods for identifying and using agents and/or targets that modulate these conditions.
• Cancers share the charactaristic of disordered control over normal cell division, growth, and differentiation. Their initial clinical manifestations are extremely heterogeneous, with over 70 types of cancer arising in virtually every organ and tissue of the body. Moreover, some of those similarly classified cancer types may represent multiple different molecular diseases. Unfortunately, some cancers may be virtually asymptomatic until late in the disease course, when treatment is more difficult, and prognosis grim. Treatment for cancer typically includes surgery, chemotherapy, and/or radiation therapy.
• antigens examples include Her2/neu and the B-cell antigen CD20.
• Humanized monclonal antibodies directed to Her2/neu are currently in use for the treatment of metastatic breast cancer. See Ross and Fletcher (1998) Stem Cells 16:413-428.
• anti-CD20 monoclonal antibodies are used to effectively treat non-Hodgkin's lymphoma. See Maloney, et al. (1997) Blood 90:2188-2195; Leget and Czuczman (1998) Curr. Opin. Oncol. 10:548-551.
• provided herein are molecular targets for therapeutic intervention in various defined cancers. Additionally, provided herein are methods that can be used in diagnosis and prognosis of cancer. Further provided are methods that can be used to screen candidate bioactive agents for the ability to modulate cancer.
• the present invention provides methods for determining the presence or absence of a pathological cell in a patient, the method comprising detecting a nucleic acid comprising a sequence at least 80% identical to a sequence as described in Tables 2A-80 in a biological sample from the patient, thereby determining the presence or absence of the pathological cell.
• the pathology is described in Table 1, including a cancer;
• the biological sample comprises isolated nucleic acids;
• the nucleic acids are mRNA;
• the biological sample is tissue from an organ which is affected by the pathology of Table 1, including a cancer;
• a further step is used of amplifying nucleic acids before the step of detecting the nucleic acid;
• the detecting is of a protein encoded by the nucleic acid;
• the nucleic acid comprises a sequence as described in Tables 2A-80;
• the detecting step is carried out by using a labeled nucleic acid probe, utilizing a biochip comprising at sequence at least 80% identical to a sequence as described in Tables 2A-80, or detecting a polypeptide encoded by the nucleic acid; or the patient is undergoing a therapeutic regimen to treat the pathology of Table 1, or is suspected of having the pathology or cancer.
• compositions are also provided, e.g., an isolated nucleic acid molecule comprising a sequence as described in Tables 2A-80, including, e.g., those which are labeled; an expression vector comprising such nucleic acid; a host cell comprising such expression vector; an isolated polypeptide which is encoded by such a nucleic acid molecule comprising a sequence as described in Tables 2A-80; or an antibody that specifically binds the polypeptide.
• the antibody is: conjugated to an effector component, is conjugated to a detectable label (including, e.g., a fluorescent label, a radioisotope, or a cytotoxic chemical), an antibody fragment, or is a humanized antibody.
• Additional methods are provided, including methods for specifically targeting a compound to a pathological cell in a patient, the method comprising administering to the patient an antibody, as described, thereby providing the targetting.
• Others include, e.g., methods for determining the presence or absence of a pathological cell in a patient, the methods comprising contacting a biological sample with an antibody, as described.
• the antibody is: conjugated to an effector component, or to a fluorescent label; or the biological sample is a blood, serum, urine, or stool sample.
• Further methods include those for identifying a compound that modulates a pathology- associated polypeptide, the method comprising steps of: contacting the compound with a pathology-associated polypeptide, the polypeptide encoded by a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as described in Tables 2A-80; and determining the functional effect of the compound upon the polypeptide.
• Another drug screening assay method comprises steps of: administering a test compound to a mammal having a pathology of Table 1 or a cell isolated therefrom; and comparing the level of gene expression of a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as described in Tables 2A-80 in a treated cell or mammal with the level of gene expression of the polynucleotide in a control cell or mammal, wherein a test compound that modulates the level of expression of the polynucleotide is a candidate for the treatment of the pathology.
• the present invention provides novel methods for diagnosis and prognosis evaluation for various disorders, e.g., angiogenesis, fibrosis, and various defined forms of cancer, including metastatic cancer, as well as methods for screening for compositions which modulate such conditions. Also provided are methods for treating such disorders or cancers. See, e.g., American Society of Clinical Oncology (ed. 2001) ASCO Curriculum: Symptom Management Kendall/Hunt, ISBN: 0787277851; Bonadonna, et al. (2001) Textbook of Breast Cancer (2d ed.) Dunitz Martin, ISBN: 1853178241; Devita and Hellman (eds.
• identification of markers selectively expressed on defined cancers allows for use of that expression in diagnostic, prognostic, or therapeutic methods.
• the invention defines various compositions, e.g., nucleic acids, polypeptides, antibodies, and small molecule agonists/antagonists, which will be useful to selectively identify those markers.
• therapeutic methods may take the form of protein therapeutics which use the marker expression for selective localization or modulation of function (for those markers which have a causative disease effect), for vaccines, identification of binding partners, or antagonism, e.g., using antisense or RNAi.
• the markers may be useful for molecular characterization of subsets of the diseases, e.g., as provided in Table 1, which subsets may actually require very different treatments. Moreover, the markers may also be important in related diseases to the specific disorders and cancers, e.g., which affect similar tissues in non-malignant diseases, or have similar mechanisms of induction/maintenance. Metastatic processes or characteristics may also be targeted. Diagnostic and prognostic uses are made available, e.g., to subset related but distinct diseases, or to determine treatment strategy. The detection methods may be based upon nucleic acid, e.g., PCR or hybridization techniques, or protein, e.g., ELISA, imaging, JJHC, etc.
• Tables 2B-76B provide unigene cluster identification numbers for the nucleotide sequence of genes that exhibit increased or decreased expression in diseased samples (see Tables 1-3), particularly sequences involved in angiogenesis, arthritis, prostate cancer, breast cancer, colorectal cancer, cervical cancer, bladder cancer, head and neck cancer, esophageal cancer, lung cancer, ovarian cancer, pancreatic cancer, renal cancer, stomach cancer, skin cancer, testicular cancer, uterine cancer, glioblastoma, Ewing sarcoma, soft tissue sarcoma, and lung fibrosis.
• Tables 2A-80 also provide an exemplar accession number that provides a nucleotide sequence that is part of the unigene cluster. Definitions
• cancer protein or “cancer polynucleotide” or “cancer-associated transcript” refers to nucleic acid and polypeptide polymo ⁇ hic variants, alleles, mutants, and interspecies homologues that: (1) have a nucleotide sequence that has greater than about 60% nucleotide sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably about 92%, 94%, 96%, 97%, 98%, or 99% or greater nucleotide sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to a nucleotide sequence of or associated with a gene of Tables 1-80; (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence encoded by a nucleotide sequence of or associated with a gene of Tables 1-80, and conservatively modified variants thereof; (3) specifically hybridize
• a polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or other mammal.
• primate e.g., human
• rodent e.g., rat, mouse, hamster
• a “cancer polypeptide” and a “cancer polynucleotide,” include both naturally occurring or recombinant forms.
• a “full length” cancer protein or nucleic acid refers to a cancer polypeptide or polynucleotide sequence, or a variant thereof, that contains elements normally contained in one or more naturally occurring, wild type cancer polynucleotide or polypeptide sequences.
• the “full length” may be prior to, or after, various stages of post-translational processing or splicing, including alternative splicing.
• Bio sample as used herein is a sample of biological tissue or fluid that contains nucleic acids or polypeptides, e.g., of a cancer protein, polynucleotide, or transcript.
• samples include, but are not limited to, tissue isolated from primates, e.g., humans, or rodents, e.g., mice, and rats.
• Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic pu ⁇ oses, archival samples, blood, plasma, serum, sputum, stool, tears, mucus, hair, skin, etc.
• Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues.
• a biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish. Livestock and domestic animals are of interest.
• Providing a biological sample means to obtain a biological sample for use in methods described in this invention. Most often, this will be done by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another pu ⁇ ose), or by performing the methods of the invention in vivo. Archival tissues or materials, having treatment or outcome history, will be particularly useful.
• nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 93%, 95%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using, e.g., a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).
• sequences are then said to be “substantially identical.”
• This definition also refers to, or may be applied to, the complement of a test sequence.
• the definition also includes sequences that have deletions and/or insertions, substitutions, and naturally occurring, e.g., polymo ⁇ hic or allelic variants, and man-made variants.
• the preferred algorithms can account for gaps and the like.
• identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is about 50-100 amino acids or nucleotides in length.
• For sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
• test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated.
• the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
• a "comparison window" includes reference to a segment of contiguous positions selected from the group consisting typically of from about 20 to 600, usually about 50 to 200, more usually about 100 to 150, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known.
• Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-489, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444-2448, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel, et al. (eds. 1995 and supplements) Current Protocols in Molecular Biology Wiley).
• BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
• This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased.
• HSPs high scoring sequence pairs
• Cumulative scores are calculated using, e.g., for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0).
• M forward score for a pair of matching residues; always > 0
• N penalty score for mismatching residues; always ⁇ 0.
• a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• W wordlength
• E expectation
• the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See, e.g., Karlin and Altschul (1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5787.
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• P(N) the smallest sum probability
• a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
• Log values may be negative large numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110, 150, 170, etc.
• nucleic acid sequences are substantially identical.
• a polypeptide is typically substantially identical to a second polypeptide, e.g., where the two peptides differ only by conservative substitutions.
• Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions.
• Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.
• a "host cell” is a naturally occurring cell or a transformed cell that contains an expression vector and supports the replication or expression of the expression vector.
• Host cells may be cultured cells, explants, cells in vivo, and the like.
• Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa, and the like (see, e.g., the American Type Culture Collection catalog or web site, www.atcc.org).
• isolated refers to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein or nucleic acid that is the predominant species present in a preparation is substantially purified. In particular, an isolated nucleic acid is separated from some open reading frames that naturally flank the gene and encode proteins other than protein encoded by the gene.
• purified in some embodiments denotes that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
• nucleic acid or protein is at least about 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.
• “Purify” or “purification” in other embodiments means removing at least one contaminant or component from the composition to be purified. In this sense, purification does not require that the purified compound be homogeneous, e.g., 100% pure.
• polypeptide polypeptide
• peptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.
• amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids.
• Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ -carboxyglutamate, and O- phosphoserine.
• Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
• Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain somebasic chemical structure as a naturally occurring amino acid.
• Amino acid mimetic refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but that functions similarly to another amino acid.
• nucleotides likewise, may be referred to by their commonly accepted single-letter codes.
• Constantly modified variant applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated, e.g., naturally contiguous, sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG, and GCU each encode the amino acid alanine.
• nucleic acid variations are "silent variations," which are one species of conservatively modified variations.
• Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid.
• each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
• TGG which is ordinarily the only codon for tryptophan
• amino acid sequences one of skill will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known. Such conservatively modified variants are in addition to and do not exclude polymo ⁇ hic variants, interspecies homologs, and alleles of the invention.
• conservative substitutions include for one another: 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, e.g., Creighton (1984) Proteins: Structure and Molecular Properties Freeman).
• Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts, ( et al. (eds. 2001) Molecular Biology of the Cell (4th ed.) Garland; and Cantor and Schimmel
• “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that often form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of ⁇ -sheet and ⁇ -helices. "Tertiary structure” refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure” refers to the three dimensional structure formed, usually by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
• Nucleic acid or “oligonucleotide” or “polynucleotide” or grammatical equivalents used herein means at least two nucleotides covalently linked together. Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50, or more nucleotides in length, up to about 100 nucleotides in length. Nucleic acids and polynucleotides are a polymers of any length, including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000, etc.
• a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkahge, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein (1992) Oligonucleotides and Analogues: A Practical Approach Oxford Univ. Press); and peptide nucleic acid backbones and linkages.
• Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Patent Nos.
• nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g., to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
• nucleic acid analogs including, e.g., phosphoramidate (Beaucage, et al. (1993) Tetrahedron 49:1925-1963 and references therein; Letsinger (1970) J. Org. Chem. 35:3800-3803; Sblul, 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.
• nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids (see Jenkins, et al. (1995) Chem. Soc. Rev, pp 169-176). Several nucleic acid analogs are described in Rawls (page 35, June 2, 1997) C&E News.
• PNA peptide nucleic acids
• These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in at least two advantages.
• the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (T m ) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4° C drop in T m for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9° C.
• PNAs are not degraded by cellular enzymes, and thus can be more stable.
• the nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
• the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence.
• the nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.
• Transcript typically refers to a naturally occurring RNA, e.g., a pre-mRNA, hnRNA, or mRNA.
• nucleoside includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides.
• nucleoside includes non-natural occurring analog structures. Thus, e.g., the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
• a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, physiological, chemical, or other physical means.
• labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies, antigens, or epitope tags; and c) colored or fluorescent dyes.
• the labels may be inco ⁇ orated into the cancer nucleic acids, proteins, and antibodies.
• the label should be capable of producing, either directly or indirectly, a detectable signal.
• the detectable moiety may be a radioisotope, such as ⁇ H, 14c, 32p s 35 ⁇ 5 or 125j 5 electron-dense reagents, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable such as alkaline phosphatase, beta-galactosidase, or horseradish peroxidase. Methods are known for conjugating the antibody to the label. See, e.g., Hunter, et al.
• effector or “effector moiety” or “effector component” is a molecule that is bound (or linked, or conjugated), either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds, to an antibody.
• the "effector” can be a variety of molecules including, e.g., detection moieties including radioactive compounds, fluorescent compounds, enzymes or substrates, tags such as epitope tags, toxins; activatable moieties, chemotherapeutic agents; lipases; antibiotics; chemoattracting moieties, immune modulators (micA/B), or radioisotopes, e.g., emitting "hard” beta, radiation.
• a "labeled nucleic acid probe or oligonucleotide” is one that is bound, e.g., covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
• methods using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin.
• nucleic acid probe or oligonucleotide is a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, e.g., through hydrogen bond formation.
• a probe may include natural (e.g., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
• the bases in a probe may be joined by a linkage other than a phosphodiester bond, preferably one that does not functionally interfere with hybridization.
• probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. Probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
• the probes are preferably directly labeled, e.g., with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled, e.g., with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
• Diagnosis or prognosis may be based at the genomic level, or at the level of RNA or protein expression.
• the term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
• recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.
• nucleic acid By the term “recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this manner, operably linkage of different sequences is achieved.
• an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined are both considered recombinant for the pu ⁇ oses of this invention.
• nucleic acid once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, e.g., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non- recombinantly, are still considered recombinant for the pu ⁇ oses of the invention.
• a "recombinant protein” is a protein made using recombinant techniques, e.g., through the expression of a recombinant nucleic acid as depicted above.
• a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics.
• the protein may be isolated or purified away from some or most of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure.
• An isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample.
• a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred.
• the definition includes the production of a cancer protein from one organism in a different organism or host cell.
• the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
• the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.
• heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not normally found in the same relationship to each other in nature.
• the nucleic acid is typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
• a heterologous protein will often refer to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
• a “promoter” is typically an array of nucleic acid control sequences that direct transcription of a nucleic acid.
• a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
• a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
• a “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
• An “inducible” promoter is active under environmental or developmental regulation.
• operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, e.g., wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
• An "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
• the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
• the expression vector includes a nucleic acid to be transcribed in operable linkage to a promoter.
• stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in "Overview of principles of hybridization and the strategy of nucleic acid assays” in Tijssen (1993) Hybridization with Nucleic Probes (Laboratory Techniques in Biochemistry and Molecular Biology) (vol. 24) Elsevier. Generally, stringent conditions are selected to be about 5-10° C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
• T m thermal melting point
• the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
• Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C for short probes (e.g., about 10-50 nucleotides) and at least about 60° C for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is typically at least two times background, preferably 10 times background hybridization.
• Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42° C, or, 5x SSC, 1% SDS, incubating at 65° C, with wash in 0.2x SSC, and 0.1% SDS at 65° C.
• a temperature of about 36° C is typical for low stringency amplification, although annealing temperatures may vary between about 32°-48° C depending on primer length.
• a temperature of about 62° C is typical, although high stringency annealing temperatures can range from about 50-65° C, depending on the primer length and specificity.
• Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90-95° C for 30-120 sec, an annealing phase lasting 30-120 sec, and an extension phase of about 72° C for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications Academic Press, NY.
• Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
• Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C, and a wash in IX SSC at 45° C. A positive hybridization is typically at least twice background. Alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous references, e.g., Ausubel, et al. (eds. 1991 and supplements) Current Protocols in Molecular Biology Wiley.
• the phrase "functional effects" in the context of assays for testing compounds that modulate activity of a cancer protein includes the determination of a parameter that is indirectly or directly under the influence of the cancer protein or nucleic acid, e.g., a physiological, functional, physical, or chemical effect, such as the ability to decrease cancer. It includes ligand binding activity; cell viability; cell growth on soft agar; anchorage dependence; contact inhibition and density limitation of growth; cellular proliferation; cellular transformation; growth factor or serum dependence; tumor specific marker levels; invasiveness into Matrigel; tumor growth and metastasis in vivo; mRNA and protein expression in cells undergoing metastasis; and other characteristics of cancer cells. "Functional effects” include in vitro, in vivo, and ex vivo activities.
• determining the functional effect is meant assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a cancer protein sequence, e.g., physiological, functional, enzymatic, physical, or chemical effects.
• Such functional effects can be measured, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein, measuring inducible markers or transcriptional activation of the cancer protein, measuring binding activity or binding assays, e.g., binding to antibodies or other ligands, and measuring growth, cellular proliferation, cell viability, cellular transformation, growth factor or serum dependence, tumor specific marker levels, invasiveness into Matrigel, tumor growth and metastasis in vivo, mRNA and protein expression, and other characteristics of cancer cells.
• spectroscopic characteristics e.g., fluorescence, absorbance, refractive index
• hydrodynamic e.g
• the functional effects can be evaluated by many means, e.g., microscopy for quantitative or qualitative measures of alterations in mo ⁇ hological features, measurement of changes in RNA or protein levels for cancer-associated sequences, measurement of RNA stability, identification of downstream or reporter gene expression (CAT, luciferase, /3-gal, GFP, and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and ligand binding assays.
• microscopy for quantitative or qualitative measures of alterations in mo ⁇ hological features
• measurement of changes in RNA or protein levels for cancer-associated sequences e.g., measurement of changes in RNA or protein levels for cancer-associated sequences
• RNA stability e.g., identification of downstream or reporter gene expression (CAT, luciferase, /3-gal, GFP, and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and
• Inhibitors are used to refer to activating, inhibitory, or modulating molecules or compounds identified using in vitro and in vivo assays of cancer polynucleotide and polypeptide sequences.
• Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of cancer proteins, e.g., antagonists.
• Antisense or inhibitory nucleic acids may seem to inhibit expression and subsequent function of the protein.
• Activators are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate cancer protein activity.
• Inhibitors, activators, or modulators also include genetically modified versions of cancer proteins, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, small chemical molecules, and the like.
• Such assays for inhibitors and activators include, e.g., expressing the cancer protein in vitro, in cells, or cell membranes, applying putative modulator compounds, and then determining the functional effects on activity, as described above.
• Activators and inhibitors of cancer can also be identified by incubating cancer cells with the test compound and determining increases or decreases in the expression of 1 or more cancer proteins, e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more cancer proteins, such as cancer proteins encoded by the sequences set out in Tables 2A-80.
• 1 or more cancer proteins e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more cancer proteins, such as cancer proteins encoded by the sequences set out in Tables 2A-80.
• Samples or assays comprising cancer proteins that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition.
• Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%.
• Inhibition of a polypeptide is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%.
• Activation of a cancer polypeptide is achieved when the activity value relative to the control (untreated with activators) is about 110%, more preferably 150%, more preferably 200-500% (e.g., two to five fold higher relative to the control), more preferably 1000-3000% higher.
• change in cell growth refers to any change in cell growth and proliferation characteristics in vitro or in vivo, such as cell viability, formation of foci, anchorage independence, semi-solid or soft agar growth, changes in contact inhibition and density limitation of growth, loss of growth factor or serum requirements, changes in cell mo ⁇ hology, gaining or losing immortalization, gaining or losing tumor specific markers, ability to form or suppress tumors when injected into suitable animal hosts, and/or immortalization of the cell.
• Tumor cell refers to precancerous, cancerous, and normal cells in a tumor.
• Cancer cells “transformed” cells or “transformation” in tissue culture, refers to spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and inco ⁇ oration of new genomic DNA, or uptake of exogenous DNA, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation is associated with phenotypic changes, such as immortalization of cells, aberrant growth control, nonmo ⁇ hological changes, and/or malignancy.
• Antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
• the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda.
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.
• IgG, IgM, IgA, IgD, and IgE immunoglobulin classes
• An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
• the terms variable light chain (Nj_) and variable heavy chain (VJJ) refer to these light and heavy chains respectively.
• Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases.
• pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)*2 a dimer of Fab which itself is a light chain joined to VJJ-CJJI by a disulfide bond.
• the F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)*2 dimer into an Fab' monomer.
• the Fab 1 monomer is essentially Fab with part of the hinge region (see Paul (ed.
• antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant D ⁇ A methodology.
• the term antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant D ⁇ A methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty, et al. (1990) Nature 348:552-554).
• phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens. See, e.g., McCafferty, et al. (1990) Nature 348:552-554; Marks, et al. (1992) Biotechnology 10:779-783.
• a "chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced, or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, effector function, chemoattractant, immune modulator, etc.; or (b) the variable region, or a portion thereof, is altered, replaced, or exchanged with a variable region having a different or altered antigen specificity.
• the expression levels of genes are determined in different patient samples for which diagnosis information is desired, to provide expression profiles.
• An expression profile of a particular sample is essentially a "finge ⁇ rint" of the state of the sample; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is characteristic of the state of the cell. That is, normal tissue may be distinguished from cancerous or metastatic cancerous tissue, or cancer tissue or metastatic cancerous tissue can be compared with tissue from surviving cancer patients. By comparing expression profiles of tissue in known different cancer states, information regarding which genes are important (including both up-and down- regulation of genes) in each of these states is obtained. Molecular profiling may distinguish subtypes of a currently collective disease designation, e.g., different forms of a cancer.
• sequences that are differentially expressed in cancer versus non- cancer tissue allows the use of this information in a number of ways. For example, a particular treatment regime may be evaluated: does a chemotherapeutic drug act to down-regulate cancer, and thus tumor growth or recurrence, in a particular patient. Alternatively, a treatment step may induce other markers which may be used as targets to destroy tumor cells. Similarly, diagnosis and treatment outcomes may be done or confirmed by comparing patient samples with the known expression profiles. Maliganant disease may be compared to non-malignant conditions. Metastatic tissue can also be analyzed to determine the stage of cancer in the tissue, or origin of primary tumor, e.g., metastasis from a remote primary site.
• these gene expression profiles allow screening of drug candidates with an eye to mimicking or altering a particular expression profile; e.g., screening can be done for drugs that suppress the cancer expression profile. This may be done by making biochips comprising sets of the important cancer genes, which can then be used in these screens. These methods can also be done on the protein basis; that is, protein expression levels of the cancer proteins can be evaluated for diagnostic pu ⁇ oses or to screen candidate agents.
• the cancer nucleic acid sequences can be administered for gene therapy pu ⁇ oses, including the administration of antisense nucleic acids, or the cancer proteins (including antibodies and other modulators thereof) administered as therapeutic drugs.
• cancer sequences include those that are up-regulated (e.g., expressed at a higher level) in cancer, as well as those that are down-regulated (e.g., expressed at a lower level).
• the cancer sequences are from humans; however, cancer sequences from other organisms may be useful in animal models of disease and drug evaluation; thus, other cancer sequences are provided, from vertebrates, including mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc.) and pets (e.g., dogs, cats, etc.). Cancer sequences from other organisms may be obtained using the techniques outlined below. Cancer sequences can include both nucleic acid and amino acid sequences. In a preferred embodiment, the skin cancer sequences are recombinant nucleic acids.
• nucleic acid sequences are useful in a variety of applications, including diagnostic applications, which will detect naturally occurring nucleic acids, as well as screening applications; e.g., biochips comprising nucleic acid probes or PCR microtiter plates with selected probes to the cancer sequences.
• a cancer sequence can be initially identified by substantial nucleic acid and/or amino acid sequence homology to the cancer sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, e.g., using homology programs or hybridization conditions.
• the cancer screen typically includes comparing genes identified in different tissues, e.g., normal and cancerous tissues, cancer and non-malignant conditions, non-malignant conditions and normal tissues, or tumor tissue samples from patients who have metastatic disease vs. non metastatic tissue.
• Other suitable tissue comparisons include comparing cancer samples with metastatic cancer samples from other cancers, such as lung, stomach, gastrointestinal cancers, etc.
• Samples of different stages of cancer e.g., survivor tissue, drug resistant states, and tissue undergoing metastasis, are applied to biochips comprising nucleic acid probes. The samples are first microdissected, if applicable, and treated for preparation of mRNA.
• Suitable biochips are commercially available, e.g., from Affymetrix, Santa Clara, CA. Gene expression profiles as described herein are generated and the data analyzed.
• the genes showing changes in expression as between normal and disease states are compared to genes expressed in other normal tissues, including, and not limited to lung, heart, brain, liver, stomach, kidney, muscle, colon, small intestine, large intestine, spleen, bone, and/or placenta.
• those genes identified during the cancer screen that are expressed in a significant amount in other tissues are removed from the profile, although in some embodiments, this is not necessary (e.g., where organs may be dispensible, e.g., female or male specific). That is, when screening for drugs, it is usually preferable that the target expression be disease specific, to minimize possible side effects on other organs were there expression.
• cancer sequences are those that are up-regulated in cancer; that is, the expression of these genes is higher in the cancer tissue as compared to non-cancer or non-malignant tissue.
• Up-regulation as used herein often means at least about a two-fold change, preferably at least about a three fold change, with at least about five-fold or higher being preferred.
• Another embodiment is directed to sequences up-regulated in non-malignant conditions relative to normal. Uniformity among relevant samples is also preferred.
• Unigene cluster identification numbers and accession numbers herein are for the GenBank sequence database and the sequences of the accession numbers are hereby expressly inco ⁇ orated by reference.
• GenBank is available, see, e.g., Benson, et al. (1998) Nuc. Acids Res. 26:1-7; and http://www.ncbi.nlm.nih.gov/.
• Sequences are also available in other databases, e.g., European Molecular Biology Laboratory (EMBL) and DNA Database of Japan (DDBJ).
• the sequences may be derived from assembly of available sequences or be predicted from genomic DNA using exon prediction algorithms, such as FGENESH. See Salamov and Solovyev (2000) Genome Res. 10:516-522.
• sequences have been derived from cloning and sequencing of isolated nucleic acids.
• cancer sequences are those that are down-regulated in the cancer; that is, the expression of these genes is lower in cancer tissue as compared to non- cancerous tissue.
• Down-regulation as used herein often means at least about a two-fold change, preferably at least about a three fold change, with at least about five-fold or higher being preferred.
• the ability to identify genes that are over or under expressed in cancer can additionally provide high-resolution, high-sensitivity datasets which can be used in the areas of diagnostics, therapeutics, drug development, pharmacogenetics, protein structure, biosensor development, and other related areas.
• the expression profiles can be used in diagnostic or prognostic evaluation of patients with cancer or related diseases. See Tables 1-3.
• subcellular toxicological information can be generated to better direct drug structure and activity correlation (see Anderson (June 11-12, 1998) Pharmaceutical Proteomics: Targets, Mechanism, and Function, paper presented at the JJ3C Proteomics conference, Coronado, CA).
• Subcellular toxicological information can also be utilized in a biological sensor device to predict the likely toxicological effect of chemical exposures and likely tolerable exposure thresholds (see U.S. Patent No. 5,811,231). Similar advantages accrue from datasets relevant to other biomolecules and bioactive agents (e.g., nucleic acids, saccharides, lipids, drugs, and the like).
• the present invention provides a database that includes at least one set of assay data.
• the data contained in the database is acquired, e.g., using array analysis either singly or in a library format.
• the database can be in a form in which data can be maintained and transmitted, but is preferably an electronic database.
• the electronic database of the invention can be maintained on any electronic device allowing for the storage of and access to the database, such as a personal computer, but is preferably distributed on a wide area network, such as the World Wide Web.
• the focus of the present section on databases that include peptide sequence data is for clarity of illustration only. Similar databases can be assembled for assay data acquired using an assay of the invention.
• compositions and methods for identifying and/or quantitating the relative and/or absolute abundance of a variety of molecular and macromolecular species from a biological sample representing cancer e.g., the identification of cancer-associated sequences described herein, provide an abundance of information which can be correlated with pathological conditions, predisposition to disease, drug testing, therapeutic monitoring, gene-disease causal linkages, identification of correlates of immunity and physiological status, among others.
• data generated from the assays of the invention is suited for manual review and analysis, in a preferred embodiment, data processing using high-speed computers is utilized.
• U.S. Patents 6,023,659 and 5,966,712 disclose a relational database system for storing biomolecular sequence information in a manner that allows sequences to be catalogued and searched according to one or more protein function hierarchies.
• U.S. Patent 5,953,727 discloses a relational database having sequence records containing information in a format that allows a collection of partial-length DNA sequences to be catalogued and searched according to association with one or more sequencing projects for obtaining full-length sequences from the collection of partial length sequences.
• Patent 5,706,498 discloses a gene database retrieval system for making a retrieval of a gene sequence similar to a sequence data item in a gene database based on the degree of similarity between a key sequence and a target sequence.
• U.S. Patent 5,538,897 discloses a method using mass spectroscopy fragmentation patterns of peptides to identify amino acid sequences in computer databases by comparison of predicted mass spectra with experimentally-derived mass spectra using a closeness-of-fit measure.
• U.S. Patent 5,926,818 discloses a multi-dimensional database comprising a functionality for multidimensional data analysis described as on-line analytical processing (OLAP), which entails the consolidation of projected and actual data according to more than one consolidation path or dimension.
• OLAP on-line analytical processing
• Patent 5,295,261 reports a hybrid database structure in which the fields of each database record are divided into two classes, navigational and informational data, with navigational fields stored in a hierarchical topological map which can be viewed as a tree structure or as the merger of two or more such tree structures. See also Baxevanis, et al. (2001) Bioinformatics: A Practical Guuide to the Analysis of Genes and Proteins Wiley; Mount (2001) Bioinformatics: Sequence and Genome Analysis CSH Press, NY; Durbin, et al. (eds. 1999) Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids Cambridge University Press; Baxevanis and Oeullette (eds.
• the present invention provides a computer database comprising a computer and software for storing in computer-retrievable form assay data records cross-tabulated, e.g., with data specifying the source of the target-containing sample from which each sequence specificity record was obtained.
• At least one of the sources of target-containing sample is from a control tissue sample known to be free of pathological disorders.
• at least one of the sources is a known pathological tissue specimen, e.g., a neoplastic lesion or another tissue specimen to be analyzed for cancer.
• the assay records cross-tabulate one or more of the following parameters for each target species in a sample: (1) a unique identification code, which can include, e.g., a target molecular structure and/or characteristic separation coordinate (e.g., electrophoretic coordinates); (2) sample source; and (3) absolute and/or relative quantity of the target species present in the sample.
• the invention also provides for the storage and retrieval of a collection of target data in a computer data storage apparatus, which can include magnetic disks, optical disks, magneto- optical disks, DRAM, SRAM, SGRAM, SDRAM, RDRAM, DDR RAM, magnetic bubble memory devices, and other data storage devices, including CPU registers and on-CPU data storage arrays.
• the target data records are stored as a bit pattern in an array of magnetic domains on a magnetizable medium or as an array of charge states or transistor gate states, such as an array of cells in a DRAM device (e.g., each cell comprised bf a transistor and a charge storage area, which may be on the transistor).
• the invention provides such storage devices, and computer systems built therewith, comprising a bit pattern encoding a protein expression finge ⁇ rint record comprising unique identifiers for at least 10 target data records cross-tabulated with target source.
• the invention preferably provides a method for identifying related peptide or nucleic acid sequences, comprising performing a computerized comparison between a peptide or nucleic acid sequence assay record stored in or retrieved from a computer storage device or database and at least one other sequence.
• the comparison can include a sequence analysis or comparison algorithm or computer program embodiment thereof (e.g., FASTA, TFASTA, GAP, BESTFIT) and/or the comparison may be of the relative amount of a peptide or nucleic acid sequence in a pool of sequences determined from a polypeptide or nucleic acid sample of a specimen.
• the invention also preferably provides a magnetic disk, such as an IBM-compatible (DOS, Windows, Windows95/98/2000, Windows NT, OS/2) or other format (e.g., Linux, SunOS, Solaris, AIX, SCO Unix, VMS, MV, Macintosh, etc.) floppy diskette or hard (fixed, Winchester) disk drive, comprising a bit pattern encoding data from an assay of the invention in a file format suitable for retrieval and processing in a computerized sequence analysis, comparison, or relative quantitation method.
• a magnetic disk such as an IBM-compatible (DOS, Windows, Windows95/98/2000, Windows NT, OS/2) or other format (e.g., Linux, SunOS, Solaris, AIX, SCO Unix, VMS, MV, Macintosh, etc.) floppy diskette or hard (fixed, Winchester) disk drive, comprising a bit pattern encoding data from an assay of the invention in a file format suitable for retrieval and processing
• the invention also provides a network, comprising a plurality of computing devices linked via a data link, such as an Ethernet cable (coax or lOBaseT), telephone line, ISDN line, wireless network, optical fiber, or other suitable signal transmission medium, whereby at least one network device (e.g., computer, disk array, etc.) comprises a pattern of magnetic domains (e.g., magnetic disk) and/or charge domains (e.g., an array of DRAM cells) composing a " bit pattern encoding data acquired from an assay of the invention.
• a network device e.g., computer, disk array, etc.
• a pattern of magnetic domains e.g., magnetic disk
• charge domains e.g., an array of DRAM cells
• the invention also provides a method for transmitting assay data that includes generating an electronic signal on an electronic communications device, such as a modem, ISDN terminal adapter, DSL, cable modem, ATM switch, or the like, wherein the signal includes (in native or encrypted format) a bit pattern encoding data from an assay or a database comprising a plurality of assay results obtained by the method of the invention.
• an electronic communications device such as a modem, ISDN terminal adapter, DSL, cable modem, ATM switch, or the like
• the signal includes (in native or encrypted format) a bit pattern encoding data from an assay or a database comprising a plurality of assay results obtained by the method of the invention.
• the invention provides a computer system for comparing a query target to a database containing an array of data structures, such as an assay result obtained by the method of the invention, and ranking database targets based on the degree of identity and gap weight to the target data.
• a central processor is preferably initialized to load and execute the computer program for alignment and/or comparison of the assay results.
• Data for a query target is entered into the central processor via an I/O device.
• Execution of the computer program results in the central processor retrieving the assay data from the data file, which comprises a binary description of an assay result.
• the target data or record and the computer program can be transferred to secondary memory, which is typically random access memory (e.g., DRAM, SRAM, SGRAM, or SDRAM).
• Targets are ranked according to the degree of correspondence between a selected assay characteristic (e.g., binding to a selected affinity moiety) and the same characteristic of the query target and results are output via an I/O device.
• a central processor can be a conventional computer (e.g., Intel Pentium, PowerPC, Alpha, PA-8000, SPARC, MIPS 4400, MIPS 10000, VAX, etc.);
• a program can be a commercial or public domain molecular biology software package (e.g., UWGCG Sequence Analysis Software, Darwin);
• a data file can be an optical or magnetic disk, a data server, a memory device (e.g., DRAM, SRAM, SGRAM, SDRAM, EPROM, bubble memory, flash memory, etc.);
• an I/O device can be a terminal comprising a video display and a keyboard, a modem, an ISDN terminal adapter, an Ethernet port, a punched card reader, a magnetic strip reader, or other suitable I/O device.
• the invention also preferably provides the use of a computer system, such as that described above, which comprises: (1) a computer; (2) a stored bit pattern encoding a collection of peptide sequence specificity records obtained by the methods of the invention, which may be stored in the computer; (3) a comparison target, such as a query target; and (4) a program for alignment and comparison, typically with rank-ordering of comparison results on the basis of computed similarity values.
• a computer system such as that described above, which comprises: (1) a computer; (2) a stored bit pattern encoding a collection of peptide sequence specificity records obtained by the methods of the invention, which may be stored in the computer; (3) a comparison target, such as a query target; and (4) a program for alignment and comparison, typically with rank-ordering of comparison results on the basis of computed similarity values.
• a computer system such as that described above, which comprises: (1) a computer; (2) a stored bit pattern encoding a collection of peptide sequence specificity records obtained by the methods of the invention,
• Cancer proteins of the present invention may be classified as secreted proteins, transmembrane proteins, or intracellular proteins.
• the cancer protein is an intracellular protein.
• Intracellular proteins may be found in the cytoplasm and/or in the nucleus. Intracellular proteins are involved in all aspects of cellular function and replication (including, e.g., signaling pathways); aberrant expression of such proteins often results in unregulated or disregulated cellular processes (see, e.g., Alberts, et al. (eds. 1994) Molecular Biology of the Cell (3d ed.) Garland).
• intracellular proteins have enzymatic activity such as protein kinase activity, protein phosphatase activity, protease activity, nucleotide cyclase activity, polymerase activity, and the like.
• Intracellular proteins also serve as docking proteins that are involved in organizing complexes of proteins, or targeting proteins to various subcellular localizations, and are involved in maintaining the structural integrity of organelles.
• Src-homology-2 (SH2) domains bind tyrosine-phosphorylated targets in a sequence dependent manner.
• PTB domains which are distinct from SH2 domains, also bind tyrosine phosphorylated targets.
• SH3 domains bind to proline-rich targets.
• PH domains, tetratricopeptide repeats and WD domains have been shown to mediate protein-protein interactions.
• Pfam protein families
• Pfam protein families
• the cancer sequences are transmembrane proteins.
• Transmembrane proteins are molecules that span a phospholipid bilayer of a cell. They may have an intracellular domain, an extracellular domain, or both. The intracellular domains of such proteins may have a number of functions including those already described for intracellular proteins. For example, the intracellular domain may have enzymatic activity and/or may serve as a binding site for additional proteins. Frequently the intracellular domain of transmembrane proteins serves both roles. For example certain receptor tyrosine kinases have both protein kinase activity and SH2 domains. In addition, autophosphorylation of tyrosines on the receptor molecule itself, creates binding sites for additional SH2 domain containing proteins.
• Transmembrane proteins may contain from one to many transmembrane domains.
• receptor tyrosine kinases certain cytokine receptors, receptor guanylyl cyclases and receptor serine/threonine protein kinases contain a single transmembrane domain.
• various other proteins including channels and adenylyl cyclases contain numerous transmembrane domains.
• Many important cell surface receptors such as G protein coupled receptors (GPCRs) are classified as "seven transmembrane domain" proteins, as they contain 7 membrane spanning regions. Characteristics of transmembrane domains include approximately 17 consecutive hydrophobic amino acids that may be followed by charged amino acids.
• transmembrane protein receptors include, but are not limited to the insulin receptor, insulin-like growth factor receptor, human growth hormone receptor, glucose transporters, transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor, epidermal growth factor receptor, leptin receptor, and interleukin receptors, e.g., IL-1 receptor, E -2 receptor, etc.
• extracellular domains of transmembrane proteins are diverse; however, conserved motifs are found repeatedly among various extracellular domains. conserveed structure and/or functions have been ascribed to different extracellular motifs. Many extracellular domains are involved in binding to other molecules. In one aspect, extracellular domains are found on receptors. Factors that bind the receptor domain include circulating ligands, which may be peptides, proteins, or small molecules such as adenosine and the like. For example, growth factors such as EGF, FGF, and PDGF are circulating growth factors that bind to their cognate receptors to initiate a variety of cellular responses. Other factors include cytokines, mitogenic factors, neurotrophic factors, and the like.
• Extracellular domains also bind to cell-associated molecules. In this respect, they may mediate cell-cell interactions.
• Cell-associated ligands can be tethered to the cell, e.g., via a glycosylphosphatidylinositol (GPI) anchor, or may themselves be transmembrane proteins.
• Extracellular domains may also associate with the extracellular matrix and contribute to the maintenance of the cell structure.
• Cancer proteins that are transmembrane are particularly preferred in the present invention as they are readily accessible targets for immunotherapeutics, as are described herein.
• transmembrane proteins can be also useful in imaging modalities. Antibodies may be used to label such readily accessible proteins in situ.
• antibodies can also label intracellular proteins, in which case samples are typically permeablized to provide access to intracellular proteins.
• membrane proteins can be processed to release a soluble protein, or to expose a residual fragment. Released soluble proteins may be useful diagnostic markers, processed residual protein fragments may be useful lung markers of disease.
• transmembrane protein can be made soluble by removing transmembrane sequences, e.g., through recombinant methods.
• transmembrane proteins that have been made soluble can be made to be secreted through recombinant means by adding an appropriate signal sequence.
• the cancer proteins are secreted proteins; the secretion of which can be either constitutive or regulated. These proteins may have a signal peptide or signal sequence that targets the molecule to the secretory pathway. Secreted proteins are involved in numerous physiological events; e.g., if circulating, they often serve to transmit signals to various other cell types.
• the secreted protein may function in an autocrine manner (acting on the cell that secreted the factor), a paracrine manner (acting on cells in close proximity to the cell that secreted the factor), an endocrine manner (acting on cells at a distance, e.g, secretion into the blood stream), or exocrine (secretion, e.g., through a duct or to adjacent epithelial surface as sweat glands, sebaceous glands, pancreatic ducts, lacrimal glands, mammary glands, wax producing glands of the ear, etc.).
• secreted molecules often find use in modulating or altering numerous aspects of physiology.
• Cancer proteins that are secreted proteins are particularly preferred in the present invention as they serve as good targets for diagnostic markers, e.g., for blood, plasma, serum, or stool tests. Those which are enzymes may be antibody or small molecule targets. Others may be useful as vaccine targets, e.g., via CTL mechanisms. Use of cancer nucleic acids
• cancer sequence is initially identified by substantial nucleic acid and/or amino acid sequence homology or linkage to the cancer sequences outlined herein.
• homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions.
• linked sequences on a mRNA are found on the same molecule.
• percent identity can be determined using an algorithm such as BLAST.
• a preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
• Alignment may include the introduction of gaps in the sequences to be aligned.
• the percentage of homology may be determined based on the number of homologous nucleosides in relation to the total number of nucleosides. Thus, e.g., homology of sequences shorter than those of the sequences identified will be determined using the number of nucleosides in the shorter sequence.
• the nucleic acid homology is determined through hybridization studies.
• nucleic acids which hybridize under high stringency to a described nucleic acid, or its complement, or is also found on naturally occurring mRNAs is considered a cancer sequence.
• less stringent hybridization conditions are used; e.g., moderate or low stringency conditions may be used; see Ausubel, supra, and Tijssen, supra.
• the cancer nucleic acid sequences of the invention e.g., the sequences in Tables 1-80, can be fragments of larger genes, e.g., they are nucleic acid segments. "Genes" in this context includes coding regions, non-coding regions, and mixtures of coding and non-coding regions.
• extended sequences, in either direction, of the cancer genes can be obtained, using techniques well known for cloning either longer sequences or the full length sequences; see Ausubel, et al., supra. Much can be done by informatics and many sequences can be clustered to include multiple sequences corresponding to a single gene, e.g., systems such as UniGene (see, http://www.ncbi.nlm.nih.gov/UniGene/).
• a cancer nucleic acid Once a cancer nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire cancer nucleic acid coding regions or the entire mRNA sequence.
• the recombinant cancer nucleic acid Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant cancer nucleic acid can be further used as a probe to identify and isolate other cancer nucleic acids, e.g., extended coding regions. It can also be used as a "precursor" nucleic acid to make modified or variant cancer nucleic acids and proteins.
• nucleic acid probes to the cancer nucleic acids are made and attached to biochips to be used in screening and diagnostic methods, as outlined below, or for administration, e.g., for gene therapy, vaccine, RNAi, and/or antisense applications.
• cancer nucleic acids that include coding regions of cancer proteins can be put into expression vectors for the expression of cancer proteins, again for screening pu ⁇ oses or for administration to a patient.
• nucleic acid probes to cancer nucleic acids are made.
• the nucleic acid probes attached to the biochip are designed to be substantially complementary to the cancer nucleic acids, e.g., the target sequence (either the target sequence of the sample or to other probe sequences, e.g., in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs.
• this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention.
• the sequence is not a complementary target sequence.
• substantially complementary herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions, as outlined herein.
• a nucleic acid probe is generally single stranded but can be partially single and partially double stranded.
• the strandedness of the probe is dictated by the structure, composition, and properties of the target sequence.
• the nucleic acid probes range from about 8-100 bases long, with from about 10-80 bases being prefened, and from about 30-50 bases being particularly preferred. That is, generally whole genes are not used. In some embodiments, much longer nucleic acids can be used, up to hundreds of bases.
• more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being preferred, are used to build in a redundancy for a particular target.
• the probes can be overlapping (e.g., have some sequence in common), or separate.
• PCR primers may be used to amplify signal for higher sensitivity.
• Nucleic acids can be attached or immobilized to a solid support in a wide variety of ways. By “immobilized” and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined.
• the binding can typically be covalent or non-covalent.
• non-covalent binding and grammatical equivalents herein is meant one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non- covalent binding is the covalent attachment of a molecule, e.g., streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin.
• covalent binding and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds, and coordination bonds.
• Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
• the probes are attached to the biochip in a wide variety of ways.
• the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.
• the biochip comprises a suitable solid substrate.
• substrate or “solid support” or other grammatical equivalents herein is meant a material that can be modified for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. Often, the substrate may contain discrete individual sites appropriate for individual partitioning and identification.
• the number of possible substrates is very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc.
• the substrates allow optical detection and do not appreciably fluoresce. See WO 0055627.
• the substrate is planar, although other configurations of substrates may be used as well.
• the probes may be placed on the inside surface of a tube for flow- through sample analysis to minimize sample volume.
• the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
• the surface of the biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two.
• the biochip is derivatized with a chemical functional group including, but not limited to, amino groups, carboxy groups, oxo groups, and thiol groups, with amino groups being particularly preferred.
• the probes can be attached using functional groups on the probes.
• nucleic acids containing amino groups can be attached to surfaces comprising amino groups, e.g., using linkers; e.g., homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-
• additional linkers such as alkyl groups (including substituted and heteroalkyl groups) may be used.
• oligonucleotides are synthesized, and then attached to the surface of the solid support. Either the 5' or 3' terminus may be attached to the solid support, or attachment may be via linkage to an internal nucleoside.
• the immobilization to the solid support may be very strong, yet non-covalent.
• biotinylated oligonucleotides can be made, which bind to surfaces covalently coated with streptavidin, resulting in attachment.
• the oligonucleotides may be synthesized on the surface.
• photoactivation techniques utilizing photopolymerization compounds and techniques are used.
• the nucleic acids can be synthesized in situ, using known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Patent Nos. 5,700,637 and 5,445,934; and references cited within, all of which are expressly inco ⁇ orated by reference; these methods of attachment form the basis of the Affymetrix GeneChipTM technology.
• amplification-based assays are performed to measure the expression level of cancer-associated sequences. These assays are typically performed in conjunction with reverse transcription.
• a cancer-associated nucleic acid sequence acts as a template in an amplification reaction (e.g., Polymerase Chain Reaction, or PCR).
• an amplification reaction e.g., Polymerase Chain Reaction, or PCR.
• the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the amount of cancer-associated RNA.
• Methods of quantitative amplification are well known. Detailed protocols for quantitative PCR are provided, e.g., in Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications Academic Press.
• a TaqMan based assay is used to measure expression.
• TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end.
• the 5' nuclease activity of the polymerase e.g., AmpliTaq
• This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification (see, e.g., literature provided by Perkin-Elmer, e.g., www2.perkin-elmer.com).
• ligase chain reaction (LCR) (see 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-sustained sequence replication (Guatelli, et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874- 1878), dot PCR, linker adapter PCR, etc. Expression of cancer proteins from nucleic acids
• cancer nucleic acids e.g., encoding cancer proteins
• expression vectors are well known (see, e.g., Ausubel, supra, and Fernandez and Hoeffier (eds. 1999) Gene Expression Systems Academic Press) to express proteins.
• the expression vectors may be either self replicating extrachromosomal vectors or vectors which integrate into a host genome.
• these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the cancer protein.
• control sequences refers to DNA sequences used for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes, e.g., include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. Nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
• DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
• "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is typically accomplished by ligation at convenient restriction sites.
• Transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the cancer protein. Numerous types of appropriate expression vectors and suitable regulatory sequences are known for a variety of host cells.
• transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
• the regulatory sequences include a promoter and transcriptional start and stop sequences.
• Promoter sequences may be either constitutive or inducible promoters.
• the promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known, and are useful in the present invention.
• An expression vector may comprise additional elements.
• the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, e.g., in mammalian or insect cells for expression and in a prokaryotic host for cloning arid amplification.
• the expression vector often contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct.
• the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are available. See, e.g., Fernandez and Hoeffler, supra; and Kitamura, et al. (1995) Proc. Nat'l Acad. Sci. USA 92:9146- 9150.
• the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known and will vary with the host cell used.
• the cancer proteins of the present invention are usually produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a cancer protein, under the appropriate conditions to induce or cause expression of the cancer protein.
• Conditions appropriate for cancer protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained through routine experimentation or optimization.
• the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction.
• the timing of the harvest is important.
• the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
• Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, HUVEC (human umbilical vein endothelial cells), THP1 cells (a macrophage cell line), and various other human cells and cell lines.
• the cancer proteins are expressed in mammalian cells.
• Mammalian expression systems may be used, and include retroviral and adenoviral systems.
• One expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048.
• mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, he ⁇ es simplex virus promoter, and the CMV promoter (see, e.g., Fernandez and Hoeffler, supra).
• transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
• transcription terminator and polyadenlyation signals include those derived from SV40.
• Methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, are available, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
• cancer proteins are expressed in bacterial systems. Promoters from bacteriophage may also be used.
• synthetic promoters and hybrid promoters are also useful; e.g., the tac promoter is a hybrid of the frp and lac promoter sequences.
• a bacterial promoter can include naturally occurring 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 efficient ribosome binding site is desirable.
• the expression vector may also include a signal peptide sequence that provides for secretion of the cancer protein in bacteria. The protein is either secreted into the growth media (gram- positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
• the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed.
• Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin, and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways. These components are assembled into expression vectors.
• Expression vectors for bacteria are well known, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others (e.g., Fernandez and Hoeffler, supra).
• the bacterial expression vectors are transformed into bacterial host cells using techniques such as calcium chloride treatment, electroporation, and others.
• cancer proteins are produced in insect cells using, e.g., expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors.
• a cancer protein is produced in yeast cells.
• yeast expression systems are well known, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymo ⁇ ha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
• the cancer protein may also be made as a fusion protein, using available techniques.
• the cancer protein may be fused to a carrier protein to form an immunogen.
• the cancer protein may be made as a fusion protein to increase expression, or for other reasons.
• the cancer protein is a cancer peptide
• the nucleic acid encoding the peptide may be linked to other nucleic acid for expression pu ⁇ oses. Fusion with detection epitope tags can be made, e.g., with FLAG, His6, myc, HA, etc.
• the cancer protein is purified or isolated after expression.
• Cancer proteins may be isolated or purified in a variety of ways depending on what other components are present in the sample and the requirements for purified product, e.g., natural conformation or denatured.
• Standard purification methods include ammonium sulfate precipitations, electrophoretic, molecular, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing.
• the cancer protein may be purified using a standard anti- cancer protein antibody column. Ultrafilfration and diafilfration techniques, in conjunction with protein concentration, are also useful.
• cancer proteins and nucleic acids are useful in a number of applications. They may be used as immunoselection reagents, as vaccine reagents, as screening agents, therapeutic entities, for production of antibodies, as transcription or translation inhibitors, etc. Variants of cancer proteins
• cancer proteins are amino acid variants " of the naturally occurring sequences, as determined herein.
• the variants are preferably greater than about 75% homologous to the wild-type sequence, more preferably greater than about 80%, even more preferably greater than about 85%, and most preferably greater than 90%.
• the homology will be as high as about 93-95% or 98%.
• nucleic acids homology in this context means sequence similarity or identity, with identity being preferred. This homology will be determined using standard techniques, as are outlined above for nucleic acid homologies.
• Cancer proteins of the present invention may be shorter or longer than the wild type amino acid sequences.
• included within the definition of cancer proteins are portions or fragments of the wild type sequences herein.
• the cancer nucleic acids of the invention may be used to obtain additional coding regions, and thus additional protein sequence.
• the cancer proteins are derivative or variant cancer proteins as compared to the wild-type sequence. That is, as outlined more fully below, the derivative cancer peptide will often contain at least one amino acid substitution, deletion, or insertion, with amino acid substitutions being particularly preferred. The amino acid substitution, insertion, or deletion may occur at many residue positions within the cancer peptide.
• amino acid sequence variants typically fall into one or more of three classes: substitutional, insertional, or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the cancer protein, using cassette or PCR mutagenesis or other techniques, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant cancer protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the cancer protein amino acid sequence.
• the variants typically exhibit a similar qualitative biological activity as a naturally occurring analogue, although variants can also be selected which have modified characteristics. While the site or region for introducing an amino acid sequence variation is often predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed cancer variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, e.g., M13 primer mutagenesis and PCR mutagenesis. Screening of mutants is often done using assays of cancer protein activities.
• Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1-20 amino acids, although considerably larger insertions maybe tolerated. Deletions generally range from about 1-20 residues, although in some cases deletions may be much larger.
• substitutions deletions, insertions, or combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the cancer protein are desired, substitutions are generally made in accordance with the amino acid substitution relationships described.
• variants typically exhibit essentially the same qualitative biological activity and will elicit the same immune response as a naturally-occurring analog, although variants also are selected to modify the characteristics of cancer proteins as needed.
• the variant may be designed such that a biological activity of the cancer protein is altered. For example, glycosylation sites may be added, altered, or removed.
• substitutions that are less conservative than those described above.
• substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain.
• substitutions which generally are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g., serine or threone is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine, or alanine; (b) a cysteine or proline is substituted for (or by) another residue; (c) a residue having an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted for (or by) an electronegative residue, e.g., glutamic or aspartic acid; (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine; or (e) a proline residue is inco ⁇ orated or substituted, which changes the degree
• Variants typically exhibit a similar qualitative biological activity and will elicit the same immune response as the naturally-occurring analog, although variants also are selected to modify the characteristics of the skin cancer proteins as needed.
• the variant may be designed such that the biological activity of the cancer protein is altered. For example, glycosylation sites may be altered or removed.
• Covalent modifications of cancer polypeptides are included within the scope of this invention.
• One type of covalent modification includes reacting targeted amino acid residues of a cancer polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of a cancer polypeptide.
• Derivatization with bifunctional agents is useful, for instance, for crosslinking cancer polypeptides to a water- insoluble support matrix or surface for use in a method for purifying anti-cancer polypeptide antibodies or screening assays, as is more fully described below.
• crosslinking agents include, e.g., l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N- hydroxysuccinimide esters, e.g., esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-l,8-octane and agents such as methyl-3-((p- azidophenyl)dithio)propioimidate.
• l,l-bis(diazoacetyl)-2-phenylethane glutaraldehyde
• N- hydroxysuccinimide esters e.g., esters with 4-azidosalicylic acid
• homobifunctional imidoesters including disuccinimidyl esters such
• Another type of covalent modification of the cancer polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
• "Altering the native glycosylation pattern” is intended for pu ⁇ oses herein to mean deleting one or more carbohydrate moieties found in native sequence cancer polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence cancer polypeptide.
• Glycosylation patterns can be altered in many ways. Different cell types to express cancer- associated sequences can result in different glycosylation patterns.
• Addition of glycosylation sites to cancer polypeptides may also be accomplished by altering the amino acid sequence thereof.
• the alteration may be made, e.g., by the addition of, or substitution by, one or more serine or threonine residues to the native sequence cancer polypeptide (for O-linked glycosylation sites).
• the cancer amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the cancer polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
• Another means of increasing the number of carbohydrate moieties on the cancer polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. See, e.g., WO 87/05330; pp. 259-306 in Aplin and Wriston (1981) CRC Crit. Rev. Biochem. Removal of carbohydrate moieties present on the cancer polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are applicable. See, e.g., Sojar and Bahl (1987) Arch. Biochem. Biophys. 259:52-57 and Edge, et al. (1981) Anal.
• Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases. See, e.g., Thotakura, et al. (1987) Meth. Enzymol. 138:350-359.
• Another type of covalent modification of cancer comprises linking the cancer polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337.
• Cancer polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising a cancer polypeptide fused to another heterologous polypeptide or amino acid sequence.
• such a chimeric molecule comprises a fusion of a cancer polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
• the epitope tag is generally placed at the amino-or carboxyl- terminus of the cancer polypeptide. The presence of such epitope-tagged forms of a cancer polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the cancer polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
• the chimeric molecule may comprise a fusion of a cancer polypeptide with an immunoglobulin or a particular region of an immunoglobulin.
• a fusion could be to the Fc region of an IgG molecule.
• tag polypeptides and their respective antibodies are available.
• Examples "' include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; HIS6 and metal chelation tags, the flu HA tag polypeptide and its antibody 12CA5 (Field, et al. (1988) Mol. Cell. Biol. 8:2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7, and 9E10 antibodies thereto (Evan, et al. (1985) Molecular and Cellular Biology 5:3610-3616); and the He ⁇ es Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky, et al.
• gD He ⁇ es Simplex virus glycoprotein D
• tag polypeptides include the Flag-peptide (Hopp, et al. (1988) BioTechnology 6:1204-1210); the KT3 epitope peptide (Martin, et al. (1992) Science 255:192-194); tubulin epitope peptide (Skinner, et al. (1991) J. Biol. Chem. 266:15163-15166); and the T7 gene 10 protein peptide tag (Lutz-Freyermuth, et al. (1990) Proc. Natl. Acad. Sci. USA 87:6393-6397).
• probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related cancer proteins from humans or other organisms.
• Particularly useful probe and/or PCR primer sequences include the unique areas of the cancer nucleic acid sequence.
• Preferred PCR primers are from about 15-35 nucleotides in length, with from about 20-30 being preferred, and may contain inosine as needed. The conditions for PCR reaction have been well described (e.g., Innis, PCR Protocols, supra).
• cancer proteins can be made that are longer than those encoded by the nucleic acids of the Tables, e.g., by the elucidation of extended sequences, the addition of epitope or purification tags, the addition of other fusion sequences, etc.
• Cancer proteins may also be identified as being encoded by cancer nucleic acids. Thus, cancer proteins are encoded by nucleic acids that will hybridize to the sequences of the sequence listings, or their complements, as outlined herein. Antibodies to cancer proteins
• the cancer protein when the cancer protein is to be used to generate antibodies, e.g., for immunotherapy or immunodiagnosis, the cancer protein should share at least one epitope or determinant with the full length protein.
• epitope or “determinant” herein is typically meant a portion of a protein which will generate and/or bind an antibody or T-cell receptor in the context of MHC.
• the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.
• the epitope is selected from a protein sequence set out in the tables. Methods of preparing polyclonal antibodies exist (e.g., Coligan, supra; and Harlow and
• Polyclonal antibodies can be raised in a mammal, e.g., by one or more injections of an immunizing agent and, if desired, an adjuvant.
• the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
• the immunizing agent may include a protein encoded by a nucleic acid of Tables 2A-80 or fragment thereof or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
• immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
• adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
• MPL-TDM adjuvant monophosphoryl Lipid A, synthetic trehalose dicorynomycolate.
• the antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495.
• a mouse, hamster, or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
• the lymphocytes may be immunized in vitro.
• the immunizing agent will typically include a polypeptide encoded by a nucleic acid of the tables or fragment thereof, or a fusion protein thereof.
• PBLs peripheral blood lymphocytes
• spleen cells or lymph node cells are used if non-human mammalian sources are desired.
• the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (e.g., pp. 59-103 in Goding (1986) Monoclonal Antibodies: Principles and Practice Academic Press).
• Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine, or human origin. Usually, rat or mouse myeloma cell lines are employed.
• the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
• the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
• HGPRT medium hypoxanthine, aminopterin, and thymidine
• the antibodies are bispecific antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens or that have binding specificities for two epitopes on the same antigen.
• one of the binding specificities is for a protein encoded by a nucleic acid of the tables or a fragment thereof, the other one is for another antigen, and preferably for a cell-surface protein or receptor or receptor subunit, preferably one that is tumor specific.
• the antibodies to cancer protein are capable of reducing or eliminating a biological function of a cancer protein, in a naked form or conjugated to an effector moiety, as is described below. That is, the addition of anti-cancer protein antibodies (either polyclonal or preferably monoclonal) to cancer tissue (or cells containing cancer) may reduce or eliminate the cancer.
• at least a 25% decrease in activity, growth, size, or the like is preferred, with at least about 50% being particularly prefened and about a 95-100% decrease being especially prefened.
• the antibodies to the cancer proteins are humanized antibodies (e.g., Xenerex Biosciences, Medarex, Inc., Abgenix, Inc., Protein Design Labs, Inc.)
• Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) wliich contain minimal sequence derived from non-human immunoglobulin.
• Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
• CDR complementary determining region
• donor antibody such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
• Fv framework residues of a human immunoglobulin are replaced by conesponding non-human residues.
• Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions conespond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody optimally also will typically comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones, et al. (1986) Nature 321 :522-525; Riechmann, et al. (1988) Nature 332:323-329; and Presta (1992) Cun. Op. Struct. Biol. 2:593-596).
• Humanization can be essentially performed following the method of Winter and co-workers (Jones, et al. (1986) Nature 321:522-525; Riechmann, et al. (1988) Nature 332:323-327; Verhoeyen, et al. (1988) Science 239:1534-1536), by substituting rodent CDRs or CDR sequences for conesponding sequences of a human antibody.
• rodent CDRs or CDR sequences for conesponding sequences of a human antibody.
• such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by conesponding sequence from a non-human species.
• Human antibodies can also be produced using phage display libraries (Hoogenboom and Winter (1992) J. Mol.
• human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
• immunotherapy is meant treatment of cancer with an antibody raised against cancer proteins.
• immunotherapy can be passive or active.
• Passive immunotherapy as defined herein is the passive transfer of antibody to a recipient (patient).
• Active immunization is the induction of antibody and/or T-cell responses in a recipient (patient).
• Induction of an immune response is the result of providing the recipient with an antigen to which antibodies are raised.
• the antigen may be provided by injecting a polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with a nucleic acid capable of expressing the antigen and under conditions for expression of the antigen, leading to an immune response.
• the cancer proteins against which antibodies are raised are secreted proteins as described above.
• antibodies used for treatment may bind and prevent the secreted protein from binding to its receptor, thereby inactivating the secreted cancer protein, e.g., in autocrine signaling.
• the cancer protein to which antibodies are raised is a transmembrane protein.
• antibodies used for treatment may bind the extracellular domain of the cancer protein and prevent it from binding to other proteins, such as circulating ligands or cell-associated molecules.
• the antibody may cause down- regulation of the transmembrane cancer protein.
• the antibody may be a competitive, non- competitive or uncompetitive inhibitor of protein binding to the extracellular domain of the cancer protein.
• the antibody may also be an antagonist of the cancer protein.
• the antibody may prevent activation of the transmembrane cancer protein, or may induce or suppress a particular cellular pathway. In one aspect, when the antibody prevents the binding of other molecules to the cancer protein, the antibody prevents growth of the cell.
• the antibody may also be used to target or sensitize the cell to cytotoxic agents, including, but not limited to TNF- ⁇ , TNF- ⁇ , IL-1, ESTF- ⁇ , and IL-2, or chemotherapeutic agents including 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like.
• cytotoxic agents including, but not limited to TNF- ⁇ , TNF- ⁇ , IL-1, ESTF- ⁇ , and IL-2, or chemotherapeutic agents including 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like.
• the antibody may belong to a sub-type that activates serum complement when complexed with the transmembrane protein thereby mediating cytotoxicity or antigen-dependent cytotoxicity (ADCC).
• ADCC antigen-dependent cytotoxicity
• cancer may be treated by administering to a patient antibodies directed against the transmembrane cancer protein.
• Antibody-labeling
• the antibody is conjugated to an effector moiety.
• the effector moiety can be various molecules, including labeling moieties such as radioactive labels or fluorescent labels, or can be a therapeutic moiety.
• the therapeutic moiety is a small molecule that modulates the activity of a cancer protein.
• the therapeutic moiety may modulate the activity of molecules associated with or in close proximity to a cancer protein.
• the therapeutic moiety may inhibit enzymatic or signaling activity such as protease or collagenase or protein kinase activity associated with cancer, or be an attractant of other cells, such as NK cells. See, e.g., USSN 09/544,494.
• the therapeutic moiety can also be a cytotoxic agent.
• targeting the cytotoxic agent to cancer tissue or cells results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with cancer.
• Cytotoxic agents are numerous and varied and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their conesponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, saporin, auristatin, and the like.
• Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against cancer proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody.
• Targeting the therapeutic moiety to transmembrane cancer proteins not only serves to increase the local concentration of therapeutic moiety in the cancer afflicted area, but also serves to reduce deleterious side effects that may be associated with the untargeted therapeutic moiety.
• Antibody fragments may be used to target toxin loaded liposomes.
• the cancer protein against which the antibodies are raised is an intracellular protein.
• the antibody may be conjugated to a protein which facilitates entry into the cell.
• the antibody enters the cell by endocytosis.
• a nucleic acid encoding the antibody is administered to the individual or cell.
• an antibody thereto may contain a signal for that target localization, e.g., a nuclear localization signal.
• the cancer antibodies of the invention specifically bind to cancer proteins.
• “specifically bind” herein is meant that the antibodies bind to the protein with a K ⁇ of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better. Selectivity of binding to the specific target and not to related sequences is often also important. Detection of cancer sequence for diagnostic and therapeutic applications
• the RNA expression levels of genes are determined for different cellular states in the cancer phenotype. Expression levels of genes in normal tissue (e.g., not undergoing cancer) and in cancer tissue (and in some cases, for varying severities of cancer that relate to prognosis, as outlined below), or in non-malignant disease are evaluated to provide expression profiles.
• a gene expression profile of a particular cell state or point of development is essentially a "finge ⁇ rint" of the state of the cell. While two states may have a particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is reflective of the state of the cell.
• differential expression refers to qualitative or quantitative differences in the temporal and/or cellular gene expression patterns within and among cells and tissue.
• a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, e.g., normal versus cancer tissue.
• Genes may be turned on or turned off in a particular state, relative to another state thus permitting comparison of two or more states.
• a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques. Some genes will be expressed in one state or cell type, but not in both.
• the difference in expression maybe quantitative, e.g., in that expression is increased or decreased; e.g., gene expression is either upregulated, resulting in an increased amount of transcript, or downregulated, resulting in a decreased amount of transcript.
• the degree to which expression differs need only be large enough to quantify via standard characterization techniques as outlined below, such as by use of Affymetrix GeneChipTM expression anays. See, Lockhart (1996) Nature Biotechnology 14:1675-1680.
• Other techniques include, but are not limited to, quantitative reverse transcriptase PCR, northern analysis, and RNase protection.
• the change in expression is at least about 50%), more preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%, with from 300 to at least 1000% being especially prefened.
• Evaluation may be at the gene transcript or the protein level.
• the amount of gene expression may be monitored using nucleic acid probes to the RNA or DNA equivalent of the gene transcript, and the quantification of gene expression levels, or, alternatively, the final gene product itself (protein) can be monitored, e.g., with antibodies to the cancer protein and standard immunoassays (ELIS As, etc.) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc.
• ELIS As standard immunoassays
• Proteins conesponding to cancer genes can be evaluated in a cancer diagnostic test.
• gene expression monitoring is performed simultaneously on a number of genes. Multiple protein expression monitoring can be performed as well.
• the cancer nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of cancer sequences in a particular cell.
• the assays are further described below in the example. PCR techniques can be used to provide greater sensitivity.
• nucleic acids encoding the cancer protein are detected.
• DNA or RNA encoding the cancer protein may be detected, of particular interest are methods wherein an mRNA encoding a cancer protein is detected.
• Probes to detect mRNA can be a nucleotide/deoxynucleotide probe that is complementary to and hybridizes with the mRNA and includes, but is not limited to, oligonucleotides, cDNA, or RNA. Probes also should contain a detectable label, as defined herein.
• the mRNA is detected after immobilizing the nucleic acid to be examined on a solid support such as nylon membranes and hybridizing the probe with the sample.
• RNA probe digoxygenin labeled riboprobe
• RNA probe that is complementary to the mRNA encoding a cancer protein is detected by binding the digoxygenin with an anti-digoxygenin secondary antibody and developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.
• various proteins from the three classes of proteins as described herein are used in diagnostic assays.
• the cancer proteins, antibodies, nucleic acids, modified proteins, and cells containing cancer sequences are used in diagnostic assays. This can be performed on an individual gene or conesponding polypeptide level.
• the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes and/or conesponding polypeptides.
• cancer proteins including intracellular, transmembrane, or secreted proteins, find use as markers of cancer, e.g., for prognostic or diagnostic pu ⁇ oses. Detection of these proteins in putative cancer tissue allows for detection, prognosis, or diagnosis of cancer or similar disease, and for selection of therapeutic strategy.
• antibodies are used to detect cancer proteins.
• a prefened method separates proteins from a sample by electrophoresis on a gel (typically a denaturing and reducing protein gel, but maybe another type of gel, including isoelectric focusing gels and the like). Following separation of proteins, the cancer protein is detected, e.g., by immunoblotting with antibodies raised against the cancer protein.
• antibodies to the cancer protein find use in in situ imaging techniques, e.g., in histology. See, e.g., Asai, et al. (eds. 1993) Methods in Cell Biology: Antibodies in Cell Biology (vol. 37) Academic Press.
• cells are contacted with from one to many antibodies to the cancer protein(s). Following washing to remove nonspecific antibody binding, the presence of the antibody or antibodies is detected.
• the antibody is detected by incubating with a secondary antibody that contains a detectable label.
• the primary antibody to the cancer protein(s) contains a detectable label, e.g., an enzyme marker that can act on a substrate.
• each one of multiple primary antibodies contains a distinct and detectable label. This method finds particular use in simultaneous screening for a plurality of cancer proteins. Many other histological imaging techniques are also provided by the invention.
• the label is detected in a fluorometer which has the ability to detect and distinguish emissions of different wavelengths.
• a fluorescence activated cell sorter FACS
• FACS fluorescence activated cell sorter
• antibodies find use in diagnosing cancer from blood, serum, plasma, stool, and other samples. Such samples, therefore, are useful as samples to be probed or tested for the presence of cancer proteins.
• Antibodies can be used to detect a cancer protein by previously described immunoassay techniques including ELISA, immunoblotting (western blotting), immunoprecipitation, BIACORE technology and the like. Conversely, the presence of antibodies may indicate an immune response against an endogenous cancer protein.
• in situ hybridization of labeled cancer nucleic acid probes to tissue anays is done. For example, anays of tissue samples, including cancer tissue and/or normal tissue, are made.
• In situ hybridization (see, e.g., Ausubel, supra) is then performed.
• a diagnosis, a prognosis, or a prediction may be based on the findings. It is further understood that the genes which indicate the diagnosis may differ from those which indicate the prognosis and molecular profiling of the condition of the cells may lead to distinctions between responsive or refractory conditions or may be predictive of outcomes.
• the cancer proteins, antibodies, nucleic acids, modified proteins, and cells containing cancer sequences are used in prognosis assays.
• gene expression profiles can be generated that conelate to cancer, clinical, pathological, or other information, in terms of long term prognosis. Again, this may be done on either a protein or gene level, with the use of genes being prefened. Single or multiple genes may be useful in various combinations.
• cancer probes may be attached to biochips for the detection and quantification of cancer sequences in a tissue or patient. The assays proceed as outlined above for diagnosis. PCR method may provide more sensitive and accurate quantification. Assays for therapeutic compounds
• the proteins, nucleic acids, and antibodies as described herein are used in drug screening assays.
• the cancer proteins, antibodies, nucleic acids, modified proteins, and cells containing cancer sequences are used in drug screening assays or by evaluating the effect of drug candidates on a "gene expression profile" or expression profile of polypeptides.
• the expression profiles are used, preferably in conjunction with high throughput screening techniques, to allow monitoring for expression profile genes after treatment with a candidate agent (e.g., Zlokarnik, et al. (1998) Science 279:84-88; Heid (1996) Genome Res. 6:986-994.
• the cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer proteins are used in screening assays. That is, the present invention provides novel methods for screening for compositions which modulate the cancer phenotype or an identified physiological function of a cancer protein. As above, this can be done on an individual gene level or by evaluating the effect of drug candidates on a "gene expression profile".
• the expression profiles are used, preferably in conjunction with high throughput screening techniques, to allow monitoring for expression profile genes after treatment with a candidate agent, see Zlokarnik, supra.
• assays may be performed.
• assays may be run on an individual gene or protein level. That is, having identified a particular gene as up regulated in cancer, test compounds can be screened for the ability to modulate gene expression or for binding to the cancer protein.
• “Modulation” thus includes both an increase and a decrease in gene expression.
• the prefened amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater.
• a gene exhibits a 4-fold increase in cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in cancer tissue compared to normal tissue often provides a target value of a 10-fold increase in expression to be induced by the test compound.
• the amount of gene expression may be monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, the gene product itself can be monitored, e.g., through the use of antibodies to the cancer protein and standard immunoassays. Proteomics and separation techniques may also allow quantification of expression.
• gene expression or protein monitoring of a number of entities e.g., an expression profile
• Such profiles will typically involve a plurality of those entities described herein.
• the cancer nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of cancer sequences in a particular cell.
• PCR may be used.
• a series e.g., of microtiter plate, may be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.
• Expression monitoring can be performed to identify compounds that modify the expression of one or more cancer-associated sequences, e.g., a polynucleotide sequence set out in the tables.
• a test modulator is added to the cells prior to analysis.
• screens are also provided to identify agents that modulate cancer, modulate cancer proteins, bind to a cancer protein, or interfere with the binding of a cancer protein and an antibody or other binding partner.
• test compound or “drug candidate” or “modulator” or grammatical equivalents as used herein describes a molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, e.g., a nucleic acid or protein sequence.
• modulators alter expression profiles, or expression profile nucleic acids or proteins provided herein.
• the modulator suppresses a cancer phenotype, e.g., to a normal or non-malignant tissue finge ⁇ rint.
• a modulator induced a cancer phenotype.
• a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
• one of these concentrations serves as a negative control, e.g., at zero concentration or below the level of detection.
• Drug candidates encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Prefened small molecules are less than 2000, or less than 1500, or less than 1000, or less than 500 D.
• Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
• the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
• Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Particularly prefened are peptides.
• a modulator will neutralize the effect of a cancer protein.
• neutralize is meant that activity of a protein is inhibited or blocked and the consequent effect on the cell.
• combinatorial libraries of potential modulators will be screened for an ability to bind to a cancer polypeptide or to modulate activity.
• new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
• HTS high throughput screening
• high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
• a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents.
• a linear combinatorial chemical library such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (e.g., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks (Gallop, et al. (1994) J. Med. Chem. 37:1233-1251).
• combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175, Furka (1991) Pept. Prot. Res. 37:487-493, Houghton, et al. (1991) Nature 354:84-88), peptoids (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat. No.
• the assays to identify modulators are amenable to high throughput screening. Prefened assays thus detect enhancement or inhibition of cancer gene transcription, inhibition, or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.
• High throughput assays for the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known.
• binding assays and reporter gene assays are similarly well known.
• U.S. Patent No. 5,559,410 discloses high throughput screening methods for proteins
• U.S. Patent No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (e.g., in anays)
• U.S. Patent Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.
• high throughput screening systems are commercially available (see, e.g., Zymark Co ⁇ ., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures, including sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay.
• These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems.
• Zymark Co ⁇ provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
• modulators are proteins, often naturally occurring proteins or fragments of naturally occurring proteins.
• cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be used.
• libraries of proteins may be made for screening in the methods of the invention.
• Particularly prefened in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being prefened, and human proteins being especially prefened.
• Particularly Useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes or ligands and receptors.
• modulators are peptides of from about 5-30 amino acids, with from about 5-20 amino acids being prefened, and from about 7-15 being particularly prefened.
• the peptides may be digests of naturally occurring proteins, random peptides, or "biased” random peptides.
• randomized or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may inco ⁇ orate a nucleotide or amino acid at any position.
• the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
• the library is fully randomized, with no sequence preferences or constants at any position.
• the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
• the nucleotides or amino acid residues are randomized within a defined class, e.g., of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines, or histidines for phosphorylation sites, etc., or to purines, etc.
• Modulators of cancer can also be nucleic acids, as defined above.
• nucleic acid modulating agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids.
• digests of prokaryotic or eukaryotic genomes maybe used as is outlined above for proteins.
• the candidate compounds are organic chemical moieties, a wide variety of which are available in the literature.
• the sample containing a target sequence to be analyzed is added to the biochip.
• the target sequence is prepared using known techniques.
• the sample may be treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate.
• an in vitro transcription with labels covalently attached to the nucleotides is performed.
• the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
• the target sequence is labeled with, e.g., a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe.
• the label also can be an enzyme, such as, alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that can be detected.
• the label can 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 also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin.
• the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.
• assays can be direct hybridization assays or can comprise "sandwich assays", which include the use of multiple probes, as is generally outlined in 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, 5,591,584,
• the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
• hybridization conditions may be used in the present invention, including high, moderate, and low stringency conditions as outlined above.
• the assays are generally run under stringency conditions which allows formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaofropic salt concentration, pH, organic solvent concentration, etc.
• the reactions outlined herein may be accomplished in a variety of ways. Components of the reaction may be added simultaneously, or sequentially, in different orders, with prefened embodiments outlined below.
• the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g., albumin, detergents, etc. which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target.
• the assay data are analyzed to determine the expression levels, and changes in expression levels as between states of individual genes, forming a gene expression profile.
• Screens are performed to identify modulators of the cancer phenotype.
• screening is performed to identify modulators that can induce or suppress a particular expression profile, thus preferably generating the associated phenotype.
• screens can be performed to identify modulators that alter expression of individual genes.
• screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.
• screens can be done for genes that are induced in response to a candidate agent or treatment process. After identifying a modulator based upon its ability to suppress a cancer expression pattern leading to a normal expression pattern (or its converse), or to modulate a single cancer gene expression profile so as to mimic the expression of the gene from normal tissue, a screen as described above can be performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent treated cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue.
• agent-specific sequences can be identified and used by methods described herein for cancer genes or proteins. In particular, these sequences and the proteins they encode find use in marking or identifying agent treated cells.
• antibodies can be raised against the agent induced proteins and used to target novel therapeutics, e.g., toxin loaded liposomes, to the treated cancer tissue sample.
• a test compound is administered to a population of cancer cells that have an associated cancer expression profile.
• administration or “contacting” herein is meant that the candidate agent is added to the cells in such a manner as to allow the agent to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface.
• nucleic acid encoding a proteinaceous candidate agent e.g., a peptide
• a viral construct such as an adenoviral or retroviral construct
• Regulatable gene therapy systems can also be used.
• cancer or non-malignant tissue may be screened for agents that modulate, e.g., induce or suppress a cancer phenotype.
• a change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity.
• screens may be done on individual genes and gene products (proteins). That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself can be done.
• the gene products of differentially expressed genes are sometimes refened to herein as "cancer proteins” or a "cancer modulatory protein".
• the cancer modulatory protein may be a fragment, or alternatively, be the full length protein to the fragment encoded by the nucleic acids of the Tables.
• the cancer modulatory protein is a fragment.
• the cancer amino acid sequence which is used to determine sequence identity or similarity is encoded by a nucleic acid of the Tables.
• the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of the Tables.
• the sequences are sequence variants as further described herein.
• the cancer modulatory protein is a fragment of about 14-24 amino acids long. More preferably the fragment is a soluble fragment. Preferably, the fragment includes a non-fransmembrane region. In a prefened embodiment, the fragment has an N-terminal Cys to aid in solubility. In one embodiment, the C-terminus of the fragment is kept as a free acid and the N-terminus is a free amine to aid in coupling, e.g., to cysteine. In one embodiment the cancer proteins are conjugated to an immunogenic agent as discussed herein. In one embodiment the cancer protein is conjugated to BSA.
• Measurements of cancer polypeptide activity, or of cancer or the cancer phenotype can be performed using a variety of assays.
• the effects of the test compounds upon the function of the cancer polypeptides can be measured by examining parameters described above.
• a suitable physiological change that affects activity can be used to assess the influence of a test compound on the polypeptides of this invention.
• cancer polypeptide is typically used, e.g., mouse, preferably human.
• Assays to identify compounds with modulating activity can be performed in vitro.
• a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5-48 hours.
• the cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA.
• the level of protein is typically measured using immunoassays such as western blotting, ELISA, and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof.
• amplification e.g., using PCR, LCR, or hybridization assays, e.g., northern hybridization, RNAse protection, dot blotting
• the level of protein or mRNA is typically detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
• a reporter gene system can be devised using a cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or ⁇ -gal. The reporter construct is typically transfected into a cell.
• the amount of reporter gene transcription, translation, or activity is measured according to standard techniques.
• screens may be done on individual genes and gene products (proteins). That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself can be done.
• the gene products of differentially expressed genes are sometimes refened to herein as "cancer proteins.”
• the cancer protein may be a fragment, or alternatively, the full length protein to a fragment shown herein.
• screening for modulators of expression of specific genes is performed. Typically, the expression of only one or a few genes are evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships. In a prefened embodiment, binding assays are done. In general, purified or isolated gene product is used; that is, the gene products of one or more differentially expressed nucleic acids are made. For example, antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present. Alternatively, cells comprising the cancer proteins can be used in the assays.
• the methods comprise combining a cancer protein and a candidate compound, and determining the binding of the compound to the cancer protein.
• Prefened embodiments utilize the human cancer protein, although other mammalian proteins may also be used, e.g., for the development of animal models of human disease.
• variant or derivative cancer proteins may be used.
• the cancer protein or the candidate agent is non-diffusably bound to an insoluble support, preferably having isolated sample receiving areas (e.g., a microtiter plate, an anay, etc.).
• the insoluble supports may be made of a composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
• the surface of such supports may be solid or porous and of a convenient shape.
• suitable insoluble supports include microtiter plates, anays, membranes, and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, teflonTM, etc. Microtiter plates and anays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.
• the particular manner of binding of the composition is typically not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition, and is nondiffusable.
• Prefened methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein, or other innocuous protein or other moiety.
• BSA bovine serum albumin
• the cancer protein is bound to the support, and a test compound is added to the assay.
• the candidate agent is bound to the support and the cancer protein is added.
• Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this pu ⁇ ose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.), and the like.
• the determination of the binding of the test modulating compound to the cancer protein may be done in a number of ways.
• the compound is labeled, and binding determined directly, e.g., by attaching all or a portion of the cancer protein to a solid support, adding a labeled candidate agent (e.g., a fluorescent label), washing off excess reagent, and determimng whether the label is present on the solid support.
• a labeled candidate agent e.g., a fluorescent label
• washing off excess reagent e.g., a fluorescent label
• determimng whether the label is present on the solid support.
• Various blocking and washing steps may be utilized as appropriate.
• the proteins can be labeled.
• more than one component can be labeled with different labels, e.g., 125j f or fl ⁇ p ro teins and a fluorophor for the compound.
• Proximity reagents e.g., quenching or energy transfer reagents are also useful.
• the binding of the test compound is determined by competitive binding assay.
• the competitor may be a binding moiety known to bind to the target molecule (e.g., a cancer protein), such as an antibody, peptide, binding partner, ligand, etc.
• the test compound is labeled. Either the compound, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present. Incubations may be performed at a temperature which facilitates optimal activity, typically between about 4-40° C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening. Typically between 0.1-1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding. In a prefened embodiment, the competitor is added first, followed by a test compound.
• Displacement of the competitor is an indication that the test compound is binding to the cancer protein and thus is capable of binding to, and potentially modulating, the activity of the cancer protein.
• either component can be labeled.
• the presence of label in the wash solution indicates displacement by the agent.
• the presence of the label on the support indicates displacement.
• test compound is added first, with incubation and washing, followed by the competitor.
• the absence of binding by the competitor may indicate that the test compound is bound to the cancer protein with a higher affinity.
• the presence of the label on the support, coupled with a lack of competitor binding may indicate that the test compound is capable of binding to the cancer protein.
• the methods comprise differential screening to identity agents that are capable of modulating the activity of the cancer proteins.
• the methods comprise combining a cancer protein and a competitor in a first sample.
• a second sample comprises a test compound, a cancer protein, and a competitor.
• the binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the cancer protein arid potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the cancer protein.
• differential screening is used to identify drug candidates that bind to the native cancer protein, but cannot bind to modified cancer proteins.
• the structure of the cancer protein may be modeled, and used in rational drug design to synthesize agents that interact with that site.
• Drug candidates that affect the activity of a cancer protein are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.
• Positive controls and negative controls may be used in the assays.
• confrol and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
• reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc., which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., maybe used. The mixture of components may be added in an order that provides for the requisite binding.
• the invention provides methods for screening for a compound capable of modulating the activity of a cancer protein.
• the methods comprise adding a test compound, as defined above, to a cell comprising cancer proteins.
• Prefened cell types include almost any cell.
• the cells contain a recombinant nucleic acid that encodes a cancer protein.
• a library of candidate agents are tested on a plurality of cells.
• the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g., hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (e.g., cell-cell contacts).
• physiological signals e.g., hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (e.g., cell-cell contacts).
• the determinations are determined at different stages of the cell cycle process.
• a method of inhibiting cancer cell division comprises administration of a cancer inhibitor.
• a method of inhibiting cancer is provided.
• the method may comprise adminisfration of a cancer inhibitor.
• methods of treating cells or individuals with cancer are provided, e.g., comprising administration of a cancer inhibitor.
• a cancer inhibitor is an antibody as discussed above. In another embodiment, the cancer inhibitor is an antisense molecule.
• a variety of cell growth, proliferation, viability, and metastasis assays are available, as described below. Soft agar growth or colony formation in suspension
• Normal cells require a solid substrate to attach and grow. When the cells are transformed, they lose this phenotype and grow detached from the substrate.
• transformed cells can grow in stined suspension culture or suspended in semi-solid media, such as semi-solid or soft agar.
• the transformed cells when transfected with tumor suppressor genes, regenerate normal phenotype and require a solid substrate to attach and grow.
• Soft agar growth or colony formation in suspension assays can be used to identify modulators of cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation.
• a therapeutic compound would reduce or eliminate the host cells' ability to grow in stined suspension culture or suspended in semi-solid media, such as semi-solid or soft.
• Normal cells typically grow in a flat and organized pattern in a petri dish until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. When cells are transformed, however, the cells are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, the transformed cells grow to a higher saturation density than normal cells. This can be detected mo ⁇ hologically by the formation of a disoriented monolayer of cells or rounded cells in foci within the regular pattern of normal sunounding cells. Alternatively, labeling index with (3H)-thymidine at saturation density can be used to measure density limitation of growth. See Freshney (2000), supra. The transformed cells, when transfected with tumor suppressor genes, regenerate a normal phenotype and become contact inhibited and would grow to a lower density.
• labeling index with (3H)-thymidine at saturation density is a prefened method of measuring density limitation of growth.
• Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions.
• the percentage of cells labeling with (3H)-thymidine is determined autoradiographically. See, Freshney (1998), supra. Growth factor or serum dependence
• Transformed cells typically have a lower serum dependence than their normal counte ⁇ arts (see, e.g., Temin (1966) J. Natl. Cancer Insti. 37:167-175; Eagle, et al.(1970) J.
• Tumor cells release an increased amount of certain factors (hereinafter “tumor specific markers”) than their normal counte ⁇ arts.
• tumor specific markers For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino
• tumor angiogenesis factor (TAF) is released at a higher level in tumor cells than their normal counte ⁇ arts. See, e.g., Folkman (1992) Sem. Cancer Biol. 3:89-96. Various techniques which measure the release of these factors are described in Freshney
• the degree of invasiveness into Matrigel or some other extracellular matrix constituent can be used as an assay to identify compounds that modulate cancer-associated sequences.
• Tumor cells exhibit a good conelation between malignancy and invasiveness of cells into Matrigel or some other exfracellular matrix constituent.
• tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells.
• the level of invasion of host cells can be measured by using filters coated with Matrigel or some other exfracellular matrix constituent. Penefration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 125j and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra. Tumor growth in vivo
• Knock-out transgenic mice can be made, in which the cancer gene is disrupted or in which a cancer gene is inserted.
• Knock-out transgenic mice can be made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in the mouse genome via homologous recombination.
• Such mice can also be made by substituting the endogenous cancer gene with a mutated version of the cancer gene, or by mutating the endogenous cancer gene, e.g., by exposure to carcinogens.
• a DNA construct is introduced into the nuclei of embryonic stem cells.
• Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re- implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells partially derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi, et al. (1989) Science 244:1288-1292).
• Chimeric targeted mice can be derived according to Hogan, et al. (1988) Manipulating the Mouse Embryo: A Laboratory Manual CSH Press; and Robertson (ed.
• Transplantable tumor cells typically about 10 ⁇ cells
• injected into isogenic hosts will produce invasive tumors in a high proportions of cases, while normal cells of similar origin will not.
• cells expressing a cancer-associated sequences are injected subcutaneously.
• tumor growth is measured (e.g., by volume or by its two largest dimensions) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth.
• the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of an inhibitory or antisense polynucleotide, e.g., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., a cancer protein mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.
• an inhibitory or antisense polynucleotide e.g., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., a cancer protein mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.
• antisense polynucleotides can comprise naturally- occurring nucleotides, or synthetic species formed from naturally-occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species. Analogs are comprehended by this invention so long as they function effectively to hybridize with the cancer protein mRNA. See, e.g., Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA. Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known.
• Antisense molecules as used herein include antisense or sense oligonucleotides.
• Sense oligonucleotides can, e.g., be employed to block transcription by binding to the anti-sense strand.
• the antisense and sense oligonucleotide comprise a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules.
• a prefened antisense molecule is for a cancer sequences in the Tables, or for a ligand or activator thereof.
• Antisense or sense oligonucleotides comprise a fragment generally at least about 14 nucleotides, preferably from about 14-30 nucleotides.
• the ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, e.g., Stein and Cohen (1988) Cancer Res. 48:2659-2668; and van der Krol, et al. (1988) BioTechniques 6:958-976.
• RNA interference is a mechanism to suppress gene expression in a sequence specific manner. See, e.g., Brumelkamp, et al.
• RNAi double stranded small interfering RNAs
• Ribozymes In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancer-associated nucleotide sequences.
• a ribozyme is an RNA molecule that catalytically cleaves other RNA molecules.
• Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hai ⁇ in ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto, et al. (1994) Adv. in Pharmacology 25: 289-317 for a general review of the properties of different ribozymes).
• hai ⁇ in ribozymes are described, e.g., in Hampel, et al. (1990) Nucl. Acids Res. 18:299-304; European Patent Publication No. 0 360257; U.S. Patent No. 5,254,678. Methods of preparation are described in, e.g., WO 94/26877; Ojwang, et al. (1993) 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 92:699-703; Leavitt, et al. (1994) Human Gene Therapy 5:1151-120; and Yamada, et al. (1994) Virology 205: 121-126.
• Polynucleotide modulators of cancer may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.
• 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.
• conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its conesponding molecule or receptor, dr block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
• a polynucleotide modulator of cancer may be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of an polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of freatment.
• methods of modulating cancer in cells or organisms comprise administering to a cell an anti-cancer antibody that reduces or eliminates the biological activity of an endogenous cancer protein.
• the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a cancer protein. This may be accomplished in any number of ways. In a prefened embodiment, e.g., when the cancer sequence is down-regulated in cancer, such state may be reversed by increasing the amount of cancer gene product in the cell. This can be accomplished, e.g., by overexpressing the endogenous cancer gene or administering a gene encoding the cancer sequence, using known gene-therapy techniques.
• the gene therapy techniques include the inco ⁇ oration of the exogenous gene using enhanced homologous recombination (EHR), e.g., as described in PCT/US93/0386.
• EHR enhanced homologous recombination
• the cancer sequence is up-regulated in cancer
• the activity of the endogenous cancer gene is decreased, e.g., by the administration of a cancer antisense or other inhibitor, e.g., RNAi.
• the cancer proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to cancer proteins.
• the cancer proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify cancer antibodies useful for production, diagnostic, or therapeutic piuposes.
• the antibodies are generated to epitopes unique to a cancer protein; that is, the antibodies show little or no cross-reactivity to other proteins.
• the cancer antibodies may be coupled to standard affinity chromatography columns and used to purify cancer proteins.
• the antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the cancer protein.
• the invention provides methods for identifying cells containing variant cancer genes, e.g., determining all or part of the sequence of at least one endogenous cancer gene in a cell.
• the invention provides methods of identifying the cancer genotype of an individual, e.g., determining all or part of the sequence of at least one cancer gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue.
• the method may include comparing the sequence of the sequenced cancer gene to a known cancer gene, e.g., a wild-type gene.
• the sequence of all or part of the cancer gene can then be compared to the sequence of a known cancer gene to determine if any differences exist. This can be done using known homology programs, such as Bestfit, etc.
• known homology programs such as Bestfit, etc.
• the presence of a difference in the sequence between the cancer gene of the patient and the known cancer gene conelates with a disease state or a propensity for a disease state, as outlined herein.
• the cancer genes are used as probes to determine the number of copies of the cancer gene in the genome.
• the cancer genes are used as probes to determine the chromosomal localization of the cancer genes.
• Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the cancer gene locus.
• a therapeutically effective dose of a cancer protein or modulator thereof is administered to a patient.
• therapeutically effective dose herein is meant a dose that produces effects for which it is administered. The exact dose will depend on the pu ⁇ ose of the freatment, and will be ascertainable using known techniques. See, e.g., 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.
• a "patient” for the pu ⁇ oses of the present invention includes both humans and other animals, particularly mammals. Thus the methods are applicable to both human therapy and veterinary applications.
• the patient is a mammal, preferably a primate, and in the most prefened embodiment the patient is human.
• the administration of the cancer proteins and modulators thereof of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or infraocularly.
• the cancer proteins and modulators may be directly applied as a solution or spray.
• the pharmaceutical compositions of the present invention comprise a cancer protein in a form suitable for administration to a patient.
• the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
• “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as 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 the like.
• inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like
• organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic
• “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Particularly prefened are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
• compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
• compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
• unit dosage forms suitable for oral adminisfration include, but are not limited to, powder, tablets, pills, capsules and lozenges.
• cancer protein modulators e.g., antibodies, antisense constructs, ribozymes, small organic molecules, etc.
• This is typically accomplished either by complexing the molecule(s) with a composition to render it resistant to acidic and enzymatic hydrolysis, or by packaging the molecule(s) in an appropriately resistant carrier, such as a liposome or a protection barrier. Means of protecting agents from digestion are available.
• compositions for administration will commonly comprise a cancer protein modulator dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
• a pharmaceutically acceptable carrier preferably an aqueous carrier.
• aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
• These compositions may be sterilized by conventional, well known sterilization techniques.
• the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.
• concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like in accordance with the particular mode of administration selected and the patient's needs (e.g., (1980) Remington's Pharmaceutical Science (18th ed.) Mack, and Hardman and Limbird (eds. 2001) Goodman and Gilman: The Pharmacological Basis of Therapeutics (10th ed.) McGraw-Hill.
• a typical pharmaceutical composition for intravenous administration would be about 0.1 to 10 mg per patient per day.
• compositions containing modulators of cancer proteins can be administered for therapeutic or prophylactic treatments.
• compositions are administered to a patient suffering from a disease (e.g., a cancer) in an amount sufficient ' to cure or at least partially anest the disease and its complications.
• a disease e.g., a cancer
• a therapeutically effective dose An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the agents of this invention to effectively treat the patient.
• An amount of modulator that is capable of preventing or slowing the development of cancer in a mammal is refened to as a "prophylactically effective dose.” The particular dose required for a prophylactic treatment will depend upon the medical condition and history of the mammal, the particular cancer being prevented, as well as other factors such as age, weight, gender, administration route, efficiency, etc.
• Such prophylactic treatments may be used, e.g., in a mammal who has previously had cancer to prevent a recunence of the cancer, or in a mammal who is suspected of having a significant likelihood of developing cancer based, at least in part, upon gene expression profiles.
• Vaccine strategies may be used, in either a DNA vaccine form, or protein vaccine.
• cancer protein-modulating compounds can be administered alone or in combination with additional cancer modulating compounds or with other therapeutic agent, e.g., other anti-cancer agents or treatments.
• one or more nucleic acids e.g., polynucleotides comprising nucleic acid sequences set forth in the Tables, such as RNAi, antisense polynucleotides or ribozymes, will be introduced into cells, in vitro or in vivo.
• the present invention provides methods, reagents, vectors, and cells useful for expression of cancer-associated polypeptides and nucleic acids using in vitro (cell-free), ex vivo or in vivo (cell or organism-based) recombinant expression systems.
• the particular procedure used to introduce the nucleic acids into a host cell for expression of a protein or nucleic acid is application specific.
• cancer proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above.
• cancer genes (including both the full-length sequence, partial sequences, or regulatory sequences of the cancer coding regions) can be administered in a gene therapy application. These cancer genes can include inhibitory applications, e.g., as inhibitory RNA, gene therapy (e.g., for inco ⁇ oration into the genome), or antisense compositions.
• Cancer polypeptides and polynucleotides can also be administered as Vaccine compositions to stimulate HTL, CTL, and antibody responses.
• Such vaccine compositions can include, e.g., lipidated peptides (see, e.g.Nitiello, et al. (1995) J. Clin. Invest. 95:341-349), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG”) microspheres (see, e.g., Eldridge, et al. (1991) Molec. hnmunol. 28:287-294,; Alonso, et al. (1994) Vaccine 12:299-306; Jones, et al.
• Vaccine 13:675-681 peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi, et al. (1990) Nature 344:873- 875; Hu, et al. (1998) Clin Exp Immunol. 113:235-243), multiple antigen peptide systems (MAPs) (see, e.g., Tarn (1988) Proc. Natl. Acad. Sci. USA 85:5409-5413; Tarn (1996) Immunol.
• ISCOMS immune stimulating complexes
• MAPs multiple antigen peptide systems
• Toxin-targeted delivery technologies also known as receptor mediated targeting, such as those of Avant hnmunotherapeutics, Inc. (Needham, Massachusetts) may also be used.
• Vaccine compositions often include adjuvants.
• Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis, or .
• lipid A lipid A
• Bortadella pertussis or .
• Mycobacterium tuberculosis derived proteins are often include adjuvants.
• adjuvants are commercially available as, e.g., 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; salts of calcium, iron, or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A.
• Cytokines such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.
• Vaccines can be administered as nucleic acid compositions wherein DNA or RNA encoding one or more of the polypeptides, or a fragment thereof, is administered to a patient.
• This approach is described, for instance, in Wolff et. al. (1990) Science 247:1465-1468, as well as U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720; and in more detail below.
• DNA-based delivery technologies include "naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).
• the peptides of the invention can be expressed by viral or bacterial vectors.
• expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, e.g., as a vector to express nucleotide sequences that encode cancer polypeptides or polypeptide fragments. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response.
• Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848.
• BCG Bacille Calmette Guerin
• BCG vectors are described in Stover, et al. (1991) Nature 351 :456-460.
• a wide variety of other vectors are availablel for therapeutic administration or immunization, e.g., adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like. See, e.g., Shata, et al. (2000) Mol Med Today 6:66-71; Shedlock, et al. (2000) J. Leukoc. Biol. 68:793- 806; Hipp, et al. (2000) In Vivo 14:571-85.
• Methods for the use of genes as DNA vaccines are well known, and include placing a cancer gene or portion of a cancer gene under the control of a regulatable promoter or a tissue- specific promoter for expression in a cancer patient.
• the cancer gene used for DNA vaccines can encode full-length cancer proteins, but more preferably encodes portions of the cancer proteins including peptides derived from the cancer protein.
• a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a cancer gene.
• cancer-associated genes or sequence encoding subfragments of a cancer protein are introduced into expression vectors and tested for their immunogenicity in the context of Class I MHC and an ability to generate cytotoxic T cell responses. This procedure provides for production of cytotoxic T cell responses against cells which present antigen, including intracellular epitopes.
• DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine.
• adjuvant molecules include cytokines that increase the immunogenic response to the cancer polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are available.
• cancer genes find use in generating animal models of cancer.
• gene therapy technology e.g., wherein inhibitory or antisense RNA directed to the cancer gene will also diminish or repress expression of the gene.
• Animal models of cancer find use in screening for modulators of a cancer-associated sequence or modulators of cancer.
• transgenic animal technology including gene knockout technology, e.g., as a result of homologous recombination with an appropriate gene targeting vector, will result in the absence or increased expression of the cancer protein.
• tissue-specific expression or knockout of the cancer protein may be necessary.
• the cancer protein is overexpressed in cancer.
• transgenic animals can be generated that overexpress the cancer protein.
• promoters of various strengths can be employed to express the fransgene.
• the number of copies of the integrated fransgene can be determined and compared for a determination of the expression level of the transgene. Animals generated by such methods will find use as animal models of cancer and are additionally useful in screening for modulators to treat cancer. Kits for Use in Diagnostic and/or Prognostic Applications
• kits are also provided by the invention.
• such kits may 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 or polynucleotides, small molecule inhibitors of cancer-associated sequences etc.
• a therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.
• kits may include instructional materials containing instructions (e.g., protocols) for the practice of the methods of this invention.
• instructional materials typically comprise written or printed materials, they are not limited to such.
• a medium capable of storing such instructions and communicating them to an end user is contemplated by this invention.
• Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
• Such media may include addresses to internet sites that provide such instructional materials.
• kits for screening for modulators of cancer- associated sequences can be prepared from readily available materials and reagents.
• such kits can comprise one or more of the following materials: a cancer-associated polypeptide or polynucleotide, reaction tubes, and instructions for testing cancer-associated activity.
• the kit contains biologically active cancer protein.
• kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user. Diagnosis would typically involve evaluation of a plurality of genes or products. The genes will typically be selected based on conelations with important parameters in disease which may be identified in historical or outcome data.
• Table 1 lists medical conditions, abnormalities, or organs affected by disease, referred to in Tables 2A and 3A for which markers have been identified, and other related medical conditions (including va ⁇ ous stages and/or metastases) in which those markers will also be useful, e g , in therapeutic, diagnostic, prognostic, subsetting, vaccine, and other uses blood vessels/angiogenesis hemangiomas, lymphangiomas, a ⁇ giosarcoma, lymphangiosarcoma, Kaposi's sarcoma, wound healing, tissue remodeling, pso ⁇ asis, ischemic, heart disease, inflammatory diseases (e g , arthntis asthma, chronic bronchitis), atherosclerosis, endomel ⁇ osis, presumed ocular histoplasmosis syndrome, hypoxia solid tumors lymphomas, lymphadenitis, lymphangitis, autoimmune diseases (e g , RA, SLE, juvenile chronic arthntis, pigmented villo ⁇ odular syn
• fibrosis lung fibrosis idiopathic pulmonary fibrosis, hypersensitivity pneumonitis.interstitial pneumonitis, nonspecific idiopathic pneumonitis), chronic obstructive pulmonary disease (e g , emphysema, chronic bronchitis), asthma, bronchiectasis, cirrhosis (liver fibrosis), renal fibrosis, scleroderma, wound healing , head and neck tumors of the nasal cavity, paranasal sinuses, nasopharynx, oral cavity, oral pharynx, lip, larynx, hypopharynx, salivary glands, paragangliomas, esophagus kidney clear cell (nonpapillary) carcinoma papillary carcinoma, chromophobe renal carcinoma, hypernephroma adenocarcinoma, sporadic renal carcinomas,
• Tables 2B-72B, 76B, and 79B list accession numbers for Pkeys lacking UmgenelD's for Tables 2A-72A, 76A, and 79A, respectively
• gene cluster number 0 from which oligonucleotides were designed
• Gene clusters were compiled using sequences denved from Genbank ESTs and mRNAs These sequences were clustered based on sequence simila ⁇ ty using Cluste ⁇ g and Alignment Tools (DoubleTwist, Oakland California)
• Genbank accession numbers for sequences comp ⁇ sing each cluster are listed in the "Accession" column
• Tables 2C-72C, 76C, and 79C list genomic positioning for Pkeys lacking Unigene ID's and accession numbers in Tables 2A-72A, 76A, and 79A, respectively
• genomic sequence source used for prediction Nucleotide locations of each predicted exon are also listed
• Table 2A provides preferred disease indications and preferred utilities for about 413 selected genes These genes were identified using Eos/Affymet ⁇ x Genechip arrays 0
• blad bladder diseases
• angio blood vessel diseases
• EWS EWS
• E angio CTL+s m 133370, AF245505 Hs 72157, Adlican, breast, lung, pane, diag 310016, AW449612, Hs 152475, ESTs, colon, GTL 5 322303, AI357412, Hs 157601, ESTs, colon, pros, fibro, breast, CTL+diag
• hypoxia-mducible protein 2 lung, renal, CTL
• Hs 42645 solute earner family 16 (mono, breast, pane, uter, mela, mAb+s m 416658, U03272, Hs 79432, fibnllin 2 (congenital contra, lung, ovar, uter, blad, angio, test, sarc, diagra
• Hs 350470 trefoil factor 1 (breast cance, breast pane, lung o uc, diag 421659, NMJ14459, Hs 106511, protocadhe ⁇ n 17, fibro, mAb 421753, BE314828, Hs 107911, ATP-binding cassette, sub-fami, lung, mAb+s m 421817, AF146074, Hs 108660, ATP-binding cassette, sub-fami, lung, cerv, headnk, blad, mAb+s m 421829, AB018330, Hs 108708, calcium/calmodulin-dependent p, pros, s m 422048, NM_012445, Hs 288126, spondm 2, extracellular mat ⁇ , pane pros sarc, diag
• Hs 111256 arachidonate 15-l ⁇ poxygenase, , pros, s m 422109, S73265, Hs 1473, gast ⁇ n releasing peptide, pane, lung, colon, fibro, diag 422158, L10343, Hs 112341, protease inhibitor 3, skin der, headnk, blad, lung, cerv, stom, esoph, diag 422192, AA305159, Hs 113019, fls485, mela, s m 422260, AA315993, Hs 105484, regenerating gene type IV, colon, omuc, stom, pane, mAb+diag
• NM_002205 Hs 149609, integ ⁇ n, alpha 5 (fibronectin, pane, pros.angio, blad, lung, mAb+s m 424620, AA101043, Hs 151254, kallikrein 7 (chymotryptic, st, ovar; diag
• bone morphogenetic protein 7 (, ovar, colon, blad, lung, cerv, mAb+diag
• Hs 171014 VGF nerve growth factor induci, mela, sarc, diag 426761, AI015709, Hs 172089, PORIMIN Prc-oncosis receptor i, lung, esoph, pros, uter, pane, colon, ovar, headnk, mAb+s m
• H55709 Hs 2250
• leukemia inhibitory factor ch, ovar, pane, leuk, lung, diag 428296, NMJ03058, Hs 183572, solute earner family 22 (orga, renal, mAb+s m
• Hs 184601 solute earner family 7 (catio, lung, blad, headnk, cerv, esoph, glio, uter, stom, colon, mela, mAb+s m
• RAIG retinoic acid induced 3
• Hs 234434 hairy/enhancer-of split relate, glio, s m 430178, AW449612, Hs 152475, 3 UTR of achaete scute comple, colon, stom, ovar; CTL
• nucleolar protein 3 apoptosis, renal, CTL 432596, AJ224741, Hs 278461, mat ⁇ lin 3, pane, breast, sarc, diag
• NM_002104 Hs 3066, granzyme K (senne protease, g, renal, breast, lung, stom, hepC, fibro, leuk, CTL
• Hs 334762 type I transmembrane protein F, pane, colon, lung ovar, renal, esoph, mela blad, stom cerv, mAb 444090, S69115, Hs 10306, natural killer cell group 7 se, fibro, renal, mela, drag 444371, BE540274, Hs 239, forkhead box M1, lung, headnk, blad, glio, test, mela, s m
• Hs 11383 small inducible cytokine subfa, fibro, esoph, drag 444781, NM_014400, Hs 11950, GPI-anchored metastasis-associ, lung, blad, headnk, cerv, mAb+diag
• ADAMTS2 a disintegnn like a, headnk, breast colon, leuk, lung, blad, esoph, stom, sarc, mAb+diag
• Table 3A provides preferred disease indications and preferred utilities for about 2709 selected genes These genes were identified using Eos/Affymetnx Genechip arrays
• chromosome segregation 1 (yeas, ovar, lung, blad , diag 102348, U37519, Hs 87539, aldehyde dehydrogenase 3 famil, lung, blad, s m
• trefoil factor 1 (breast cance, breast pane, diag 103021, BE001596, Hs 85266, mteg ⁇ n, beta 4, lung blad, mAb
• ADAMTS2 a disinteg ⁇ n-like a, breast, colon, leuk, lung, blad, mAb+diag
• Hs 288969 Hs 288969, HSCARG protein, breast, diag 111900, AF131784, Hs 25318, Homo sapiens clone 25194 mRNA , breast, diag 111929, AF027208, Hs 112360, promimn (mouse)-l ⁇ ke 1, colon, breast, fibro, mAb _ _ 112134, 41823, Hs 7413, ESTs, calsy ⁇ ten ⁇ n-2, breast, EWS, diag 5 112244, AB029000, Hs 70823, KIAA1077 protein, breast colon, blad, lung, diag
• Hs 104800 hypothetical protein FLJ10134, breast, drag 121748, BE536911, Hs 234545, hypothetical protein NUF2R, breast, drag
• Hs 18212 DNA segment on chromosome X (u, breast, diag 130760, AW379130, Hs 18953, phosphodiesterase 9A, pros, CTL+s m 130800, AI187292, Hs 19574, hypothetical protein MGC5469, colon, lung, diag 130839, AB011169, Hs 380875, similar to S cerevisiae SSM4, angio, diag 130844, U76248, Hs 20191, seven in absentia (Drasophila), breast, diag
• Hs 82772 collagen, type XI, alpha 1, breast, lung, ovar, headnk, CTL 134470, X54942, Hs 83758, CDC28 protein kinase 2, lung, blad, headnk, s m 134520, BE091005, Hs 349506, activated RNA polymerase II t ⁇ ovar, s m 134529, AW411479, Hs 848, FK506-b ⁇ nd ⁇ ng protein 4 (59kD), breast, diag 5 134570, U66615, Hs 172280, SWI/SNF related, matrix associ, EWS, CTL+s m

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