EP1163524A2 - Screeningverfahren für modulatoren von darmkrebs - Google Patents

Screeningverfahren für modulatoren von darmkrebs

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Publication number
EP1163524A2
EP1163524A2 EP00916451A EP00916451A EP1163524A2 EP 1163524 A2 EP1163524 A2 EP 1163524A2 EP 00916451 A EP00916451 A EP 00916451A EP 00916451 A EP00916451 A EP 00916451A EP 1163524 A2 EP1163524 A2 EP 1163524A2
Authority
EP
European Patent Office
Prior art keywords
crc
protein
antibody
fragment
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00916451A
Other languages
English (en)
French (fr)
Inventor
David Mack
Kurt C. Gish
Keith E. Wilson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
EOS Biotechnology Inc
Original Assignee
EOS Biotechnology Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US09/268,866 external-priority patent/US6316272B1/en
Priority claimed from US09/436,983 external-priority patent/US6294343B1/en
Priority claimed from US09/435,945 external-priority patent/US6773878B1/en
Application filed by EOS Biotechnology Inc filed Critical EOS Biotechnology Inc
Publication of EP1163524A2 publication Critical patent/EP1163524A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the invention relates to the identification of expression profiles and the nucleic acids involved in colorectal cancer, and to the use of such expression profiles and nucleic acids in diagnosis and prognosis of colorectal cancer.
  • the invention further relates to methods for identifying and using candidate agents and/or targets which modulate colorectal cancer.
  • Colorectal cancer is a significant cancer in Western populations. It develops as the result of a pathologic transformation of normal colon epithelium to an invasive cancer.
  • mutations inactivating mutations of both alleles of the adenomatous polyposis coli (APC) gene, a tumor suppressor gene appears to be one of the earliest events in colorectal cancer, and may even be the initiating event.
  • 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- ⁇ signaling pathway.
  • methods that can be used for diagnosis and prognosis of colorectal cancer would be desirable Accordingly, provided herein are methods that can be used in diagnosis and prognosis of colorectal cancer Further provided are methods that can be used to screen candidate bioactive agents for the ability to modulate colorectal cancer Additionally, provided herein are molecular targets for therapeutic intervention in colorectal and other cancers
  • the present invention provides methods for screening for compositions which modulate colorectal cancer Also provided herein are methods of inhibiting proliferation of cell, preferably a colorectal cancer cell Methods of treatment of cancer, as well as compositions, are also provided herein
  • 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 are genes which are differentially expressed in cancer cells, preferably colorectal cancer cells, compared to other cells
  • Preferred embodiments of expression profile genes used in the methods herein include but are not limited to the group consisting of CZA8, BCX2, CBC2, CBC1 , CBC3, CJA8, CJA9, CGA7, BCN5, CQA1 , BCN7, CQA2, CGA8, CAA7 and CAA9, fragments of the proteins of this group are also preferred
  • molecules for use in the present invention may be from any figure or any subset of listed molecules Therefore, for example, any one or more of the genes listed above can be used in the methods herein
  • a nucleic acid is selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14
  • Preferred nucleic acids are in Figure 12, more preferably Figure 13, and most preferably in Figure 14 The method
  • 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
  • a method of screening for a bioactive agent capable of binding to a colorectal cancer modulator protein (CCMP) the method comprising combining the CCMP and a candidate bioactive agent, and determining the binding of the candidate agent to the CCMP
  • the CCMP is a protein or fragment thereof selected from the group consisting of CZA8, BCX2, CBC2, CBC1 , CBC3, CJA8, CJA9, CGA7, BCN5, CQA1 , BCN7, CQA2, CGA8, CAA7 and CAA9
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2,
  • the method comprises combining the CCMP and a candidate bioactive agent, and determining the effect of the candidate agent on the bioactivity of the CCMP
  • the CCMP is a protein or fragment thereof selected from the group consisting of CZA8, BCX2, CBC2, CBC1 , CBC3, CJA8, CJA9, CGA7, BCN5, CQA1 , BCN7, CQA2, CGA8, CAA7 and CAA9
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 Preferred nucleic acids are in Figure 12, more preferably Figure
  • Also provided is a method of evaluating the effect of a candidate colorectal cancer drug comprising administering the drug to a transgenic animal expressing or over-expressing the CCMP, or an animal lacking the CCMP, for example as a result of a gene knockout
  • a method of evaluating the effect of a candidate colorectal cancer drug comprising administering the drug to a patient and removing a cell sample from the patient The expression profile of the cell is then determined This method may further comprise comparing the expression profile to an expression profile of a healthy individual
  • a biochip comprising a nucleic acid segment which encodes a colorectal cancer protein, preferably selected from the group consisting of CZA8, BCX2, CBC2, CBC1 , CBC3,
  • the biochip comprises fewer than 1000 nucleic acid probes
  • the nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or 14 Preferred nucleic acids are in Figure 12, more preferably Figure 13, and most preferably in Figure 14
  • a method of diagnosing a disorder associated with colorectal cancer comprises determining the expression of a gene which encodes a colorectal cancer protein preferably selected from the group consisting of CZA8, BCX2, CBC2, CBC1 , CBC3, CJA8, CJA9, CGA7, BCN5, CQA1 , BCN7, CQA2, CGA8, CAA7 and CAA9 or a fragment thereof in a first tissue type of a first individual, and comparing the distribution to the
  • the present invention provides an antibody which specifically binds to a colorectal cancer protein, preferably selected from the group consisting of CZA8, BCX2, CBC2, CBC1 , CBC3, CJA8, CJA9, CGA7, BCN5, CQA1 , BCN7, CQA2, CGA8, CAA7 and CAA9, or a fragment thereof
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 Preferred nucleic acids are in Figure 12, more preferably Figure 13, and most preferably in Figure 14
  • the fragment of CAA9 is selected from CAA9p1 , CAA9p2, CAA9p3, CAA9p4, CAA9p4MAPS, CAA9p5 and CAA9p5MAPS
  • the antibody is a monoclonal antibody
  • the antibody can be a fragment of an antibody such as a single a single antigen IMAPS, or a single-associated antigen IMAPS, or
  • a method for screening for a bioactive agent capable of interfering with the binding of a colorectal cancer protein (CCMP) or a fragment thereof and an antibody which binds to said CCMP or fragment thereof comprises combining a CCMP or fragment thereof, a candidate bioactive agent and an antibody which binds to said CCMP or fragment thereof
  • the method further includes determining the binding of said CCMP or fragment thereof and said antibody Wherein there is a change in binding, 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 colorectal cancer
  • a method for inhibiting colorectal cancer comprises administering to a cell a composition comprising an antibody to a colorectal modulating protein, preferably selected from the group consisting of CZA8, BCX2, CBC2, CBC1 , CBC3, CJA8, CJA9, CGA7, BCN5, CQA1 , BCN7, CQA2, CGA8, CAA7 and CAA9, or a fragment thereof.
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14. Preferred nucleic acids are in Figure 12, more preferably Figure 13, and most preferably in Figure 14.
  • the method can be performed in vitro or in vivo, preferably in vivo to an individual.
  • the method of inhibiting colorectal cancer is provided to an individual with cancer.
  • methods of inhibiting colorectal cancer can be performed by administering an inhibitor of colorectal cancer protein activity, including antisense molecules, and preferably small molecules.
  • a method provided herein comprises administering to an individual a composition comprising a colorectal modulating protein, preferably selected from the group consisting of CZA8, BCX2, CBC2,
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14.
  • Preferred nucleic acids are in Figure 12, more preferably Figure 13, and most preferably in Figure 14.
  • said composition comprises a nucleic acid comprising a sequence encoding a colorectal cancer modulating protein, preferably selected from the group consisting of CZA8, BCX2, CBC2, CBC1 , CBC3, CJA8, CJA9, CGA7, BCN5, CQA1 , BCN7, CQA2, CGA8, CAA7 and CAA9, or a fragment thereof.
  • the nucleic acid is selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14.
  • Preferred nucleic acids are in Figure 12, more preferably Figure 13, and most preferably in Figure 14.
  • compositions capable of eliciting an immune response in an individual.
  • a composition provided herein comprises a colorectal cancer modulating protein, preferably selected from the group consisting of CZA8, BCX2, CBC2, CBC1 , CBC3, CJA8, CJA9, CGA7, BCN5, CQA1 , BCN7, CQA2, CGA8, CAA7 and CAA9, or a fragment thereof, and a pharmaceutically acceptable carrier.
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14. Preferred nucleic acids are in
  • said composition comprises a nucleic acid comprising a sequence encoding a colorectal cancer modulating protein, preferably selected from the group consisting of CZA8, BCX2, CBC2, CBC1 , CBC3, CJA8, CJA9, CGA7, BCN5, CQA1 , BCN7, CQA2, CGA8, CAA7 and CAA9, or a fragment thereof, and a pharmaceutically acceptable carrier.
  • the nucleic acid is selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14.
  • Preferred nucleic acids are in Figure 12, more preferably Figure 13, and most preferably in Figure 14.
  • a method of neutralizing the effect of a colorectal cancer protein preferably selected from the group consisting of CZA8, BCX2, CBC2, CBC1 , CBC3, CJA8, CJA9, CGA7, BCN5, CQA1 , BCN7, CQA2, CGA8, CAA7 and CAA9, or a fragment thereof, comprising contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14
  • Preferred nucleic acids are in Figure 12, more preferably Figure 13, and most preferably in Figure 14
  • a method of treating an individual for colorectal cancer comprises administering to said individual an inhibitor of CJA8 In another embodiment, the method comprises administering to a patient having colorectal cancer an antibody to CJA8 conjugated to a therapeutic moiety Such a therapeutic moiety can be a cytotoxic agent or a radioisotope
  • Also provided herein is a method for determining the prognosis of an individual with colorectal cancer comprising determining the level of CJA8 in a sample, wherein a high level of CJA8 indicates a poor prognosis
  • Figure 1 provides the Accession numbers for genes, including expression sequence tags, (incorporated in their entirety here and throughout the application where Accession numbers are provided), upregulated in tumor tissue compared to normal colon tissue
  • Figure 2 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal colon tissue
  • Figure 3 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal colon tissue
  • Figure 4 provides the Accession numbers for genes including expression sequence tags, upregulated in tumor tissue compared to normal colon tissue
  • Figure 5 provides the Accession numbers for genes, including expression sequence tags, downregulated in tumor tissue compared to normal colon tissue
  • Figure 6 provides the Accession numbers for genes, including expression sequence tags, downregulated in tumor tissue compared to normal colon tissue.
  • Figure 7 provides the Accession numbers for genes, including expression sequence tags, downregulated in tumor tissue compared to normal colon tissue.
  • Figure 8 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal colon tissue. Open reading frames in the sequences have been characterized as having a signal sequence (SS), a transmembrane domain (TM) or other.
  • SS signal sequence
  • TM transmembrane domain
  • Figure 9 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal colon tissue. Open reading frames in the sequences have been characterized as having a signal sequence (SS), a transmembrane domain (TM) or other.
  • SS signal sequence
  • TM transmembrane domain
  • Figure 10 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal colon tissue. Open reading frames have been characterized as having a signal sequence (SS), a transmembrane domain (TM) or other.
  • SS signal sequence
  • TM transmembrane domain
  • Figure 11 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal colon tissue Open reading frames have been characterized as having a signal sequence (SS), a transmembrane domain (TM) or other
  • Figure 12 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal colon tissue Open reading frames have been characterized as having a signal sequence (SS), a transmembrane domain (TM) or other.
  • SS signal sequence
  • TM transmembrane domain
  • Figure 13 provides the Accession numbers for genes or fragments thereof, including descriptions of the gene or encoded protein, upregulated in tumor tissue compared to normal colon tissue.
  • Figure 14 provides a list of proteins, including Accession numbers for nucleic acid sequences associated with the encoding genes thereof, upregulated in tumor tissue compared to normal colon tissue.
  • Figure 15 shows an embodiment of a nucleic acid which includes a sequence which encodes a colorectal protein provided herein, CAA2. The start and stop codon are shaded. The sequence within the two cross marks indicates a preferred novel fragment of CAA2 provided herein, referred to herein as the "CAA2 5' end". Preferred embodiments of CAA2 include at least a portion of the CAA2 5'. The sequence in bold and indicated with a bar at the bottom right beginning with "GGC" and ending with
  • AAA can be found in Accession no. AA505133.
  • Figure 16 shows an embodiment of a nucleic acid encoding CAA2, wherein the start and stop codons are shaded.
  • Figure 17 shows an embodiment of an amino acid sequence of CAA2.
  • Preferred fragments include at least about 10 amino acids in the N-terminal end.
  • the N-terminus as defined herein includes an embodiment beginning at the first amino acid until about any one of the three amino acids marked with a dot above them.
  • the N-terminus of CAA2 is defined as the amino acid sequence encoded by the CAA2 5' end.
  • Figure 18 shows the amino acid sequence of CAA2p1 , a preferred CAA2 fragment provided herein.
  • Figure 19 shows the amino acid sequence of CAA2p2, a preferred CAA2 fragment provided herein.
  • Figure 20 shows an alignment of the human and mouse CAA2 polypeptides provided herein.
  • the mouse polypeptide contains at least some of the sequence of each of the following Accession numbers: AA386837; AI508773; AA505293; and AA636546.
  • Figure 21 shows the relative amount of expression of CAA2 in various samples of colon cancer tissue (dark bars) and many normal tissue types (light bars).
  • Figure 22 shows an embodiment of a colorectal cancer nucleic acid, CAA9 mRNA.
  • the start and stop codons are underlined.
  • Figure 23 shows the open reading frame of the CAA9 gene wherein the start and stop codons are underlined.
  • Figure 24 shows an embodiment of the amino acid sequence of a colorectal cancer protein, CAA9, wherein putative transmembrane sequences are underlined.
  • CAA9 or fragments of CAA9 are soluble, therefore, the transmembrane domains are deleted, inactivated, and/or the peptide is truncated (with or without re-ligation) to form soluble CAA9.
  • Figure 25 shows embodiments of colorectal cancer proteins (also termed colorectal cancer modulator proteins). Specifically, Figure 25 shows CAA9p1 , CAA9p2, CAA9p3, CAA9p4, CAA9p4MAPS, CAA9p5 and CAA9p5MAPS and their respective solubilities.
  • Figure 26 shows the relative amount of CAA9 expression in several different samples of colon cancer tissue (dark bars) and normal tissues (light bars).
  • Figure 27 shows the nucleic acid sequence for the gene encoding CGA7. Start (ATG) and stop (TAG) codons are indicated by shaded boxes. In bold is the sequence of Accession No. AA331393. Underlined is the consensus sequence derived from the compilation and alignment of published est sequences.
  • Figures 28A and 28B show the alignment summary and descriptions, respectively, of the various est's (by accession number) compiled to generate the consensus sequence of figure 1.
  • Figure 29 shows the amino acid sequence of CGA7.
  • Figures 30A and 30B show the relative expression of CGA7 in normal tissue and colon cancer tissue, respectively.
  • Figure 31 shows the nucleic acid sequence for the mRNA encoding CGA8. Start (ATG) and stop (TAG) codons are indicated by shaded boxes. In bold is the sequence of Accession No. AA2786503. Underlined is the consensus sequence derived from the compilation and alignment of published est sequences.
  • Figures 32A and 32B show the alignment summary and descriptions, respectively, of the various est's (by accession number) compiled to generate the consensus sequence of figure 1.
  • Figure 33 shows the amino acid sequence of CGA8.
  • Figure 34 shows the relative expression of CGA8 in breast cancer tissue, colon cancer tissue, normal tissue and fetal tissue.
  • Figure 35 shows the sequence for the mRNA encoding CJA8 Start (ATG) and stop (TAA) codons are indicated by shaded boxes
  • Figure 36 shows the ammo acid sequence for CJA8 A putative transmembrane region is designated by shading A mouse homolog of this human protein is found at Accession Number AAF21308 1
  • Figure 37 shows the relative amount of expression of CJA8 in several different samples of colon tissues (dark bars) and normal tissues (light bars)
  • Figure 38 shows the relative amount of expression of BCN7 in several different samples of colon tissues (dark bars) and normal tissues (light bars), as determined using the sequence of Accession Number N22107 as a probe
  • Figure 39 shows an embodiment of a nucleic acid which includes a sequence which encodes a colorectal cancer protein provided herein, BCN7
  • Figure 40 shows the sequence for the mRNA encoding CZA8 Start (ATG) and stop (TGA) codons are indicated by underlining
  • Figure 41 shows the sequence for the mRNA encoding BCX2 Start (ATG) and stop (TGA) codons are indicated by underlining
  • Figure 42 shows the sequence for the mRNA encoding CBC2 Start (ATG) and stop (TAA) codons are indicated by underlining
  • Figure 43 shows the sequence for the mRNA encoding CBC1 Start (ATG) and stop (TGA) codons are indicated by underlining
  • Figure 44 shows the sequence for the mRNA encoding CBC3 Start (ATG) and stop (TGA) codons are indicated by underlining
  • Figure 45 shows the sequence for the mRNA encoding BCN5 Start (ATG) and stop (TAA) codons are indicated by underlining
  • Figure 46 shows an embodiment of a nucleic acid which includes a sequence which encodes a colorectal cancer protein provided herein
  • CJA9 Figure 47 shows an embodiment of a nucleic acid which includes a sequence which encodes a colorectal cancer protein provided herein
  • CQA1 CQA1
  • Figure 48 shows an embodiment of a nucleic acid which includes a sequence which encodes a colorectal cancer protein provided herein, CQA2
  • the present invention provides novel methods for diagnosis and prognosis evaluation for colorectal cancer (CRC), as well as methods for screening for compositions which modulate CRC
  • 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 That is, normal tissue may be distinguished from CRC tissue, and within CRC tissue, different prognosis states (good or poor long term survival prospects, for example) may be determined
  • By comparing expression profiles of colon tissue in known 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 CRC versus normal colon tissue, as well as differential expression resulting in different prognostic outcomes, allows the use of this information
  • CRC genes which can then be used in these screens These methods can also be done on the protein basis, that is, protein expression levels of the CRC proteins can be evaluated for diagnostic and prognostic purposes or to screen candidate agents
  • the CRC nucleic acid sequences can be administered for gene therapy purposes, including the administration of antisense nucleic acids, or the CRC proteins (including antibodies and other modulators thereof) administered as therapeutic drugs
  • CRC sequences include those that are up-regulated (i e expressed at a higher level) in CRC, as well as those that are down-regulated (i e expressed at a lower level) in CRC
  • the CRC sequences are from humans, however, as will be appreciated by those in the art, CRC sequences from other organisms may be useful in animal models of disease and drug evaluation, thus, other CRC sequences are provided, from vertebrates, including
  • CRC sequences can include both nucleic acid and ammo acid sequences
  • the CRC 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
  • 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 CRC protein from one organism in a different organism or host cell Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of an ducible promoter or high
  • the CRC sequences are nucleic acids
  • CRC 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 CRC 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), Sp ⁇ nzl et al , Eur J Biochem 81 579
  • PNA peptide nucleic acids
  • PNA backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids.
  • Tm melting temperature
  • RNA typically exhibit a 2-4 ° C drop in Tm for an internal mismatch
  • 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 As will be appreciated by those in the art, the depiction of a single strand (“Watson") 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 deoxynbo- and ⁇ bo-nucleotides, and any combination of bases, including uracil, adenine, thymme, cytosine, guanme, mosine, xanthme hypoxanthine, isocytosine, isoguanme, etc
  • the term "nucleoside ' includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as ammo modified nucle
  • a CRC sequence can be initially identified by substantial nucleic acid and/or am o acid sequence homology to the CRC sequences outlined herein Such homology can be based upon the overall nucleic acid or ammo acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions
  • the CRC 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 Affymet ⁇ x 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 CRC 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
  • CRC sequences are those that are up-regulated in CRC, that is, the expression of these genes is higher in colorectal carcinoma as compared to normal colon tissue
  • Up- 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
  • 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 ,
  • CRC sequences are those that are down-regulated in CRC, that is, the expression of these genes is lower in colorectal carcinoma as compared to normal colon 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
  • CRC proteins of the present invention may be classified as secreted proteins, transmembrane proteins or intracellular proteins
  • the CRC protein is an mtracellular protein
  • Intracellular proteins may be found in the cytoplasm and/or in the nucleus Intracellular proteins are involved in all aspects of cellular function and replication (including, for example, signaling pathways), aberrant expression of such proteins results in unregulated or deregulated cellular processes
  • many intracellular proteins have enzymatic activity such as protein kmase 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
  • An increasingly appreciated concept in characterizing intracellular proteins is the presence in the proteins of one or more motifs for which defined functions have been attributed.
  • 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. Some of these may also be involved in binding to phospholipids or other second messengers.
  • these motifs can be identified on the basis of primary sequence; thus, an analysis of the sequence of proteins may provide insight into both the enzymatic potential of the molecule and/or molecules with which the protein may associate.
  • the CRC 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, interleukin receptors, e.g. IL-1 receptor,
  • 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.
  • 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.
  • Other factors 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. Extracellular domains also associate with the extracellular matrix and contribute to the maintenance of the cell structure.
  • GPI glycosylphosphatidylinositol
  • CRC proteins that are transmembrane 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 CRC 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
  • CRC 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
  • a CRC sequence is initially identified by substantial nucleic acid and/or ammo acid sequence homology to the CRC sequences outlined herein Such homology can be based upon the overall nucleic acid or ammo acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions
  • a nucleic acid is a "CRC nucleic acid” if the overall homology of the nucleic acid sequence to the nucleic acid sequences encoding the ammo 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 2 482 (1981 ), by the homology alignment algorithm of Needleman & Wunsch, J Mol Biool 48 443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT,
  • sequences which are used to determine sequence identity or similarity are selected from the sequences set forth in the figures, preferably those represented in Figure 12, more preferably those represented in Figures 13A and 13B, still more preferably those of Figures 14- 20, 22-25, 27-29, 31-33, 35-37 and 39-48, and fragments thereof
  • 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 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 5 151-153 (1989)
  • Useful PILEUP parameters including a default gap weight of 3 00, a default gap length weight of 0 10, and weighted end gaps
  • 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 % ammo 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 the sequences of the figures
  • 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
  • sequences which contain either more or fewer nucleosides than those of the figures it is understood that 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
  • 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 CRC sequence
  • High stringency conditions are known in the art, see for example Maniatis et al , Molecular Cloning A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed Ausubel et al , both 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 Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993)
  • stringent conditions are selected to be about 5-10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH
  • the Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T
  • less stringent hybridization conditions are used, for example, moderate or low stringency conditions may be used, as are known in the art, see Maniatis and Ausubel, supra, and Tijssen, supra
  • the CRC 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 CRC 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 , and Ausubel, et al , supra, hereby expressly incorporated by reference
  • the CRC nucleic acid Once the CRC nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire CRC nucleic acid
  • the recombinant CRC 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 CRC nucleic acid can be further-used as a probe to identify and isolate other CRC nucleic acids, for example additional coding regions It can also be used as a "precursor" nucleic acid to make modified or variant CRC nucleic acids and proteins
  • the CRC nucleic acids of the present invention are used in several ways
  • nucleic acid probes to the CRC 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 CRC nucleic acids that include coding regions of CRC proteins can be put into expression vectors for the expression of CRC proteins, again either for screening purposes or for administration to a patient
  • nucleic acid probes to CRC nucleic acids are made
  • the nucleic acid probes attached to the biochip are designed to be substantially complementary to the CRC 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
  • 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
  • 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
  • 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, hydrophi c, 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 fluoresce.
  • a preferred substrate is described in copending application entitled
  • 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.
  • photoactivation techniques utilizing photopolymerization compounds and techniques are used.
  • the 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
  • CRC nucleic acids encoding CRC proteins are used to make a variety of expression vectors to express CRC 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 CRC 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 nbosome 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
  • enhancers do not have to be contiguous Linking is accomplished by hgation at convenient restriction sites if such sites do not exist, the synthetic o gonucleotide adaptors or linkers are used in accordance with conventional practice The transcnptional and translational regulatory nucleic acid
  • transcnptional and translational regulatory sequences may include, but are not limited to, promoter sequences, nbosomal binding sites, transcnptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences
  • the regulatory sequences include a promoter and transcnptional start and stop sequences
  • Promoter sequences encode either constitutive or ducible 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
  • the CRC proteins of the present invention are produced by cultu ⁇ ng a host cell transformed with an expression vector containing nucleic acid encoding a CRC protein, under the appropriate conditions to induce or cause expression of the CRC protein
  • the conditions appropriate for CRC 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 mducible 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, archaebacte ⁇ a, fungi, and insect and animal cells, including mammalian cells Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E coll, 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 CRC 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
  • 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 Typically, 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 Examples of transcription terminator and polyadenlytion signals include those derived form SV40
  • CRC proteins are expressed in bacterial systems Bacterial expression systems are well known in the art Promoters from bactenophage 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 Furthermore, 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 CRC 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.
  • CRC 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.
  • CRC 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 CRC protein may also be made as a fusion protein, using techniques well known in the art.
  • the CRC protein may be fused to a carrier protein to form an immunogen.
  • the CRC protein may be made as a fusion protein to increase expression, or for other reasons.
  • the CRC protein is a CRC peptide
  • the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes.
  • the CRC 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 CRC 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 CRC protein sequences.
  • a CRC 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 CRC protein has homology to some protein in the database being used.
  • the nucleic acid sequences are input into a program that will search all three frames for homology. This is done in a preferred embodiment using the following 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.
  • CRC proteins are amino acid variants of the naturally occurring sequences, as determined herein.
  • the variants are preferably greater than about
  • 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.
  • CRC proteins of the present invention may be shorter or longer than the wild type amino acid sequences.
  • included within the definition of CRC proteins are portions or fragments of the wild type sequences, herein.
  • the CRC 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 CRC proteins are derivative or variant CRC proteins as compared to the wild-type sequence. That is, as outlined more fully below, the derivative CRC 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 CRC peptide.
  • amino acid sequence variants are also included in an embodiment of CRC proteins of the present invention. 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
  • variant CRC 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 CRC 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 CRC 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 CRC 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 deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the CRC protein are desired, substitutions are generally made in accordance with the following chart:
  • 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 isoieucyl, 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.
  • the 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 CRC proteins as needed.
  • the variant may be designed such that the biological activity of the CRC protein is altered. For example, glycosylation sites may be altered or removed.
  • Covalent modifications of CRC polypeptides are included within the scope of this invention.
  • One type of covalent modification includes reacting targeted amino acid residues of a CRC polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of a CRC polypeptide.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking CRC to a water-insoluble support matrix or surface for use in the method for purifying anti-CRC antibodies or screening assays, as is more fully described below.
  • Commonly used crosslinking agents include, e.g., 1 ,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxy- succinimide 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)dithio]propioimi- date.
  • Another type of covalent modification of the CRC 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 CRC polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence CRC polypeptide.
  • Addition of glycosylation sites to CRC 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 CRC polypeptide (for O-linked glycosylation sites).
  • the CRC amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the CRC 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 CRC polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981 ). Removal of carbohydrate moieties present on the CRC 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.
  • 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).
  • CRC polypeptide comprises linking the CRC 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;
  • CRC polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising a CRC polypeptide fused to another, heterologous polypeptide or amino acid sequence.
  • a chimeric molecule comprises a fusion of a CRC 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 CRC polypeptide. The presence of such epitope-tagged forms of a CRC polypeptide can be detected using an antibody against the tag polypeptide.
  • the epitope tag enables the CRC 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 CRC polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule.
  • tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7,
  • 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.
  • CRC protein in one embodiment are other CRC proteins of the CRC family, and CRC 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 CRC proteins from humans or other organisms.
  • particularly useful probe and/or PCR primer sequences include the unique areas of the CRC 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.
  • CRC 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.
  • CRC proteins may also be identified as being encoded by CRC nucleic acids.
  • CRC proteins are encoded by nucleic acids that will hybridize to the sequences of the sequence listings, or their complements, as outlined herein.
  • the CRC protein when the CRC protein is to be used to generate antibodies, for example for immunotherapy, the CRC 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 CRC 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.
  • the epitope is selected from CAA2p1 and CAA2p2.
  • the epitope is selected from CAA9p1 , CAA9p2, CAA9p3, CAAQ9p4, CAA9p4MAPS, CAA89p5 and CAA9p5MAPS.
  • 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 the CAA2 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. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants examples 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, Nature, 256:495 (1975).
  • a hybridoma method 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 CAA2 polypeptide or fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice. Academic Press, (1986) pp. 59-103].
  • 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 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.
  • one of the binding specificities is for a CRC protein or a fragment thereof, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit, preferably one that is tumor specific.
  • the antibodies to CRC are capable of reducing or eliminating the biological function of CRC, as is described below. That is, the addition of anti-CRC antibodies (either polyclonal or preferably monoclonal) to CRC (or cells containing CRC) may reduce or eliminate the CRC 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 CRC 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, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.. 2:593-596 (1992)].
  • 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, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • 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 ); Marks et al., J. Mol. Biol., 222:581 (1991 )].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1 ):86-95 (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. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
  • immunotherapy is meant treatment of CRC with an antibody raised against CRC 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.
  • the CRC 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 CRC protein.
  • the CRC protein to which antibodies are raised is a transmembrane protein.
  • antibodies used for treatment bind the extracellular domain of the CRC 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 CRC protein.
  • the antibody may be a competitive, non- competitive or uncompetitive inhibitor of protein binding to the extracellular domain of the CRC protein.
  • the antibody is also an antagonist of the CRC protein. Further, the antibody prevents activation of the transmembrane CRC protein. In one aspect, when the antibody prevents the binding of other molecules to the CRC 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. In some instances the antibody belongs to a sub-type that activates serum complement when complexed with the transmembrane protein thereby mediating cytotoxicity. Thus, CRC is treated by administering to a patient antibodies directed against the transmembrane CRC protein.
  • the antibody is conjugated to a therapeutic moiety.
  • the therapeutic moiety is a small molecule that modulates the activity of the CRC protein.
  • the therapeutic moiety modulates the activity of molecules associated with or in close proximity to the CRC protein.
  • the therapeutic moiety may inhibit enzymatic activity such as protease or protein kinase activity associated with CRC.
  • 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 CRC.
  • 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 CRC proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody.
  • Targeting the therapeutic moiety to transmembrane CRC proteins not only serves to increase the local concentration of therapeutic moiety in the CRC afflicted area, but also serves to reduce deleterious side effects that may be associated with the therapeutic moiety.
  • the CRC 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.
  • CRC protein can be targeted within a cell, i.e., the nucleus, an antibody thereto contains a signal for that target localization, i.e., a nuclear localization signal.
  • the CRC antibodies of the invention specifically bind to CRC 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 “1 , with a preferred range being 10 "7 - 10 "9 M “1 .
  • the CRC protein is purified or isolated after expression
  • CRC 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 chromatofocusmg
  • the CRC protein may be purified using a standard anti-CRC 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 CRC protein In some instances no purification will be necessary
  • the CRC proteins and nucleic acids are useful in a number of applications
  • the expression levels of genes are determined for different cellular states in the CRC phenotype, that is, the expression levels of genes in normal colon tissue and in CRC tissue (and in some cases, for varying seventies of CRC 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
  • information regarding which genes are important (including both up- and down- regulation of genes) in each of these states is obtained Then, diagnosis may be done or confirmed does tissue from a particular patient have the gene expression profile of normal or CRC 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 CRC tissue That is, genes may be turned on or turned off in a particular state, relative to another state
  • 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 Alternatively, 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 Affymet ⁇ x GeneChipTM expression array
  • 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 (protein) can be monitored, for example through the use of antibodies to the CRC 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 i e those identified as being important in a CRC phenotype
  • 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
  • the CRC nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of CRC sequences in a particular cell
  • the assays are further described below in the example
  • nucleic acids encoding the CRC protein are detected
  • DNA or RNA encoding the CRC protein may be detected, of particular interest are methods wherein the mRNA encoding a CRC protein is detected
  • the presence of mRNA in a sample is an indication that the CRC 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- spe fically 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.
  • RNA probe digoxygenin labeled riboprobe (RNA probe) that is complementary to the mRNA encoding a CRC 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 are used in diagnostic assays.
  • the CRC proteins, antibodies, nucleic acids, modified proteins and cells containing CRC 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.
  • CRC proteins including intracellular, transmembrane or secreted proteins, find use as markers of CRC. Detection of these proteins in putative CRC tissue or patients allows for a determination or diagnosis of CRC. Numerous methods known to those of ordinary skill in the art find use in detecting CRC.
  • antibodies are used to detect CRC proteins.
  • 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). Following separation of proteins, the CRC protein is detected by immunoblotting with antibodies raised against the CRC protein. Methods of immunoblotting are well known to those of ordinary skill in the art.
  • antibodies to the CRC protein find use in in situ imaging techniques.
  • cells are contacted with from one to many antibodies to the CRC protein(s). Following washing to remove non-specific 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 CRC protein(s) contains a detectable label.
  • each one of multiple primary antibodies contains a distinct and detectable label.
  • 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 CRC from blood samples.
  • certain CRC proteins are secreted/circulating molecules. Blood samples, therefore, are useful as samples to be probed or tested for the presence of secreted CRC proteins.
  • Antibodies can be used to detect the CRC 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 CRC nucleic acid probes to tissue arrays is done.
  • arrays of tissue samples, including CRC tissue and/or normal tissue are made.
  • In situ hybridization as is known in the art can then be done.
  • the CRC proteins, antibodies, nucleic acids, modified proteins and cells containing CRC sequences are used in prognosis assays.
  • gene expression profiles can be generated that correlate to CRC 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 CRC probes are attached to biochips for the detection and quantification of CRC 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 CRC proteins, antibodies, nucleic acids, modified proteins and cells containing CRC 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 CRC proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified CRC proteins are used in screening assays. That is, the present invention provides novel methods for screening for compositions which modulate the CRC 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.
  • assays may be executed.
  • assays may be run on an individual gene or protein level. That is, having identified a particular gene as up regulated in CRC, 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. Thus, if a gene exhibits a 4 fold increase in tumor compared to normal tissue, a decrease of about four fold is desired; a 10 fold decrease in tumor compared to normal tissue gives 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 CRC 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.
  • the CRC nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of CRC 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 colorectal cancer, modulates CRC proteins, binds to a CRC protein, or interferes between the binding of a CRC protein and an antibody.
  • the term “candidate bioactive agent” or “drug candidate” or grammatical equivalents as used herein describes any molecule, e g , protein, o gopeptide, small organic molecule, polysacchande, polynucleotide, etc , to be tested for bioactive agents that are capable of directly or indirectly altering either the CRC phenotype or the expression of a CRC 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 CRC phenotype, for example to a normal colon tissue fingerprint
  • the candidate agent preferably suppresses a severe CRC 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 heterocyciic 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, sacchandes, fatty acids, steroids, pu ⁇ nes, pynmidmes, 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 Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, estenfication, amidification to produce structural analogs
  • the candidate bioactive agents are proteins
  • protein herein is meant at least two covalently attached ammo acids, which includes proteins, polypeptides, oligopeptides and peptides
  • the protein may be made up of naturally occurring am o acids and peptide bonds, or synthetic peptidomimetic structures
  • “ammo acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic ammo acids
  • homo-phenylalanme, citrulline and noreleucme are considered ammo acids for the purposes of the invention
  • Ammo acid also includes imino acid residues such as prolme and hydroxyprohne
  • the side chains may be in either the (R) or the (S) configuration
  • the ammo acids are in the (S) or L-configuration
  • non-ammo 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 protemaceous 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 ammo acids, with from about 5 to about 20 am o 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 ammo 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 protemaceous 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 ammo 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, pralines 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 CRC 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 CRC similar to the expression profile of normal colon tissue is expected to result in a suppression of the CRC 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.
  • 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.
  • 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.
  • 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 CRC 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 CRC tissue reveals genes that are not expressed in normal tissue or CRC tissue, but are expressed in agent treated tissue.
  • agent specific sequences can be identified and used by any of the methods described herein for CRC 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 CRC tissue sample.
  • a candidate agent is administered to a population of CRC cells, that thus has an associated CRC 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
  • a viral construct such as a retroviral construct and added to the cell, such that expression of the peptide agent is accomplished; see PCT US97/01019, hereby expressly incorporated by reference.
  • CRC tissue may be screened for agents that reduce or suppress the CRC phenotype
  • a change in at least one gene of the expression profile indicates that the agent has an effect on CRC 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 "CRC proteins” or a "CCMP"
  • the CCMP is as depicted in Figures 17-20, 24, 25, 29, 33 and 36, more preferably the protein having the sequence shown in
  • the CCMP may be a fragment, or alternatively, be the full length protein to a fragment shown herein
  • the CCMP is a fragment of approximately 14 to 24 ammo acids long More preferably the fragment is a soluble fragment
  • the fragment is from CAA9
  • the fragment includes a non- transmenbrane region
  • the CAA9 fragment has an N-termmal Cys to aid in solubility
  • the fragment is selected from CAA9p1 , Caa9p2, CAA9p3, CAA9p4, CAA9p4MAPS, CAA9p5 and CAA9p5MAPS
  • the fragment is charged and from the c-termmus of CAA2
  • the c-termmus of the fragment is kept as a free acid and the n-terminus is a free amine to aid in coupling, i e , to cysteme
  • the fragment is an internal peptide overlapping hydrophilic stretch of CAA2
  • the termini is blocked
  • the fragment of CAA2 is selected from CAA2p1 or CAA2p2
  • the fragment is a novel fragment from the N-termmal
  • the fragment excludes sequence outside of the N-termmal, in another embodiment, the fragment includes at least a portion of the N-termmal "N-term ⁇ nal" is used interchangeably herein with "N-terminus" which is further described above
  • the CRC proteins are conjugated to an immunogenic agent as discussed herein. In one embodiment the CRC 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 CRC proteins can be used in the assays.
  • the methods comprise combining a CRC protein and a candidate bioactive agent, and determining the binding of the candidate agent to the CRC protein.
  • Preferred embodiments utilize the human CRC protein, although other mammalian proteins may also be used, for example for the development of animal models of human disease.
  • variant or derivative CRC proteins may be used.
  • the CRC 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.).
  • 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.
  • suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, teflonTM, etc.
  • 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 CRC 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 CRC 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 CRC 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 CRC 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, fluorescers, 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.
  • the proteins may be labeled at tyrosine positions using 125 l, or with fluorophores.
  • 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 CRC), such as an antibody, peptide, binding partner, ligand, etc
  • the target molecule i e CRC
  • 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 CRC protein and thus is capable of binding to, and potentially modulating, the activity of the CRC 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 CRC protein with a higher affinity
  • 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 CRC protein
  • the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the CRC proteins
  • the methods comprise combining a CRC protein and a competitor in a first sample
  • a second sample comprises a candidate bioactive agent, a CRC 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 CRC 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 CRC protein
  • a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native CRC protein, but cannot bind to modified CRC proteins
  • the structure of the CRC protein may be modeled, and used in rational drug design to synthesize agents that interact with that site Drug candidates that affect CRC 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
  • 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
  • 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 CRC proteins comprise the steps of adding a candidate bioactive agent to a sample of CRC proteins, as above, and determining an alteration in the biological activity of CRC proteins "Modulating the activity of CRC" 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 CRC 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 CRC proteins
  • the methods comprise combining a CRC sample and a candidate bioactive agent, and evaluating the effect on CRC activity
  • CRC activity or grammatical equivalents herein is meant one of the CRC's biological activities, including, but not limited to, cell division, preferably in colon tissue, cell proliferation, tumor growth, transformation of cells
  • CRC activity includes activation of CZA8, BCX2, CBC2, CBC1 , CBC3, CJA9, BCN5, CQA1 , BCN7, CQA2, CJA8, CAA2, CAA9, CGA7 and/or CGA8*, preferably one of the CRC proteins listed in Figure 14.
  • An inhibitor of CRC activity is the inhibition of any one or more CRC activities.
  • the activity of the CRC protein is increased; in another preferred embodiment, the activity of the CRC 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 CRC protein.
  • the methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising CRC proteins.
  • Preferred cell types include almost any cell.
  • the cells contain a recombinant nucleic acid that encodes a CRC 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.
  • colonal cancer protein activity includes at least one of the following: colorectal cancer activity, binding to CJA8, activation of CJA8 or activation of substrates of CJA8 by CJA8.
  • colorectal cancer activity is defined as the unregulated proliferation of colon tissue, or the growth of cancer in colon tissue.
  • colorectal cancer activity as defined herein is related to the activity of CJA8 in the upregulation of CJA8 in colon cancer tissue.
  • colorectal cancer protein activity includes at least one of the following: colorectal cancer activity, binding to one of CAA2, CAA9, CGA7 and CGA8, activation of one of
  • CAA2 CAA2, CAA9, CGA7, and CGA8 or activation of substrates of CAA2, CAA9, CGA7 or CGA8 by CAA2, CAA9, CGA7 or CGA8, respectively.
  • CAA2 comprises its N-terminal end.
  • colorectal cancer activity as defined herein is related to the activity of CAA2, CAA9, CGA7 and/or CGA8 in the upregulation of CAA2, CAA9, CGA7 and/or CGA8, respectively, in colon cancer tissue.
  • a method of inhibiting colon cancer cell division is provided The method comprises administration of a colorectal cancer inhibitor
  • a method of inhibiting tumor growth comprises administration of a colorectal cancer inhibitor
  • methods of treating cells or individuals with cancer comprises administration of a colorectal cancer inhibitor
  • a colorectal cancer inhibitor is an antibody as discussed above
  • the colorectal cancer inhibitor is an antisense molecule
  • Antisense molecules as used herein include antisense or sense oligonucleotides composing a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for colorectal cancer molecules
  • a preferred antisense molecule is for CZA8, BCX2, CBC2, CBC1 , CBC3, CJA8, CJA9, BCN5, CQA1 , BCN7, CQA2, CAA2, CAA9, CGA7 or CGA ⁇ , more preferably for the CRC sequences referenced in Figure 14, or for a ligand or activator thereof
  • a most preferred antisense molecule is for CJA8 or for a ligand or activator thereof
  • Antisense or sense oligonucleotides, according to the present invention comprise a fragment generally at least about 14 nucleo
  • 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 cytokmes, 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 o gonucleotide- 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
  • 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, intrape ⁇ toneally, 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 CRC genes comprising determining all or part of the sequence of at least one endogeneous CRC genes in a cell
  • the invention provides methods of identifying the CRC genotype of an individual comprising determining all or part of the sequence of at least one CRC 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 CRC gene to a known CRC gene, i e a wild-type gene
  • the sequence of all or part of the CRC gene can then be compared to the sequence of a known CRC 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 a difference in the sequence between the CRC gene of the patient and the known CRC gene is indicative of a disease state or a propensity for a disease state, as outlined herein
  • the CRC genes are used as probes to determine the number of copies of the CRC gene in the genome
  • CRC genes are used as probed to determine the chromosomal localization of the CRC 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 CRC gene loci.
  • methods of modulating CRC in cells or organisms comprise administering to a cell an anti-CRC antibody that reduces or eliminates the biological activity of an endogeneous CRC protein.
  • the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a CRC protein.
  • this may be accomplished in any number of ways.
  • the activity of the CRC gene is increased by increasing the amount of CRC in the cell, for example by overexpressing the endogeneous CRC or by administering a gene encoding the CRC sequence, using known gene- therapy techniques, for example.
  • the gene therapy techniques include the incorporation of the erogenous 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 CRC gene is decreased, for example by the administration of a CRC antisense nucleic acid.
  • the CRC proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to CRC proteins, which are useful as described herein.
  • the CRC proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify CRC antibodies.
  • the antibodies are generated to epitopes unique to a CRC protein; that is, the antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications.
  • the CRC antibodies may be coupled to standard affinity chromatography columns and used to purify CRC proteins.
  • the antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the CRC protein.
  • a therapeutically effective dose of a CRC 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 CRC 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. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.
  • the administration of the CRC 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 CRC proteins and modulators may be directly applied as a solution or spray.
  • compositions of the present invention comprise a CRC 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 aod 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 aod the like
  • organic acids such as acetic acid, propionic acid, glycolic acid,
  • “Pharmaceutically acceptable base addition salts” include those derived from ioorganic 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
  • CRC proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above.
  • CRC genes (including both the full-length sequence, partial sequences, or regulatory sequences of the CRC coding regions) can be administered in gene therapy applications, as is known in the art
  • These CRC 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
  • CRC genes are administered as DNA vaccines, either single genes or combinations of CRC genes Naked DNA vaccines are generally known in the art Brower, Nature Biotechnology, 16 1304-1305 (1998)
  • CRC 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 CRC gene or portion of a CRC gene under the control of a promoter for expression in a CRC patient
  • the CRC gene used for DNA vaccines can encode full-length CRC proteins, but more preferably encodes portions of the CRC proteins including peptides derived from the CRC protein
  • a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a CRC gene
  • a DNA vaccine comprising a plurality of nucleotide sequences derived from a CRC gene
  • expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helper T-cells and antibodies are induced which recognize and destroy or eliminate cells expressing CRC proteins
  • the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine
  • adjuvant molecules include cytokmes that increase the immunogenic response to the CRC 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
  • CRC genes find use in generating animal models of CRC
  • gene therapy technology wherein antisense RNA directed to the CRC gene will also dimmish or repress expression of the gene
  • An animal generated as such serves as an animal model of CRC 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 CRC protein
  • tissue-specific expression or knockout of the CRC protein may be necessary
  • CRC protein is overexpressed in CRC
  • transgenic animals can be generated that overexpress the CRC 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 CRC and are additionally useful in screening for bioactive molecules to treat CRC
  • tissue weight Homogenize tissue samples in 1 ml of TRIzol per 50mg of tissue using a Polytron 3100 homogenizer
  • the generator/probe used depends upon the tissue size A generator that is too large for the amount of tissue to be homogenized will cause a loss of sample and lower RNA yield
  • RNA PRECIPITATION Transfer the aqueous phase to a fresh tube. Save the organic phase if isolation of DNA or protein is desired. Add 0.5ml of isopropyl alcohol per 1 ml of TRIzol reagent used in the original homogenization. Cap tubes securely and invert to mix. Incubate samples at room temp, for 10 minutes. Centrifuge samples at 6500rpm in Sorvall for 20min. at 4°C.
  • RNA WASH Pour off the supernate. Wash pellet with cold 75% ethanol. Use 1 ml of 75% ethanol per 1 ml of
  • TRIzol reagent used in the initial homogenization. Cap tubes securely and invert several times to loosen pellet. (Do not vortex). Centrifuge at ⁇ 8000rpm ( ⁇ 7500 x g) for 5 minutes at 4°C. Pour off the wash. Carefully transfer pellet to an eppendorf tube (let it slide down the tube into the new tube and use a pipet tip to help guide it in if necessary). Depending on the volumes you are working with, you can decide what size tube(s) you want to precipitate the RNA in. When I tried leaving the RNA in the large 15ml tube, it took so long to dry (i.e. it did not dry) that I eventually had to transfer it to a smaller tube. Let pellet dry in hood. Resuspend RNA in an appropriate volume of DEPC H 2 0. Try for 2-5ug/ul. Take absorbance readings.
  • centrifuge for 2 minutes at 14,000 to 18,000 g. If centrifuge has a "soft setting,” then use it. Remove supernatant without disturbing Oligotex pellet. A little bit of solution can be left behind to reduce the loss of Oligotex. Save sup until certain that satisfactory binding and elution of poly A + mRNA has occurred.
  • Second Strand Synthesis Place 1 st strand reactions on ice. Add: 91 ul DEPC H20 30ul 5X 2 nd Strand Buffer
  • IVTT In vitro Transcription
  • biotin Pipet 1.5ul of cDNA into a thin-wall PCR tube.
  • cRNA will most likely need to be ethanol precipitated Resuspend in a volume compatible with the fragmentation step
  • Fragment RNA by incubation at 94 C for 35 minutes in 1 x Fragmentation buffer
  • RNA transcript can be analyzed before and after fragmentation Samples can be heated to 65C for 15 minutes and electrophoresed on 1 % agarose/TBE gels to get an approximate idea of the transcript size range
  • Hybridization 200 ul (10ug cRNA) of a hybridization mix is put on the chip If multiple hybridizations are to be done (such as cycling through a 5 chip set), then it is recommended that an initial hybridization mix of 300 ul or more be made Hybrization Mix: fragment labeled RNA (50ng/ul final cone.) 50 pM 948-b control oligo 1.5 pM BioB 5 pM BioC 25 pM BioD
  • IVT antisense RNA 4 ⁇ g: ⁇ l
  • Na pyro phosphate 7.5 ⁇ l 10mg/ml Herring sperm DNA 1ul of 1/10 dilution
  • the results are shown in Figures 1 through 11.
  • the lists of genes come from colorectal tumors from a variety of stages of the disease.
  • the genes that are up regulated in the tumors (overall) were also found to be expressed at a limited amount or not at all in the body map.
  • the body map for the colorectal project consists of ten tissues: Heart, Brain, Lung, Liver, Breast, Kidney, Prostrate, Small Intestine, Spleen, and Colon.
  • the down regulated genes in tumors (overall) versus normal colon were not selected for their expression or lack of expression in the body map.
  • some of the Accession numbers include expression sequence tags (ESTs).
  • genes within an expression profile include ESTs and are not necessarily full length.
  • Figure 1 shows 51 upregulated genes;
  • Figure 2 shows 194 upregulated genes;
  • Figure 3 shows 1144 upregulated genes and
  • Figure 4 shows 1815 upregulated genes.
  • the genes shown in Figures 1 and 5 are particularly preferred.
  • Figure 5 shows 54 downregulated genes;
  • Figure 6 shows 558 downregulated genes; and
  • Figure 7 shows
  • Figures 8, 9, 10 and 11 provide the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal colon tissue.
  • CAA2 is upregulated in colon cancer tissue.
  • CAA2 is found in chromosome 15, cytoband 15q15-22, interval D15S146-D15S117.
  • CAA2 has N-myristoylation sites and a C-terminal microbody targeting signal.
  • the preferred fragments shown in Figures 18 and 19 have a solubility of 1 mg/ 1 ml H20.
  • CAA9 is upregulated in colon cancer tissue.
  • CAA9 is found in chromosome 5, cytoband 5q23.3, interval D5S471-D5S393.
  • CGA7 is upregulated in colon cancer tissue.
  • CGA7 is found in chromosome 2.
  • CGA8 is upregulated in colon cancer tissue.
  • CJA8 is upregulated in colon cancer tissue.
  • CJA8 is found in chromosome 11.
  • BCN7 is upregulated in colon cancer tissue.
  • BCN7 is found in chromosome 5, cytoband 5q22, interval D5S471-D5S393.

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US450857 1982-12-17
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US268866 1999-03-15
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US09/436,983 US6294343B1 (en) 1999-11-09 1999-11-09 Methods of diagnosing colorectal cancer, compositions, and methods of screening for colorectal cancer modulators
US09/435,945 US6773878B1 (en) 1999-11-09 1999-11-09 Methods of diagnosing of colorectal cancer, compositions, and methods of screening for colorectal cancer modulators
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