Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57419—Specifically defined cancers of colon
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57415—Specifically defined cancers of breast

Definitions

• the invention relates to the identification of expression profiles and the nucleic acids involved in breast and/or colorectal cancer, and to the use of such expression profiles and nucleic acids in diagnosis and prognosis of such cancers.
• the invention further relates to methods for identifying and using candidate agents and/or targets which modulate certain cancers.
• Antigens suitable for immunotherapeutic strategies should be highly expressed in cancer tissues and ideally not expressed in normal adult tissues. Expression in tissues that are dispensable for life, however, may be tolerated. Examples of such antigens include Her2/neu and the B-cell antigen CD20. Humanized monoclonal antibodies directed to Her2/neu (Herceptin) are currently in use for the treatment of metastatic breast cancer (Ross and Fletcher, 1998, Stem Cells 16:413-428).
• anti-CD20 monoclonal antibodies are used to effectively treat non-Hodgekin's lymphoma (Maloney et al., 1997, Blood 90:2188-2195; Leget and Czuczman, 1998, Curr. Opin. Oncol. 10:548-551).
• Breast cancer is a significant cancer in Western populations. It develops as the result of a pathologic transformation of normal breast epithelium to an invasive cancer. There have been a number of recently characterized genetic alterations that have been implicated in breast cancer. However, identification of additional genetic alterations involved in the development of breast cancer is needed.
• colon cancer Another disease state which requires more attention is colon cancer (used interchangeably herein with “colorectal cancer”).
• colon cancer has been a number of recently characterized genetic alterations that have been implicated in colorectal cancer, including mutations in two classes of genes, tumor-suppressor genes and proto-oncogenes, with recent work suggesting that mutations in DNA repair genes may also be involved in tumorigenesis.
• APC adenomatous polyposis coli
• genes implicated in colorectal cancer include the MCC gene, the p53 gene, the DCC (deleted in colorectal carcinoma) gene and other chromosome 18q genes, and genes in the TGF- ⁇ signalling pathway.
• Ephrin-A3 (Kozlosky et al., Oncogene 10(2):299-306 (1995)) is found at accession number P52797 (SEQ ID NO:5), which is nearly identical to the amino acid sequence for EHK1 (Davis et al., Science 266(5186):816-819 (1994)), deduced from the cDNA sequence shown in accession number L37360 (SEQ ID NO:6). These two proteins are possibly the result of mRNA splice variants of the same gene.
• the amino acid sequence of a mouse homolog of Ephrin-A3 is shown in accession number 008545 (SEQ ID NO:7).
• Ephrin-A3 has not been associated with any disease state.
• provided herein are methods that can be used in diagnosis and prognosis of breast and colorectal cancer. Further provided are methods that can be used to screen candidate bioactive agents for the ability to modulate breast and/or colon cancer. Additionally, provided herein are molecular targets for therapeutic intervention in certain cancers.
• a method of screening drug candidates comprises providing a cell that expresses an expression profile gene or fragments thereof.
• Preferred embodiments of the expression profile gene as described herein include the sequence comprising SEQ ID NO:8 (encoding CHA4) or SEQ ID NO:9 (encoding CBK8), or a fragment thereof.
• the method further includes adding a drug candidate to the cell and determining the effect of the drug candidate on the expression of the expression profile gene.
• the method of screening drug candidates includes comparing the level of expression in the absence of the drug candidate to the level of expression in the presence of the drug candidate, wherein the concentration of the drug candidate can vary when present, and wherein the comparison can occur after addition or removal of the drug candidate.
• the cell expresses at least two expression profile genes. The profile genes may show an increase or decrease.
• Also provided herein is a method of screening for a bioactive agent capable of binding to CHA4 or CBK8, or a fragment thereof, the method comprising combining CHA4, CBK8, or fragment thereof and a candidate bioactive agent, and determining the binding of the candidate agent to the CHA4, CBK8, or fragment thereof.
• a method for screening for a bioactive agent capable of modulating the bioactivity of CHA4 or CBK8, or a fragment thereof comprises combining CHA4 or CBK8 or fragment thereof and a candidate bioactive agent, and determining the effect of the candidate agent on the bioactivity of CHA4 or CBK8 or the fragment thereof.
• CHA4 has the bioactivity of a breast cancer modulating protein.
• CHA4 or CBK8 has the bioactivity of a colorectal cancer modulating protein.
• CHA4 has the bioactivity of a breast cancer modulating protein and a colorectal cancer modulating protein.
• Also provided herein is a method of evaluating the effect of a candidate cancer drug comprising administering the drug to a transgenic animal expressing or over-expressing CHA4 or CBK8, or a fragment thereof, or an animal lacking CHA4 or CBK8 for example as a result of a gene knockout.
• a method of evaluating the effect of a candidate cancer drug comprising administering the drug to a patient and removing a cell sample from the patient.
• the expression profile of the ⁇ ell is then determined.
• the determination of the expression profile comprises determining the expression of CHA4 and/or CBK8.
• This method may further comprise comparing the expression profile to an expression profile of a healthy individual.
• the method comprises determining the expression of a gene which comprising SEQ ID NO:8 or SEQ ID NO: 9, or a fragment thereof, in a first tissue sample of a first individual, and comparing this to the expression of the gene from a second normal tissue sample of the first individual or a tissue sample of a second individual, the comparison of the expression indicates that the first individual has cancer.
• the first tissue sample is breast tissue and the cancer is breast cancer.
• the first tissue sample is colorectal tissue and the cancer is colorectal cancer.
• the present invention provides an antibody which specifically binds to CHA4 or CBK8, or a fragment thereof.
• the antibody is a monoclonal antibody.
• the antibody can be a fragment of an antibody such as a single stranded antibody as further described herein, or can be conjugated to another molecule.
• the antibody is a humanized antibody.
• a method for screening for a bioactive agent capable of interfering with the binding of CHA4 or a fragment thereof, and an antibody which binds to said CHA4 or fragment thereof comprises combining
• a method for screening for a bioactive agent capable of interfering with the binding of CBK8 or a fragment thereof, and an antibody which binds to said CBK8 or fragment thereof is provided.
• the method comprises combining CBK8 or a fragment thereof, a candidate bioactive agent and an antibody which binds to said CBK8 or fragment thereof.
• the method further includes determining the binding of said CBK8 or fragment thereof and said antibody.
• an agent is identified as an interfering agent.
• the interfering agent can be an agonist or an antagonist.
• the antibody as well as the agent inhibits breast cancer and/or colorectal cancer.
• a method for inhibiting the activity of a breast cancer or colorectal cancer modulating protein comprises binding an inhibitor to the protein.
• the protein is CHA4.
• the protein is CBK8.
• the invention provides a method for neutralizing the effect of a breast cancer or colorectal cancer modulating protein.
• the method comprises contacting an agent specific for the protein with the protein in an amount sufficient to effect neutralization.
• the protein is CHA4.
• the protein is a preferred embodiment, CHA4.
• a method for inhibiting breast cancer and/or colorectal cancer comprises administering to a cell a composition comprising an antibody to CHA4 or a fragment thereof. In another embodiment, the method comprises administering to a cell a composition comprising an antibody to CBK8 or a fragment thereof. In one embodiment, the antibody is conjugated to a therapeutic moiety. Such therapeutic moieties include a cytotoxic agent and a radioisotope.
• the method can be performed in vitro or in vivo, preferably in vivo to an individual. In a preferred embodiment the method of inhibiting breast cancer and/or colorectal cancer is provided to an individual with such cancer.
• methods of inhibiting breast cancer and/or colorectal cancer can be performed by administering any inhibitor of CHA4 or CBK8 activity to a cell or individual.
• a CHA4 inhibitor is an antisense molecule to CHA4.
• a CBK8 inhibitor is an antisense molecule to CBK8.
• biochip comprising a nucleic acid segment which encodes
• a method provided herein comprises administering to an individual a composition comprising CHA4 or CBK8, or a fragment thereof.
• said composition comprises a nucleic acid comprising a sequence encoding CHA4 or CBK8, or a fragment thereof.
• a composition comprising CHA4 or CBK8, or a fragment thereof is provided.
• the composition comprises a pharmaceutically acceptable carrier.
• Figure 1 shows an embodiment of a nucleic acid (SEQ ID NO:8) (mRNA) which includes a sequence which encodes a differentially expressed protein provided herein, CHA4. Start (ATG) and stop (TAG) codons are underlined.
• Figure 2 shows an embodiment of the amino acid sequence of CHA4 (SEQ ID NO: 10).
• Figures 3A-3D show the relative amount of expression of CHA4 in various samples of breast cancer tissue (3A), colorectal cancer tissue (3B), including primary tumors (dark bars) and metastatic tissue (light bars), and several normal tissue types (3C-3D).
• CHA4 is upregulated in both breast cancer tissue and colorectal cancer tissue as compared with normal tissues.
• Figure 4 shows an embodiment of a nucleic acid (SEQ ID NO:9) (mRNA) which includes a sequence which encodes a colorectal cancer protein provided herein, CBK8. The start
• accession no. AW136973 SEQ ID NO:2.
• Figure 5 shows an embodiment of an amino acid sequence of CBK8 (SEQ ID NO:11). each of the two sequences in bold corresponds to a Band 4.1 domain. The sequence underlined corresponds to a Pleckstrin domain.
• Figures 6A-6C show the relative amounts of expression of CBK8 in several different colorectal cancer tissue samples (Figure 6A), including primary tumor samples (solid bars) and samples of metastatic tissue (light bars), and various normal tissue types (Figure 6B and 6C).
• CBK8 is overexpressed in coloroectal cancer tissue as compared to normal tissues.
• the present invention provides novel methods for diagnosis and prognosis evaluation for breast and colorectal cancer, as well as methods for screening for compositions which modulate breast and colorectal cancer and compositions which bind to modulators of breast and colorectal cancer.
• the expression levels of genes are determined in different patient samples for which either diagnosis or prognosis information is desired, to provide expression profiles.
• An expression profile of a particular sample is essentially a "fingerprint" 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 unique to the state of the cell.
• normal tissue may be distinguished from cancer tissue, and within cancer tissue, different prognosis states (good or poor long term survival prospects, for example) may be determined.
• prognosis states good or poor long term survival prospects, for example
• By comparing expression profiles of cancer tissue in different states information regarding which genes are important (including both up- and down-regulation of genes) in each of these states is obtained.
• the identification of sequences that are differentially expressed in cancer tissue versus normal tissue, as well as differential expression resulting in different prognostic outcomes, allows the use of this information in a number of ways. For example, the evaluation of a particular treatment regime may be evaluated: does a chemotherapeutic drug act to improve the long-term prognosis in a particular patient. Similarly, diagnosis may be done or confirmed by comparing patient samples with the known expression profiles.
• these gene expression profiles allow screening of drug candidates with an eye to mimicking or altering a particular expression profile; for example, screening can be done for drugs that suppress the expression profile gene or convert a poor prognosis profile to a better prognosis profile. This may be done by making biochips comprising sets of the important cancer genes, which can then be used in these screens.
• cancer nucleic acid sequences can be administered for gene therapy purposes, including the administration of antisense nucleic acids, or the cancer proteins (including antibodies and other modulators thereof) administered as therapeutic drugs.
• the methods of screening, diagnosis, prognosis and treatment provided herein relate to cancer.
• the cancer is breast cancer and/or colorectal cancer.
• sequences that are differentially expressed in breast cancer and/or colorectal cancer when compared to normal tissue.
• sequences include those that are up-regulated (i.e. expressed at a higher level) in breast cancer and/or colorectal cancer, as well as those that are down-regulated (i.e. expressed at a lower level) in breast cancer and/or colorectal cancer.
• differentially expressed sequences are from humans; however, as will be appreciated by those in the art, differentially expressed sequences from other organisms may be useful in animal models of disease and drug evaluation; thus, other differentially expressed 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). Differentially expressed sequences from other organisms may be obtained using the techniques outlined below.
• the differentially expressed sequences are those of nucleic acids encoding CHA4 or fragments thereof.
• the differentially expressed sequence is that depicted in Figure 1 , or a fragment thereof.
• the differentially expressed sequences encode a protein having the amino acid sequence depicted in Figure 2, or a fragment thereof.
• CHA4 is human Ephrin-A3.
• the differentially expressed sequences are those of nucleic acids encoding CBK8 or fragments thereof.
• the differentially expressed sequence is that depicted in Figure 4, or a fragment thereof.
• the differentially expressed sequences encode a protein having the amino acid sequence depicted in Figure 5, or a fragment thereof.
• Differentially expressed sequences can include both nucleic acid and amino acid sequences.
• the differentially expressed sequences are recombinant nucleic acids.
• recombinant nucleic acid herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by polymerases and endonucleases, in a form not normally found in nature.
• 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 purposes 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, i.e. 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 purposes of the invention.
• a "recombinant protein” is a protein made using recombinant techniques, i.e. 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 all 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 differentially expressed 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.
• the differentially expressed sequences are nucleic acids.
• differentially expressed sequences are useful in a variety of applications, including diagnostic applications, which will detect naturally occurring nucleic acids, as well as screening applications; for example, biochips comprising nucleic acid probes to the differentially expressed sequences can be generated.
• diagnostic applications which will detect naturally occurring nucleic acids, as well as screening applications; for example, biochips comprising nucleic acid probes to the differentially expressed sequences can be generated.
• nucleic acid or oligonucleotide or grammatical equivalents herein means at least two nucleotides covalently linked together.
• a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); SRocl et al., Eur. J. Biochem. 81 :579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc.
• nucleic acid analogs may find use in the present invention.
• 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.
• 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 two advantages.
• the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (Tm) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4 ° C drop in Tm for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9°C.
• Tm melting temperature
• RNA typically exhibit a 2-4 ° C drop in Tm for an internal mismatch.
• the non-ionic PNA backbone the drop is closer to 7-9°C.
• hybridization of the bases attached to these backbones is relatively insensitive to salt concentration.
• 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 other strand (“Crick"); thus the sequences described herein also includes the complement of the sequence.
• the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.
• nucleoside includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides.
• nucleoside includes non-naturally occurring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
• a differentially expressed sequence can be initially identified by substantial nucleic acid and/or amino acid sequence homology to the differentially expressed sequences outlined herein. Such 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.
• differentially expressed sequences of the invention can be identified as follows. Samples of normal and tumor tissue are applied to biochips comprising nucleic acid probes.
• the samples are first microdissected, if applicable, and treated as is know in the art for the preparation of mRNA.
• Suitable biochips are commercially available, for example from Affymetrix. 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, but not limited to lung, heart, brain, liver, breast, kidney, muscle, prostate, small intestine, large intestine, spleen, bone, and placenta.
• those genes identified during the cancer screen that are expressed in any significant amount in other tissues are removed from the profile, although in some embodiments, this is not necessary. That is, when screening for drugs, it is preferable that the target be disease specific, to minimize possible side effects.
• differentially expressed sequences are those that are upregulated in breast cancer and/or colorectal cancer; that is, the expression of these genes is higher in carcinoma as compared to normal breast or colon tissue.
• Up-regulation as used herein means at least about a 50% increase, preferably a two-fold change, more preferably at least about a three fold change, with at least about five-fold or higher being preferred. All accession numbers herein are for the GenBank sequence database and the sequences of the accession numbers are hereby expressly incorporated by reference.
• GenBank is known in the art, see, e.g., Benson, DA, et al., Nucleic Acids Research 26:1-7 (1998) and the NCBI web site (www.ncbi.nlm.nih.gov). In addition, these genes were found to be expressed in a limited amount or not at all in heart, brain, lung, liver, kidney, muscle, pancreas, testes, stomach, small intestine and spleen.
• CHA4 is upregulated in breast cancer.
• CHA4 and/or CBK8 is upregulated in colorectal cancer.
• differentially expressed sequences are those that are down- regulated in breast or colorectal cancer; that is, the expression of these genes is lower in, for example, carcinoma as compared to normal tissue.
• Down-regulation as used herein 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.
• Differentially expressed proteins of the present invention may be classified as secreted proteins, transmembrane proteins or intracellular proteins.
• the differentially expressed protein is an intracellular protein.
• Intracellular proteins may be found in the cytoplasm and/or in the nucleus and may be associated with the plasma membrane. Intracellular proteins are involved in all aspects of cellular function and replication (including, for example, signaling pathways); aberrant expression of such proteins results in unregulated or disregulated cellular processes. For example, many 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.
• cytoskeleton-associated proteins have been found to contain certain motifs, for example, the Band 4.1 domain has been identified in a number of proteins that associate with other proteins at the interface between the plasma membrane and the cytoskeleton. (See, Rees et al., Nature 347:685-689 (1990); Funayama et al., J. Cell Biol. 115:1039-1048 (1991); Takeuchi et al., J. Cell Sci. 107:1921-1928 (1994)) .
• Proteins which contain a Band 4.1 domain included Band 4.1 , Ezrin, Moesin, Radixin, Talin, Filopodin and Merlin, which all have been shown or are suspected to bind cytoskeletal proteins, and many of which are associated with cell motility.
• Several protein tyrosine phosphatases e.g. PTPN3, PTPN4, PTPN14, PTP-D1 , PTP-RL10 and PTP2E
• PH pleckstrin homology
• Proteins which have been identified as possessing at least one PH domain include Pleckstrin, Ser/Thr protein kinases (Act/Rac family; beta-adrenergic receptor kinases), Tyrosine protein kinases (Btk/ltk/Tec subfamily),
• Insulin Receptor Substrate 1 Regulators of small G-proteins (guanine nucleotide releasing factor, guanine nucleotide exchange proteins, GTPase activating proteins), cytoskeletal proteins (dynamin, spectrin beta-chain, syntrophin), mammalian phosphatidylinositol-specific phospholipase C, oxysterol binding proteins (OSDBP, OSH1 , YHR073w), and Mouse protein citron (a putative rho/rac effector). Putative functions which have been suggested for the PH domain include binding to G-proteins, binding lipids, binding to phosphorylated Ser/Tyr residues and attachment to membranes by an unknown mechanism.
• G-proteins guanine nucleotide releasing factor, guanine nucleotide exchange proteins, GTPase activating proteins
• cytoskeletal proteins dynamin, spectrin beta-chain, syntrophin
• CBK8 is an intracellular protein. In a preferred embodiment, CBK8 is associated with the plasma membrane and/or the cytoskeleton of a cell in which it is expressed. In one embodiment, CBK8 is involved in intracellular signaling.
• the differentially expressed sequences are transmembrane proteins.
• Transmembrane proteins are molecules that span the 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.
• the intracellular domain may have enzymatic activity and/or may serve as a binding site for additional proteins.
• the intracellular domain of transmembrane proteins serves both roles.
• certain receptor tyrosine kinases have both protein kinase activity and SH2 domains.
• 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 are classified as "seven transmembrane domain" proteins, as they contain 7 membrane spanning regions.
• transmembrane protein receptors include, but are not limited to 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, interieukin receptors, e.g. IL-1 receptor, IL-2 receptor, etc.
• Characteristics of transmembrane domains include approximately 20 consecutive hydrophobic amino acids that may be followed by charged amino acids. Therefore, upon analysis of the amino acid sequence of a particular protein, the localization and number of transmembrane domains within the protein may be predicted.
• Immunoglobulin-like domains are highly conserved. Mucin-like domains may be involved in cell adhesion and leucine-rich repeats participate in protein-protein interactions.
• extracellular domains are involved in binding to other molecules.
• extracellular domains are receptors.
• Factors that bind the receptor domain include circulating ligands, which may be peptides, proteins, or small molecules such as adenosine and the like.
• 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.
• cytokines include cytokines, mitogenic factors, neurotrophic factors and the like.
• Extracellular domains also bind to cell-associated molecules. In this respect, they mediate cell-cell interactions.
• Cell-associated ligands can be tethered to the cell for example via a glycosylphosphatidylinositol (GPI) anchor, or may themselves be transmembrane proteins.
• GPI glycosylphosphatidylinositol
• Extracellular domains also associate with the extracellular matrix and contribute to the maintenance of the cell structure.
• transmembrane proteins are particularly preferred in the present invention as they are good targets for immunotherapeutics, as are described herein.
• transmembrane proteins can be also useful in imaging modalities.
• transmembrane protein can be made soluble by removing transmembrane sequences, for example 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 differentially expressed proteins are secreted proteins; the secretion of which can be either constitutive or regulated. These proteins have a signal peptide or signal sequence that targets the molecule to the secretory pathway.
• Secreted proteins are involved in numerous physiological events; by virtue of their circulating nature, they 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) or an endocrine manner (acting on cells at a distance).
• secreted molecules find use in modulating or altering numerous aspects of physiology.
• Differentially expressed proteins that are secreted proteins are particularly preferred in the present invention as they serve as good targets for diagnostic markers, for example for blood tests.
• CHA4 is a secreted protein.
• a differentially expressed sequence is initially identified by substantial nucleic acid and/or amino acid sequence homology to the differentially expressed 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.
• a nucleic acid is a "differentially expressed nucleic acid” if the overall homology of the nucleic acid sequence to the nucleic acid sequences encoding the amino acid sequences of the figures is preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90%. In some embodiments the homology will be as high as about 93 to 95 or 98%. Homology in this context means sequence similarity or identity, with identity being preferred. A preferred comparison for homology purposes is to compare the sequence containing sequencing errors to the correct sequence. This homology will be determined using standard techniques known in the art, including, but not limited to, the local homology algorithm of Smith & Waterman, Adv. Appl. Math.
• sequences which are used to determine sequence identity or similarity are selected from the sequences set forth in the figures.
• sequences utilized herein are those set forth in the figures.
• sequences are naturally occurring allelic variants of the sequences set forth in the figures.
• sequences are sequence variants as further described herein.
• PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by Higgins & Sharp CABIOS
• Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
• BLAST algorithm described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993).
• WU-BLAST-2 program is the WU-BLAST-2 program which was obtained from
• WU-BLAST-2 uses several search parameters, most of which are set to the default values.
• the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
• a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
• percent (%) nucleic acid sequence identity is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues of Figure 1 (SEQ ID NO:8) or Figure 4 (SEQ ID NO:9).
• 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.
• the alignment may include the introduction of gaps in the sequences to be aligned.
• the percentage of homology will be determined based on the number of homologous nucleosides in relation to the total number of nucleosides.
• homology of sequences shorter than those of the sequences identified herein and as discussed below 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 the nucleic acid sequences which encode the peptides identified in the figures, or their complements are considered a differentially expressed sequence.
• High stringency conditions are known in the art; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition, 2001 , and Short Protocols in Molecular Biology, Third Edition (ed. Ausubel, et al.) 1995, each of which are hereby incorporated by reference.
• 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 Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid
• Tm thermal melting point
• Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 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. 10 to 50 nucleotides) and at least about 60 ° C for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
• the degree of stringency can be based, for example, on the calculated (estimated) melting temperature (Tm) of the nucleic acid sequence binding complex or probe. Calculation of Tm is well known in the art (see, e.g. page 9.50-9.51 of Sambrook (1989), below). For example,
• “maximum stringency” typically occurs at about Tm-5°C (5° below the Tm of the probe); “high stringency” at about 5-10° below the Tm; “intermediate stringency” at about 10-20° below the Tm of the probe; and “low stringency” at about 20-25° below the Tm.
• hybridization conditions are carried out under high ionic strength conditions, for example, using 6XSSC or 6XSSPE. Under high stringency conditions, hybridization is followed by two washes with low salt solution, for example 0.5XSSC, at the calculated temperature. Under medium stringency conditions, hybridization is followed by two washes with medium salt solution, for example 2XSSC. Under low stringency conditions, hybridization is followed by two washes with high salt solution, for example 6XSSC.
• less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art; see Maniatis, supra, Sambrook, supra and Ausubel, supra, and Tijssen, supra.
• differentially expressed nucleic acid sequences of the invention are fragments of larger genes, i.e. they are nucleic acid segments. "Genes" in this context includes coding regions, non-coding regions, and mixtures of coding and non-coding regions. Accordingly, as will be appreciated by those in the art, using the sequences provided herein, additional sequences of the differentially expressed genes can be obtained, using techniques well known in the art for cloning either longer sequences or the full length sequences; see Maniatis et al., Sambrook and Russell and Ausubel, et al., supra, hereby expressly incorporated by reference.
• differentially expressed nucleic acid Once the differentially expressed nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire differentially expressed 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 differentially expressed nucleic acid can be further-used as a probe to identify and isolate other differentially expressed nucleic acids, for example additional coding regions. It can also be used as a
• precursor nucleic acid to make modified or variant differentially expressed nucleic acids and proteins.
• differentially expressed nucleic acids of the present invention are used in several ways.
• nucleic acid probes to the differentially expressed nucleic acids are made and attached to biochips to be used in screening and diagnostic methods, as outlined below, or for administration, for example for gene therapy and/or antisense applications.
• the differentially expressed nucleic acids that include coding regions of differentially expressed proteins can be put into expression vectors for the expression of differentially expressed proteins, again either for screening purposes or for administration to a patient.
• nucleic acid probes to differentially expressed nucleic acids are made.
• the nucleic acid probes attached to the biochip are designed to be substantially complementary to the differentially expressed nucleic acids, i.e. the target sequence (either the target sequence of the sample or to other probe sequences, for example 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. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, 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 to about 100 bases long, with from about 10 to about 80 bases being preferred, and from about 30 to about 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 (i.e. have some sequence in common), or separate.
• nucleic acids can be attached or immobilized to a solid support in a wide variety of ways.
• 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 below.
• the binding can be covalent or non-covalent.
• non-covalent binding and grammatical equivalents herein is meant one or more of either electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, 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, as will be appreciated by those in the art.
• 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 any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method.
• the number of possible substrates are 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 fluorescese.
• a preferred substrate is described in PCT publication WO
• the substrate is planar, although as will be appreciated by those in the art, 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, for example using linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
• additional linkers such as alkyl groups (including substituted and heteroalkyl groups) may be used.
• the oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5' or 3' terminus may be attached to the solid support, or attachment may be via 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, as is known in the art.
• nucleic acids can be synthesized in situ, using well 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 incorporated by reference; these methods of attachment form the basis of the Affimetrix GeneChipTM technology.
• differentially expressed nucleic acids encoding differentially expressed proteins are used to make a variety of expression vectors to express differentially expressed proteins which can then be used in screening assays, as described below.
• 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 differentially expressed protein.
• control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
• the control sequences that are suitable for prokaryotes 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.
• transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the differentially expressed protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the differentially expressed protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
• the 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 encode 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 in the art, and are useful in the present invention.
• the expression vector may comprise additional elements.
• the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification.
• the expression vector 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 well known in the art.
• the expression vector contains a selectable marker gene to allow the selection of transformed host cells.
• Selection genes are well known in the art and will vary with the host cell used.
• the differentially expressed proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a differentially expressed protein, under the appropriate conditions to induce or cause expression of the differentially expressed protein.
• the conditions appropriate for differentially expressed protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
• 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 Drosophila melangaster cells,
• Saccharomyces cerevisiae and other yeasts E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, THP1 cell line (a macrophage cell line) and human cells and cell lines.
• the differentially expressed proteins are expressed in mammalian cells.
• Mammalian expression systems are also known in the art, and include retroviral systems.
• a preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference.
• 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, herpes simplex virus promoter, and the CMV promoter.
• 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 polyadenlytion signals include those derived form SV40.
• differentially expressed proteins are expressed in bacterial systems.
• Bacterial expression systems are well known in the art. Promoters from bacteriophage may also be used and are known in the art.
• synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp 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 differentially expressed 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 in the art, and include vectors for Bacillus subtilis, E.
• the bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.
• differentially expressed proteins are produced in insect cells.
• Expression vectors for the transformation of insect cells and in particular, baculovirus-based expression vectors, are well known in the art.
• differentially expressed protein is produced in yeast cells.
• Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
• the differentially expressed protein may also be made as a fusion protein, using techniques well known in the art.
• the differentially expressed protein may be fused to a carrier protein to form an immunogen.
• the differentially expressed protein may be made as a fusion protein to increase expression, or for other reasons.
• the differentially expressed protein is a differentially expressed peptide
• the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes.
• the differentially expressed nucleic acids, proteins and antibodies of the invention are labeled.
• labeled herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound.
• labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes.
• the labels may be incorporated into the differentially expressed nucleic acids, proteins and antibodies at any position.
• the label should be capable of producing, either directly or indirectly, a detectable signal.
• the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 l, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
• a radioisotope such as 3 H, 14 C, 32 P, 35 S, or 125 l
• a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin
• an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
• Any method known in the art for conjugating the antibody to the label may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al
• the present invention also provides differentially expressed protein sequences.
• a differentially expressed protein of the present invention may be identified in several ways. "Protein” in this sense includes proteins, polypeptides, and peptides.
• the nucleic acid sequences of the invention can be used to generate protein sequences. There are a variety of ways to do this, including cloning the entire gene and verifying its frame and amino acid sequence, or by comparing it to known sequences to search for homology to provide a frame, assuming the differentially expressed protein has homology to some protein in the database being used. Generally, the nucleic acid sequences are input into a program that will search all three frames for homology.
• NCBI Advanced BLAST parameters The program is blastx or blastn.
• the database is nr.
• the input data is as "Sequence in FASTA format”.
• the organism list is "none”.
• the “expect” is 10; the filter is default.
• the “descriptions” is 500, the “alignments” is 500, and the “alignment view” is pairwise.
• the "Query Genetic Codes” is standard (1 ).
• the matrix is BLOSUM62; gap existence cost is 11 , per residue gap cost is 1 ; and the lambda ratio is .85 default. This results in the generation of a putative protein sequence.
• differentially expressed 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 to 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 known in the art as are outlined above for the nucleic acid homologies. Differentially expressed proteins of the present invention may be shorter or longer than the wild type amino acid sequences.
• differentially expressed proteins include portions or fragments of the wild type sequences, herein.
• the differentially expressed nucleic acids of the invention may be used to obtain additional coding regions, and thus additional protein sequence, using techniques known in the art.
• the differentially expressed proteins are derivative or variant differentially expressed proteins as compared to the wild-type sequence. That is, as outlined more fully below, the derivative differentially expressed peptide will 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 any residue within the differentially expressed peptide.
• differentially expressed proteins of the present invention are amino acid sequence variants. These variants 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 differentially expressed protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant differentially expressed 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 differentially expressed protein amino acid sequence.
• the variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
• the mutation per se need not be predetermined.
• random mutagenesis may be conducted at the target codon or region and the expressed differentially expressed 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, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of differentially expressed protein activities.
• Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.
• substitutions that are less conservative than those shown in Chart I.
• 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.
• the substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl is substituted for (or by) a hydrophobic residue, e.g.
• leucyl isoleucyl, phenylalanyl, valyl or alanyl
• a cysteine or proline is substituted for (or by) any other residue
• a residue having an electropositive side chain e.g. lysyl, arginyl, or histidyl
• an electronegative residue e.g. glutamyl or aspartyl
• a residue having a bulky side chain e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
• variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the differentially expressed proteins as needed.
• the variant may be designed such that the biological activity of the differentially expressed protein is altered. For example, glycosylation sites may be altered or removed.
• Covalent modifications of differentially expressed polypeptides are included within the scope of this invention.
• One type of covalent modification includes reacting targeted amino acid residues of a differentially expressed polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of a differentially expressed polypeptide.
• Derivatization with bifunctional agents is useful, for instance, for crosslinking differentially expressed to a water-insoluble support matrix or surface for use in the method for purifying a nti-d iff e rentia I ly expressed antibodies or screening assays, as is more fully described below.
• crosslinking agents include, e.g., 1 ,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1 ,8-octane and agents such as methyl-3-[(p-azidophenyl)- dithiojpropioimidate.
• Another type of covalent modification of the differentially expressed 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 purposes herein to mean deleting one or more carbohydrate moieties found in native sequence differentially expressed polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence differentially expressed polypeptide.
• Addition of glycosylation sites to differentially expressed polypeptides may be accomplished by altering the amino acid sequence thereof.
• the alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence differentially expressed polypeptide (for O-linked glycosylation sites).
• the differentially expressed amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the differentially expressed 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 differentially expressed polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide.
• Removal of carbohydrate moieties present on the differentially expressed 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 known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981 ).
• Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
• Another type of covalent modification of differentially expressed comprises linking the differentially expressed 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.
• nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes
• Differentially expressed polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising a differentially expressed polypeptide fused to another, heterologous polypeptide or amino acid sequence.
• a chimeric molecule comprises a fusion of a differentially expressed 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 differentially expressed polypeptide. The presence of such epitope-tagged forms of a differentially expressed polypeptide can be detected using an antibody against the tag polypeptide.
• the epitope tag enables the differentially expressed 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 differentially expressed 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 well known in the art.
• poly-histidine poly-his
• poly-histidine-glycine poly-his-glycine tags
• flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]
• c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]
• Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)].
• tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-
• differentially expressed protein in one embodiment are other differentially expressed proteins of the differentially expressed family, and differentially expressed proteins from other organisms, which are cloned and expressed as outlined below.
• probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related differentially expressed proteins from humans or other organisms.
• particularly useful probe and/or PCR primer sequences include the unique areas of the differentially expressed nucleic acid sequence.
• preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. The conditions for the PCR reaction are well known in the art.
• differentially expressed proteins can be made that are longer than those depicted in the figures, for example, by the elucidation of additional sequences, the addition of epitope or purification tags, the addition of other fusion sequences, etc.
• differentially expressed proteins may also be identified as being encoded by differentially expressed nucleic acids.
• differentially expressed proteins are encoded by nucleic acids that will hybridize to the sequences of the sequence listings, or their complements, as outlined herein.
• the differentially expressed protein when the differentially expressed protein is to be used to generate antibodies, for example for immunotherapy, the differentially expressed protein should share at least one epitope or determinant with the full length protein.
• epitope or “determinant” herein is meant a portion of a protein which will generate and/or bind an antibody or T-cell receptor in the context of MHC. Thus, in most instances, antibodies made to a smaller differentially expressed protein will be able to bind to the full length protein.
• the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.
• antibody includes antibody fragments, as are known in the art, including Fab, Fab 2 , single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
• Polyclonal antibodies can be raised in a mammal, for example, 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 CHA4 or CBK8, 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).
• the immunization protocol may be selected by one skilled in the art without undue experimentation.
• the antibodies may, alternatively, be monoclonal antibodies.
• Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein,
• 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 the CHA4 or CBK8 polypeptide, 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.
• lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].
• a suitable fusing agent such as polyethylene glycol
• Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and 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.
• a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
• the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
• HGPRT or HPRT hypoxanthine guanine phosphoribosyl transferase
• the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
• the antibodies are bispecific antibodies.
• Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the
• the antibodies to differentially expressed are capable of reducing or eliminating the biological function of differentially expressed, as is described below. That is, the addition of anti-differentially expressed antibodies (either polyclonal or preferably monoclonal) to differentially expressed (or cells containing differentially expressed) may reduce or eliminate the differentially expressed activity. Generally, at least a 25% decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95- 100% decrease being especially preferred.
• the antibodies to the differentially expressed proteins are humanized antibodies.
• 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) which contain minimal sequence derived from non-human immunoglobulin.
• Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form 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
• Fv framework residues of the human immunoglobulin are replaced by corresponding 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.
• the 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 correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature.
• Fc immunoglobulin constant region
• a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature,
• 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 the corresponding sequence from a non-human species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol.. 227:381 (1991 );
• human antibodies can be made by introducing of 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 differentially expressed 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.
• differentially expressed proteins against which antibodies are raised are secreted proteins as described above.
• antibodies used for treatment bind and prevent the secreted protein from binding to its receptor, thereby inactivating the secreted differentially expressed protein.
• the differentially expressed protein to which antibodies are raised is a transmembrane protein.
• antibodies used for treatment bind the extracellular domain of the differentially expressed 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 differentially expressed protein.
• the antibody may be a competitive, non-competitive or uncompetitive inhibitor of protein binding to the extracellular domain of the differentially expressed protein.
• the antibody is also an antagonist of the differentially expressed protein. Further, the antibody prevents activation of the transmembrane differentially expressed protein.
• the antibody when the antibody prevents the binding of other molecules to the differentially expressed protein, the antibody prevents growth of the cell.
• the antibody also sensitizes the cell to cytotoxic agents, including, but not limited to TNF-a, TNF-b, IL-1 , INF-g and IL-2, or chemotherapeutic agents including 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like.
• the antibody belongs to a sub-type that activates serum complement when complexed with the transmembrane protein thereby mediating cytotoxicity.
• differentially expressed is treated by administering to a patient antibodies directed against the transmembrane differentially expressed protein.
• the antibody is conjugated to a therapeutic moiety.
• the therapeutic moiety is a small molecule that modulates the activity of the differentially expressed protein.
• the therapeutic moiety modulates the activity of molecules associated with or in close proximity to the differentially expressed protein.
• the therapeutic moiety may inhibit enzymatic activity such as protease or protein kinase activity associated with cancer.
• the therapeutic moiety may also be a cytotoxic agent.
• targeting the cytotoxic agent to tumor 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 corresponding fragments include diptheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like.
• Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against differentially expressed proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody.
• Targeting the therapeutic moiety to transmembrane differentially expressed proteins not only serves to increase the local concentration of therapeutic moiety in the differentially expressed afflicted area, but also serves to reduce deleterious side effects that may be associated with the therapeutic moiety.
• the PC 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 contains a signal for that target localization, i.e., a nuclear localization signal.
• differentially expressed antibodies of the invention specifically bind to differentially expressed proteins.
• specifically bind herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10 "4 - 10 "6 M " ⁇ with a preferred range being 10 "7 - 10 "9 M “1 .
• the differentially expressed protein is purified or isolated after expression.
• Differentially expressed proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample.
• Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse- phase HPLC chromatography, and chromatofocusing.
• the differentially expressed protein may be purified using a standard anti-differentially expressed antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, NY (1982). The degree of purification necessary will vary depending on the use of the differentially expressed protein. In some instances no purification will be necessary.
• the differentially expressed proteins and nucleic acids are useful in a number of applications.
• the expression levels of genes are determined for different cellular states in the cancer phenotype; that is, the expression levels of genes in normal tissue and in cancer tissue (and in some cases, for varying severities of cancer that relate to prognosis, as outlined below) are evaluated to provide expression profiles.
• An expression profile of a particular cell state or point of development is essentially a "fingerprint" of the state; 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 unique to the state of the cell.
• tissue from a particular patient have the gene expression profile of normal or cancer tissue.
• differential expression refers to both qualitative as well as quantitative differences in the genes' temporal and/or cellular expression patterns within and among the cells.
• a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, for example, normal versus, for example, cancer tissue. That is, genes may be turned on or turned off in a particular state, relative to another state. As is apparent to the skilled artisan, any comparison of two or more states can be made. Such a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques in one such state or cell type, but is not detectable in both.
• the determination is quantitative in that expression is increased or decreased; that is, the expression of the gene 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 arrays,
• 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 preferred.
• this may be done by evaluation at either the gene transcript, or the protein level; that is, the amount of gene expression may be monitored using nucleic acid probes to the DNA or RNA equivalent of the gene transcript, and the quantification of gene expression levels, or, alternatively, the final gene product itself
• proteins can be monitored, for example through the use of antibodies to the differentially expressed protein and standard immunoassays (ELISAs,e tc.) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc.
• ELISAs,e tc. standard immunoassays
• mass spectroscopy assays mass spectroscopy assays
• 2D gel electrophoresis assays etc.
• the proteins corresponding to breast or colorectal cancer genes i.e. those identified as being important in a breast or colorectal cancer phenotype, can be evaluated in a breast or colorectal cancer diagnostic test.
• gene expression monitoring is done and a number of genes, i.e. an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well. Similarly, these assays may be done on an individual basis as well.
• differentially expressed nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of differentially expressed sequences in a particular cell.
• the assays are further described below in the example.
• nucleic acids encoding the differentially expressed protein are detected.
• DNA or RNA encoding the differentially expressed protein may be detected, of particular interest are methods wherein the mRNA encoding a differentially expressed protein is detected.
• the presence of mRNA in a sample is an indication that the differentially expressed gene has been transcribed to form the mRNA, and suggests that the protein is expressed.
• Probes to detect the mRNA can be any nucleotide/deoxynucleotide probe that is complementary to and base pairs 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. Following washing to remove the non-specifically bound probe, the label is detected.
• detection of the mRNA is performed in situ. In this method permeabilized cells or tissue samples are contacted with a detectably labeled nucleic acid probe for sufficient time to allow the probe to hybridize with the target mRNA. Following washing to remove the non-specifically bound probe, the label is detected.
• RNA probe for example a digoxygenin labeled riboprobe (RNA probe) that is complementary to the mRNA encoding a differentially expressed 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.
• any of the three classes of proteins as described herein secreted, transmembrane or intracellular proteins are used in diagnostic assays.
• the differentially expressed proteins, antibodies, nucleic acids, modified proteins and cells containing differentially expressed sequences are used in diagnostic assays. This can be done on an individual gene or corresponding polypeptide level.
• the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes and/or corresponding polypeptides.
• differentially expressed proteins find use as markers of breast cancer and colorectal cancer. Detection of these proteins in putative cancer tissue of patients allows for a determination or diagnosis of cancer. Numerous methods known to those of ordinary skill in the art find use in detecting cancer.
• antibodies are used to detect cancer.
• a preferred method separates proteins from a sample or patient by electrophoresis on a gel (typically a denaturing and reducing protein gel, but may be any other type of gel including isoelectric focusing gels and the like).
• the breast or colorectal cancer protein is detected by immunoblotting with antibodies raised against the cancer protein. Methods of immunoblotting are well known to those of ordinary skill in the art.
• antibodies to the differentially expressed protein find use in in situ imaging techniques.
• cells are contacted with from one to many antibodies to the differentially expressed 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 differentially expressed protein(s) contains a detectable label.
• each one of multiple primary antibodies contains a distinct and detectable label. This method finds particular use in simultaneous screening for a pluralilty of differentially expressed proteins. As will be appreciated by one of ordinary skill in the art, numerous other histological imaging techniques are useful in 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 differentially expressed from blood samples and other bodily secretions.
• certain differentially expressed proteins are secreted/circulating molecules. Blood samples and other bodily secretions, therefore, are useful as samples to be probed or tested for the presence of secreted differentially expressed proteins.
• Antibodies can be used to detect the differentially expressed by any of the previously described immunoassay techniques including ELISA, immunoblotting (Western blotting), immunoprecipitation, BIACORE technology and the like, as will be appreciated by one of ordinary skill in the art.
• in situ hybridization of labeled differentially expressed nucleic acid probes to tissue arrays is done.
• arrays of tissue samples including breast or colorectal cancer tissue and/or normal tissue, are made.
• In situ hybridization as is known in the art can then be done.
• differentially expressed proteins, antibodies, nucleic acids, modified proteins and cells containing differentially expressed sequences are used in prognosis assays.
• gene expression profiles can be generated that correlate to breast and/or colorectal cancer severity, in terms of long term prognosis. Again, this may be done on either a protein or gene level, with the use of genes being preferred.
• the differentially expressed probes are attached to biochips for the detection and quantification of differentially expressed sequences in a tissue or patient. The assays proceed as outlined for diagnosis.
• any of the three classes of proteins as described herein are used in drug screening assays.
• the differentially expressed proteins, antibodies, nucleic acids, modified proteins and cells containing differentially expressed 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, Zlokarnik, et al., Science 279, 84-8 (1998), Heid, 1996 #69.
• the differentially expressed proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified differentially expressed proteins are used in screening assays. That is, the present invention provides novel methods for screening for compositions which modulate the breast or colorectal cancer phenotype. 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. Having identified the differentially expressed genes herein, a variety of assays may be executed. In a preferred embodiment, assays may be run on an individual gene or protein level.
• candidate bioactive agents may be screened to modulate this gene's response; preferably to down regulate the gene, although in some circumstances to up regulate the gene.
• Modulation thus includes both an increase and a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tumor tissue, 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 tumor compared to normal tissue, a decrease of about four fold is desired; in the case of a 10 fold decrease in tumor compared to normal tissue, a 10 fold increase in expression for a candidate agent is desired.
• this may be done by evaluation at either the gene or the protein level; that is, 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, for example through the use of antibodies to the differentially expressed protein and standard immunoassays.
• gene expression monitoring is done and a number of genes, i.e. an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well.
• differentially expressed nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of differentially expressed sequences in a particular cell.
• the assays are further described below.
• a candidate bioactive agent is added to the cells prior to analysis.
• screens are provided to identify a candidate bioactive agent which modulates cancer, modulates cancer proteins, binds to a cancer protein, or interferes between the binding of a cancer protein and an antibody.
• cancer bioactive agent or “drug candidate” or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for bioactive agents that are capable of directly or indirectly altering the cancer phenotype or the expression of a differentially expressed sequence, including both nucleic acid sequences and protein sequences.
• the bioactive agents modulate the expression profiles, or expression profile nucleic acids or proteins provided herein.
• the candidate agent suppresses a cancer phenotype, for example to a normal tissue fingerprint.
• the candidate agent preferably suppresses a severe cancer phenotype.
• a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
• a candidate agent will neutralize the effect of a CRC protein.
• neutralize is meant that activity of a protein is either inhibited or counter acted against so as to have substantially no effect on a cell.
• Candidate agents 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. Preferred 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 preferred are peptides.
• Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
• the candidate bioactive agents are proteins.
• protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
• the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
• amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
• Amino acid also includes imino acid residues such as proline and hydroxyproline.
• the side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
• the candidate bioactive agents are 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 procaryotic and eucaryotic proteins may be made for screening in the methods of the invention.
• Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
• the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred.
• the peptides may be digests of naturally occurring proteins as is outlined above, 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 incorporate any 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, for example, 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.
• the candidate bioactive agents are nucleic acids, as defined above.
• nucleic acid candidate bioactive agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids.
• digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.
• the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.
• the sample containing the target sequences 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 occurring as needed, as will be appreciated by those in the art.
• an in vitro transcription with labels covalently attached to the nucleosides is done.
• the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
• the target sequence is labeled with, for example, 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 removed prior to analysis.
• these 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, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681 ,697, all of which are hereby incorporated by reference.
• 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, chaotropic salt concentration pH, organic solvent concentration, etc.
• reaction may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below.
• the reaction may include a variety of other reagents may be included in the assays. These include reagents like 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. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample preparation methods and purity of the target.
• the data is analyzed to determine the expression levels, and changes in expression levels as between states, of individual genes, forming a gene expression profile.
• the screens are done to identify drugs or bioactive agents that modulate the cancer phenotype.
• a preferred embodiment is in the screening of candidate agents that can induce or suppress a particular expression profile, thus preferably generating the associated phenotype. That is, candidate agents that can mimic or produce an expression profile in, for example, breast or colorectal cancer similar to the expression profile of normal breast or colon tissue is expected to result in a suppression of the breast or colorectal cancer phenotype.
• mimicking an expression profile, or changing one profile to another is the goal.
• screens can be run to alter the expression of the genes individually. That is, screening for modulation of regulation of expression of a single gene can be done; that is, rather than try to mimic all or part of an expression profile, screening for regulation of individual genes can be done. Thus, for example, particularly in the case of target genes whose presence or absence is unique between two states, screening is done for modulators of the target gene expression.
• screening is done to alter the biological function of the expression product of the differentially expressed gene. Again, having identified the importance of a gene in a particular state, screening for agents that bind and/or modulate the biological activity of the gene product can be run as is more fully outlined below.
• screening of candidate agents that modulate the cancer phenotype either at the gene expression level or the protein level can be done.
• screens can be done for novel genes that are induced in response to a candidate agent.
• 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 any of the methods described herein for differentially expressed 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 to the treated cancer tissue sample.
• a candidate agent is administered to a population of breast or colorectal cancer cells, that thus has an associated breast or colorectal 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 i.e. a peptide
• the cells can be washed if desired and are allowed to incubate under preferably physiological conditions for some period of time.
• the cells are then harvested and a new gene expression profile is generated, as outlined herein.
• breast or colorectal cancer tissue may be screened for agents that reduce or suppress the breast or colorectal cancer phenotype.
• a change in at least one gene of the expression profile indicates that the agent has an effect on breast or colorectal 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 referred to herein as “differentially expressed proteins” or “cancer modulating proteins”. Additionally, “modulator” and “modulating” proteins are sometimes used interchangeably herein.
• the differentially expressed protein is termed CHA4.
• the diferentially expressed protein is termed CBK8.
• CBK8 CBK8 sequences can be identified as described herein for differentially expressed sequences.
• CHA4 sequences are depicted in Figures 1 (SEQ ID NO:8) and 2 (SEQ ID NO:10).
• CBK8 sequences are depicted in Figures 4 (SEQ ID NO:9) and 5 (SEQ ID NO:11).
• the differentially expressed protein may be a fragment, or alternatively, be the full length protein to the fragment shown herein.
• the differentially expressed protein is a fragment.
• the amino acid sequence which is used to determine sequence identity or similarity is that depicted in Figure 2 or Figure 5.
• the sequences are naturally occurring allelic variants of a protein having the sequence depicted in Figure 2 or Figure 5.
• the sequences are sequence variants as further described herein.
• the differentially expressed protein is a fragment of approximately 14 to 24 amino acids long. More preferably the fragment is a soluble fragment.
• the fragment includes a non-transmembrane region.
• the fragment has an N- terminal Cys to aid in solubility.
• the c-terminus of the fragment is kept as a free acid and the n-terminus is a free amine to aid in coupling, i.e., to cysteine.
• a CHA4 fragment has at least one CHA4 bioactivity or at least one CBK8 bioactivity, as defined below.
• the differentially expressed proteins are conjugated to an immunogenic agent as discussed herein.
• the differentially expressed protein is conjugated to BSA.
• screening for modulators of expression of specific genes can be done. This will be done as outlined above, but in general the expression of only one or a few genes are evaluated.
• screens are designed to first find candidate agents that can bind to differentially expressed proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate differentially expressed activity.
• assays there are a number of different assays which may be run; binding assays and activity assays.
• binding assays are done.
• purified or isolated gene product is used; that is, the gene products of one or more differentially expressed nucleic acids are made. In general, this is done as is known in the art.
• antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present.
• cells comprising the differentially expressed proteins can be used in the assays.
• the methods comprise combining a differentially expressed protein and a candidate bioactive agent, and determining the binding of the candidate agent to the differentially expressed protein.
• Preferred embodiments utilize the human differentially expressed protein, although other mammalian proteins may also be used, for example for the development of animal models of human disease.
• variant or derivative differentially expressed proteins may be used.
• the differentially expressed protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.).
• isolated sample receiving areas e.g. a microtiter plate, an array, etc.
• soluble assays known in the art may be performed.
• the insoluble supports may be made of any 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 any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads.
• Microtiter plates and arrays 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 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.
• Preferred 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 differentially expressed protein is bound to the support, and a candidate bioactive agent is added to the assay.
• the candidate agent is bound to the support and the differentially expressed 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 purpose, 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 candidate bioactive agent to the differentially expressed protein may be done in a number of ways.
• the candidate bioactive agent is labeled, and binding determined directly. For example, this may be done by attaching all or a portion of the differentially expressed protein to a solid support, adding a labeled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support.
• a labeled candidate agent for example a fluorescent label
• washing off excess reagent for example a fluorescent label
• determining whether the label is present on the solid support Various blocking and washing steps may be utilized as is known in the art.
• label herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fiuorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc.
• a detectable signal e.g. radioisotope, fiuorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc.
• Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
• the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above.
• the label can directly or indirectly provide a detectable signal.
• only one of the components is labeled.
• the proteins or proteinaceous candidate agents
• more than one component may be labeled with different labels; using 125 l for the proteins, for example, and a fluorophor for the candidate agents.
• the binding of the candidate bioactive agent is determined through the use of competitive binding assays.
• the competitor is a binding moiety known to bind to the target molecule (i.e. breast or colorectal cancer), such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent.
• the candidate bioactive agent is labeled.
• Either the candidate bioactive agent, 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 any temperature which facilitates optimal activity, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 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.
• the competitor is added first, followed by the candidate bioactive agent.
• Displacement of the competitor is an indication that the candidate bioactive agent is binding to the differentially expressed protein and thus is capable of binding to, and potentially modulating, the activity of the differentially expressed protein.
• either component can be labeled.
• the presence of label in the wash solution indicates displacement by the agent.
• the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.
• the candidate bioactive agent is added first, with incubation and washing, followed by the competitor.
• the absence of binding by the competitor may indicate that the bioactive agent is bound to the differentially expressed protein with a higher affinity.
• the candidate bioactive agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the candidate agent is capable of binding to the differentially expressed protein.
• the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the differentially expressed proteins.
• the methods comprise combining a differentially expressed protein and a competitor in a first sample.
• a second sample comprises a candidate bioactive agent, a differentially expressed 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 differentially expressed protein and 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 differentially expressed protein.
• a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native differentially expressed protein, but cannot bind to modified differentially expressed proteins.
• the structure of the differentially expressed protein may be modeled, and used in rational drug design to synthesize agents that interact with that site.
• Drug candidates that affect breast or colorectal cancer bioactivity 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.
• Preferably all control 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, all 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., may be used. The mixture of components may be added in any order that provides for the requisite binding.
• methods for screening for a bioactive agent capable of modulating the activity of differentially expressed proteins comprise the steps of adding a candidate bioactive agent to a sample of differentially expressed proteins, as above, and determining an alteration in the biological activity of differentially expressed proteins.
• “Modulating the activity" of breast and/or colorectal cancer includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present.
• the candidate agent should both bind to cancer proteins (although this may not be necessary), and alter its biological or biochemical activity as defined herein.
• the methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of differentially expressed proteins.
• the methods comprise combining a breast or colorectal cancer sample and a candidate bioactive agent, and evaluating the effect on breast or colorectal cancer activity, respectively.
• cancer activity or grammatical equivalents herein is meant at least one of cancer's biological activities, including, but not limited to, cell division, preferably in breast or colon tissue, cell proliferation, tumor growth, and transformation of cells.
• cancer activity includes activation of CHA4 or a substrate thereof by CHA4.
• cancer activity includes activation of CBK8 or a substrate thereof by CBK8.
• An inhibitor of cancer activity is an agent which inhibits any one or more cancer activities.
• the activity of the differentially expressed protein is increased; in another preferred embodiment, the activity of the differentially expressed protein is decreased.
• bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments.
• the invention provides methods for screening for bioactive agents capable of modulating the activity of a differentially expressed protein.
• the methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising differentially expressed proteins.
• Preferred cell types include almost any cell.
• the cells contain a recombinant nucleic acid that encodes a differentially expressed 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, for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts).
• physiological signals for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts).
• the determinations are determined at different stages of the cell cycle process.
• CH4 protein activity includes at least one of the following: cancer activity, binding to hek, elk or another Eph family receptor tyrosine kinase, binding to CHA4, activation of CHA4 or activation of substrates of CHA4 by CHA4.
• An inhibitor of CHA4 inhibits at least one of CHA4's bioactivities.
• CBK8 protein activity includes at least one of the following: cancer activity, binding to a plasma membrane-associated protein, binding to a cytoskeletal protein, binding to CBK8, activation of CBK8 or activation of substrates of CBK8 by CBK8.
• An inhibitor of CBK8 inhibits at least one of CBK ⁇ 's bioactivities.
• a method of inhibiting breast cancer cell division comprises administration of a breast cancer inhibitor.
• a method of inhibiting colorectal cancer cell division is provided. The method comprises administration of a colorectal cancer inhibitor.
• a method of inhibiting tumor growth comprises administration of a breast and/or colorectal cancer inhibitor.
• the inhibitor is an inhibitor of CHA4.
• the inhibitor is an inhibitor of CBK8.
• methods of treating cells or individuals with cancer comprise administration of a breast and/or colorectal cancer inhibitor.
• the inhibitor is an inhibitor of CHA4.
• the inhibitor is an inhibitor of CBK8.
• a differentially expressed protein inhibitor is an antibody as discussed above.
• the inhibitor is an antisense molecule.
• Antisense molecules as used herein include antisense or sense oligonucleotides comprising a singe- stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for differentially expressed molecules.
• a preferred antisense molecule is for CHA4 or CBK8, or for a ligand or activator thereof.
• Antisense or sense oligonucleotides, according to the present invention comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
• Antisense molecules 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 corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
• a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-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 treatment.
• the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described.
• the agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways.
• the concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%.
• the agents may be administered alone or in combination with other treatments, i.e., radiation.
• compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
• Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds.
• Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
• the invention provides methods for identifying cells containing variant cancer genes comprising determining all or part of the sequence of at least one endogeneous cancer gene in a cell. As will be appreciated by those in the art, this may be done using any number of sequencing techniques. In a preferred embodiment, the invention provides methods of identifying the cancer genotype of an individual comprising 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 gene to a known gene, i.e. a wild-type gene.
• the sequence of all or part of the differentially expressed gene can then be compared to the sequence of a known differentially expressed gene to determine if any differences exist. This can be done using any number of known homology programs, such as Bestfit, etc.
• the presence of a difference in the sequence between the differentially expressed gene of the patient and the known differentially expressed gene is indicative of a disease state or a propensity for a disease state, as outlined herein.
• the differentially expressed genes are used as probes to determine the number of copies of the differentially expressed gene in the genome.
• differentially expressed genes are used as probed to determine the chromosomal localization of the differentially expressed 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 differentially expressed gene loci.
• methods of modulating breast cancer and/or colorectal cancer in cells or organisms comprise administering to a cell an antibody that reduces or eliminates the biological activity of an endogenous differentially expressed protein.
• the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a differentially expressed protein.
• this may be accomplished in any number of ways.
• the activity of the differentially expressed gene is increased by increasing the amount of differentially expressed protein in the cell, for example by overexpressing the endogenous protein or by administering a gene encoding the sequence, using known gene-therapy techniques.
• the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entirety.
• EHR enhanced homologous recombination
• the activity of the endogeneous gene is decreased, for example by the administration of an inhibitor of cancer, such as an antisense nucleic acid.
• the differentially expressed proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to differentially expressed proteins, which are useful as described herein.
• the differentially expressed proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify differentially expressed antibodies.
• the antibodies are generated to epitopes unique to a differentially expressed protein; that is, the antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications.
• the differentially expressed antibodies may be coupled to standard affinity chromatography columns and used to purify differentially expressed proteins.
• the antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the differentially expressed protein.
• a therapeutically effective dose of a differentially expressed nucleic acid or differentially expressed protein or modulator thereof is administered to a patient.
• therapeutically effective dose herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
• a "patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications.
• the patient is a mammal, and in the most preferred embodiment the patient is human.
• differentially expressed proteins and modulators of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
• the differentially expressed proteins and modulators may be directly applied as a solution or spray.
• compositions of the present invention comprise a differentially expressed 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 acid,
• “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 preferred 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.
• carrier proteins such as serum albumin
• buffers such as buffers
• fillers such as microcrystalline cellulose, lactose, corn and other starches
• binding agents such as microcrystalline cellulose, lactose, corn
• differentially expressed proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above.
• differentially expressed genes (including both the full-length sequence, partial sequences, or regulatory sequences of the differentially expressed coding regions) can be administered in gene therapy applications, as is known in the art.
• differentially expressed genes can include antisense applications, either as gene therapy (i.e. for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
• differentially expressed genes are administered as DNA vaccines, either single genes or combinations of differentially expressed genes. Naked DNA vaccines are generally known in the art. Brower, Nature Biotechnology, 16:1304-1305 (1998).
• differentially expressed genes of the present invention are used as DNA vaccines.
• Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a differentially expressed gene or portion of a differentially expressed gene under the control of a promoter for expression in a patient with breast cancer or cancer.
• the differentially expressed gene used for DNA vaccines can encode full- length differentially expressed proteins, but more preferably encodes portions of the differentially expressed proteins including peptides derived from the differentially expressed protein.
• a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a differentially expressed gene.
• the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine.
• adjuvant molecules include cytokines that increase the immunogenic response to the differentially expressed polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are known to those of ordinary skill in the art and find use in the invention.
• differentially expressed genes find use in generating animal models of cancer.
• gene therapy technology wherein antisense RNA directed to the cancer gene will also diminish or repress expression of the gene.
• An animal generated as such serves as an animal model of cancer that finds use in screening bioactive drug candidates.
• gene knockout technology for example as a result of homologous recombination with an appropriate gene targeting vector, will result in the absence of the cancer protein.
• tissue-specific expression or knockout of the cancer protein may be necessary.
• differentially expressed protein is overexpressed in breast and/or coloretal cancer.
• transgenic animals can be generated that overexpress the differentially expressed protein.
• promoters of various strengths can be employed to express the transgene.
• the number of copies of the integrated transgene can be determined and compared for a determination of the expression level of the transgene. Animals generated by such methods find use as animal models of differentially expressed and are additionally useful in screening for bioactive molecules to treat disorders related to the differentially expressed protein.
• RNA is purified from cells or tissue
• double-stranded cDNA is prepared from the RNA
• the cDNA is purified
• the cDNA is then labeled with biotin during an in vitro transcription (IVT) reaction
• the cRNA prepared in the IVT reaction is purified, fragmented, and hybridized to an oligonucleotide array.
• tissue homogenizer Polytron PT3100 fitted with probe 9100072, Kinematica
• Sterilize by running the homogenizer in ethanol, and then run the homogenizer in at least 3 mL of TRIzol reagent (Life Technology/GibcoBRL).
• tissue weight Homogenize tissue samples in 1 mL of TRIzol per 50 mg of tissue. If cells derived from experimental model systems are used as the source of RNA, use 1 mL of TRIzol per 5-10 x 106 cells. Homogenize tissue or cells thoroughly.
• the probe After each sample homogenization run the probe in at least 3 mL fresh TRIzol, and then add this TRIzol back to the homogenized sample. Wash the probe with at least 50 mL fresh RNase-free water before proceeding to the next sample.
• centrifuge sample in a microfuge at 12 OOOg for 10 min at 4°C (microfuge tubes) or in a Sorvall centrifuge (Sorvall Centrifuge RT7 Plus) at 4000 RPM for 60 min at 4°C (15 mL conical tubes).
• RNA pellet Resuspend RNA pellet in 50 uL RNase-free water. Vortex. Incubate at 65°C for 10 minutes, vortex for 3 seconds to resuspend pellet, and spin briefly to collect sample in the bottom of the microcentrifuge tube.
• RNA sample 1 uL of RNA sample to quantify RNA in a spectrometer.
• the ratio of the optical density readings at 260 and 280 nm should be between 1.4 and 2.0 OD.
• Use 250-500 ng of RNA sample to run on a 1 % agarose electrophoretic gel to check integrity of 28S, 18S and 5S RNAs. Smearing of the RNA should be minimal and not biased toward RNAs of lower molecular weight.
• RNA purification no more than 100 ug of RNA on an individual RNeasy column (Qiagen).
• manufacturer's instructions for RNA purification Adjust the sample to a volume of 100 uL with RNase-free water. Add 350 uL Buffer RLT and then 250 uL ethanol to the sample. Mix gently by pipetting and then apply sample to the RNeasy column. Centrifuge in a microcentrifuge for 15 seconds at 10 000 RPM.
• PolyA+ RNA can be purified from total RNA if desired using the Oligotex mRNA Purification System (Qiagen) by following the manufacturer's instructions. Before proceeding with cDNA synthesis the polyA ⁇ RNA must be ethanol precipitated and resuspended as the Oligotex procedure leaves a reagent in the polyA+RNA which inhibits downstream reactions.
• Oligotex mRNA Purification System Qiagen
• Reagents for cDNA synthesis are obtained from the Superscript Choice System for cDNA Synthesis kit (GibcoBRL).
• RNA Before aliquoting RNA to use in cDNA synthesis, heat RNA at 70°C for 2 minutes to disloge RNA that is adhering to the plastic tube. Vortex, spin briefly in microcentrifuge, and then keep RNA at room temperature until aliquot is taken.
• Total volume of second strand reaction mix per sample is 130 u L. Add mix to first strand cDNA synthesis sample.
• DNA by adding 1 volume of 5 M NH40Ac and 2.5 volumes of cold (-20°C) 100%) ethanol . Vortex and then centrifuge at 16°C for 30 minutes at 15 000 RPM.
• IVTT In vitro Transcription
• T7 Megascript kit Ambion
• T7 10XATP (75 mM) 2 uL T7 10XGTP (75 mM) 2 uL T7 10XCTP (75 mM) 1.5 uL T7 10XUTP (75 mM) 1.5 uL Bio-11-UTP (10 mM) 3.75 uL (Boehringer Mannheim or Enzo Diagnostics) Bio-16-CTP (10 mM) 3.75 uL (Enzo Diagnostics) T7 buffer (1 OX) 2 uL T7 enzyme mix (1 OX) 2 uL
• RNeasy columns Qiagen
• RNA purification using RNeasy Kit RNA purification using RNeasy Kit
• BioB control cRNA (Affymetrix) 1.5 pM
• BioC control cRNA (Affymetrix) 5 pM
• BioD control cRNA (Affymetrix) 25 pM
• Bovine serum albumin (50 mg/mL) 3 uL 2X MES 150 uL
• RNA prepared from tumor tissue versus RNA prepared from normal tissue
• the starting material for this method is IVT product prepared as described in Example 1 , above.
• the cRNA is reverse transcribed in the presence of either Cy3 (sample 1) or Cy5 (sample 2) conjugated dUTP.
• the RNA is degraded and the samples are purified to recover the Cy3 and Cy5 dUTP.
• the differentially labelled samples are combined and the cDNA is further purified to remove fragments less than 100 bp in length. The sample is then fragmented and hybridized to oligonucleotide arrays. Labeling of cRNA
• Reverse transcription is performed on the IVT product by adding the following reagents from the Superscript Choice System for cDNA Synthesis kit (GibcoBRL) to the IVT-random hexamer mixture.
• cDNA is purified using the Qiaquick PCR Purification Kit (Qiagen), following the manufacturer's directions.
• Second wash Wash slides for 5 minutes at room temperature in Buffer 2
• CHA4 is upregulated in breast cancer tissue (3A) and colon cancer tissue (3B) compared with expression in adrenal gland, aorta, aortic valve, artery, bladder, bone marrow, brain, breast, CD14 + monocytes, CD14 " cells, colonic epithelial cells, cervix, colon, diaphragm, esophagus, gallbladder, heart, kidney, liver, lungs, lymph node, muscle, vagus nerve, omentum, ovary, pancreas, prostate, rectum, salivary gland, skin, small intestine, ileum, jejunum, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, uterus, and vein/vena cava (3C-3D).
• CBK8 is upregulated in colon cancer tissue (6A), including primary tumor tissue (dark bars) and metastatic tissue (light bars), compared with expression in normal adrenal gland, aorta, aortic valve, artery, bladder, bone marrow, brain, breast, colonic epithelial cells (CEP), cervix, colon, diaphragm, esophagus, gallbladder, heart, kidney, liver, lungs, lymph node, muscle, ovary, pancreas, prostate, rectum, salivary gland, skin, small intestine, ileum, jejunum, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, ureter and uterus (6B-6C).
• colon cancer tissue (6A), including primary tumor tissue (dark bars) and metastatic tissue (light bars), compared with expression in normal adrenal gland, aorta, aortic valve, artery, bladder, bone marrow, brain, breast, colonic epithelial

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