WO2006021874A2 - Nouvelles sequences de nucleotides et d'acides amines; essais et methodes d'utilisation pour le diagnostic du cancer de a la prostate - Google Patents

Nouvelles sequences de nucleotides et d'acides amines; essais et methodes d'utilisation pour le diagnostic du cancer de a la prostate Download PDF

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WO2006021874A2
WO2006021874A2 PCT/IB2005/002560 IB2005002560W WO2006021874A2 WO 2006021874 A2 WO2006021874 A2 WO 2006021874A2 IB 2005002560 W IB2005002560 W IB 2005002560W WO 2006021874 A2 WO2006021874 A2 WO 2006021874A2
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pea
amino acid
amino acids
homologous
sequence
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PCT/IB2005/002560
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English (en)
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WO2006021874A8 (fr
WO2006021874A3 (fr
Inventor
Dvir Dahary
Sarah Pollock
Zurit Levine
Rotem Sorek
Michal Ayalon-Soffer
Pinchas Akiva
Amir Toporik
Osnat Sella-Tavor
Shirley Sameah-Greenwald
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Compugen Usa, Inc.
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Priority to CA002554707A priority Critical patent/CA2554707A1/fr
Priority to EP05805032A priority patent/EP1735468A2/fr
Priority claimed from US11/043,806 external-priority patent/US7368548B2/en
Publication of WO2006021874A2 publication Critical patent/WO2006021874A2/fr
Publication of WO2006021874A8 publication Critical patent/WO2006021874A8/fr
Publication of WO2006021874A3 publication Critical patent/WO2006021874A3/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention is related to novel nucleotide and protein sequences that are diagnostic markers for prostate cancer, and assays and methods of use thereof.
  • Prostate cancer is the most commonly diagnosed malignancy and the second most frequent cause of cancer-related deaths in the western male population.
  • Prostate cancer therapies are most effective in the earlier stages of the disease, before metastasis has occurred. Treatment is expected to be even more effective before significant local growth of the cancerous tissue has taken place. Therefore, efforts to control the disease (i.e., to decrease prostate cancer mortality) have focused on increasing detection of the cancer while it is still locally confined and potentially curable, through diagnostic assays that are suitable for early detection of prostate cancer. Unfortunately, such detection also has significant drawbacks, because diagnostic assays that use currently available prostate cancer markers lead to high numbers of false positive diagnoses, and/or are not sufficiently sensitive (potentially leading to high numbers of false negative diagnoses).
  • prostatic marker enzymes have recognized value in the clinical detection, diagnosis and management of prostate cancer.
  • the two most widely used prostatic marker enzymes are prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA).
  • PAP prostatic acid phosphatase
  • PSA prostate-specific antigen
  • both enzymes are secreted from the prostatic epithelial cells into the seminal fluid, but in patients with prostatic disease they leak into the circulation, where they can be detected by means of immunological assays (Armbruster, Clin. Che. 39:181-95 (1993)).
  • Prostatic acid phosphatase one of the earliest serum markers for prostate, has an as yet undetermined function and is one of the most predominant protein components in human prostatic secretions.
  • the use of PAP as a marker for prostatic tumors is complicated by the reported structural similarities between the prostate-specific acid phosphatase and the lysosomal acid phosphatase occurring in all tissues. Furthermore, there is a tendency towards lower PAP mRNA and protein levels in prostate cancer in comparison with benign prostatic hyperplasia (BPH). In recent years, PAP measurements were superseded by serum PSA measurements in the clinical management of prostate cancer.
  • PSA Prostate- specific antigen
  • Prostate-specific membrane antigen was originally identified using an antibody developed by immunizing mice with the membrane fraction of LNCaP human prostatic adenocarcinoma cells. Like PAP and PSA, PSM can be detected in normal prostate, BPH and prostate cancer and is absent from most other tissues. However, the usefulness of PSM as marker for prostatic cancer has not been fully established.
  • PCA3 DD3 is a new marker from DiagnoCure, which has been described as being useful in a urine-based test (PCA3 itself is described in PCT Application Nos. WO 98/45420 and WO 2000/123550). This marker is apparently only expressed in prostate cancer, and therefore nay be used to distinguish between BPH and prostate cancer. However, as described in greater detail below, the sensitivity and accuracy of this marker may be improved when used in combination with one or more additional markers.
  • PSA is recognized as the best available marker for prostate cancer, being useful for screening selected populations of patients with symptoms indicative of prostate cancer and for monitoring patients after therapy, especially after surgical prostatectomy.
  • PSA has significant drawbacks in terms of false positive measurements, since it cannot distinguish prostate cancer from BPH. It may also lead to false negative measurements, since de-differentiation of prostate cancerous tissue (which may occur with some types of prostate cancers) also leads to decreased expression of this marker.
  • New markers are currently being developed to overcome this problem, but these markers have their own drawbacks. Clearly, new markers are required.
  • the background art does not teach or suggest markers for prostate cancer that are sufficiently sensitive and/or accurate, alone or in combination.
  • the present invention overcomes these deficiencies of the background art by providing novel markers for prostate cancer that are both sensitive and accurate. Furthermore, at least some of these markers are able to distinguish between prostate cancer and benign prostate hyperplasia ("BPH"). These markers are differentially expressed, and preferably overexpressed in prostate cancer specifically, as opposed to normal prostate tissue and/or BPH.
  • BPH benign prostate hyperplasia
  • These markers are differentially expressed, and preferably overexpressed in prostate cancer specifically, as opposed to normal prostate tissue and/or BPH.
  • the measurement of these markers, alone or in combination, in patient samples (biological samples) provides information that the diagnostician can correlate with a probable diagnosis of prostate cancer.
  • the markers of the present invention alone or in combination, show a high degree of differential detection between prostate cancer and non-cancerous states.
  • suitable biological samples include but are not limited to blood, serum, plasma, blood cells, urine, sputum, saliva, stool, spinal fluid or CSF, lymph fluid, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, milk, neuronal tissue, prostate tissue or mucous and any human organ or tissue, or any sample obtained by lavage (for example of the bronchial system), and also samples of in vivo cell culture constituents.
  • the biological sample comprises prostate tissue and/or other tissues of the male genitalia, or reproductive or urinary tracts, and/or a serum (and/or any blood) sample and/or a urine sample and/or a semen sample and/or any other tissue or liquid sample.
  • the sample can optionally be diluted with a suitable eluant before contacting the sample to an antibody and/or performing any other diagnostic assay.
  • signalp_hmm and “signalp_nn” refer to two modes of operation for the program SignalP: hmm refers to Hidden Markov Model, while nn refers to neural networks. Localization was also determined through manual inspection of known protein localization and/or gene structure, and the use of heuristics by the individual inventor.
  • T - > C means that the SNP results in a change at the position given in the table from T to C.
  • M - > Q means that the SNP has caused a change in the corresponding amino acid sequence, from methionine (M) to glutamine (Q). If, in place of a letter at the right hand side for the nucleotide sequence SNP, there is a space, it indicates that a frameshift has occurred. A frameshift may also be indicated with a hyphen (-). A stop codon is indicated with an asterisk at the right hand side (*).
  • a comment may be found in parentheses after the above description of the SNP itself.
  • This comment may include an FTId, which is an identifier to a SwissProt entry that was created with the indicated SNP.
  • An FTId is a unique and stable feature identifier, which allows construction of links directly from position- specific annotation in the feature table to specialized protein-related databases.
  • the header of the first column is "SNP position(s) on amino acid sequence", representing a position of a known mutation on amino acid sequence.
  • SNPs may optionally be used as diagnostic markers according to the present invention, alone or in combination with one or more other SNPs and/or any other diagnostic marker.
  • Preferred embodiments of the present invention comprise such SNPs, including but not limited to novel SNPs on the known (WT or wild type) protein sequences given below, as well as novel nucleic acid and/or amino acid sequences formed through such SNPs, and/or any SNP on a variant amino acid and/or nucleic acid sequence described herein.
  • P- value including the level of expression in cell- lines (P2)
  • EST clone statistics predicted overexpression ratio including the level of expression in cell- lines (R4)
  • Library-based statistics refer to statistics over an entire library, while EST clone statistics refer to expression only for ESTs from a particular tissue or cancer.
  • microarrays As a microarray reference, in the specific segment paragraphs, the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured. There are two types of microarray results: those from microarrays prepared according to a design by the present inventors, for which the microarray fabrication procedure is described in detail in Materials and Experimental Procedures section herein; and those results from microarrays using Affymetrix technology. As a microarray reference, in the specific segment paragraphs, the unabbreviated tissue name was used as the reference to the type of chip for which expression was measured.
  • the probe name begins with the name of the cluster (gene), followed by an identifying number.
  • Oligonucleotide microarray results taken from Affymetrix data were from chips available from Affymetrix Inc, Santa Clara, CA, USA (see for example data regarding the Human Genome U133 (HG-U133) Set at www.affymetrix.com/products/arrays/specific/hgul33.affx; GeneChip Human Genome U133A 2.0 Array at www.affymetrix.com/products/arrays/specific/hgul33av2.affx; and Human Genome U133 Plus 2.0 Array at www.affymetrix.com/products/arrays/specific/hgul33plus.affx).
  • the probe names follow the Affymetrix naming convention.
  • the data is available from NCBI Gene Expression Omnibus (see www.ncbi.nlm.nih.gov/projects/geo/ and Edgar et al, Nucleic Acids Research, 2002, Vol. 30, No. 1 207-210).
  • TAA histograms The following list of abbreviations for tissues was used in the TAA histograms.
  • TAA Tumor Associated Antigen
  • TAA histograms represent the cancerous tissue expression pattern as predicted by the biomarkers selection engine, as described in detail in examples 1-5 below: "BONE" for "bone”;
  • OVA for "ovary”
  • PANCREAS for “pancreas”
  • PRO for “prostate”
  • STOMACH for "stomach”
  • TELL for “T cells”
  • TTYROID for "Thyroid”
  • nucleic acid sequences of the present invention refer to portions of nucleic acid sequences that were shown to have one or more properties as described below. They are also the building blocks that were used to construct complete nucleic acid sequences as described in greater detail below.
  • oligonucleotides which are embodiments of the present invention, for example as amplicons, hybridization units and/or from which primers and/or complementary oligonucleotides may optionally be derived, and/or for any other use.
  • prostate cancer refers to cancers of the prostate tissue and/or other tissues of the male genitalia, or reproductive or urinary tracts.
  • marker in the context of the present invention refers to a nucleic acid fragment, a peptide, or a polypeptide, which is differentially present in a sample taken from subjects (patients) having prostate cancer as compared to a comparable sample taken from subjects who do not have prostate cancer.
  • differentially present refers to differences in the quantity of a marker present in a sample taken from patients having prostate cancer as compared to a comparable sample taken from patients who do not have prostate cancer.
  • a nucleic acid fragment may optionally be differentially present between the two samples if the amount of the nucleic acid fragment in one sample is significantly different from the amount of the nucleic acid fragment in the other sample, for example as measured by hybridization and/or NAT-based assays.
  • a polypeptide is differentially present between the two samples if the amount of the polypeptide in one sample is significantly different from the amount of the polypeptide in the other sample. It should be noted that if the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present.
  • diagnostic means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives”). Diseased individuals not detected by the assay are “false negatives.” Subjects who are not diseased and who test negative in the assay are termed “true negatives.”
  • the "specificity” of a diagnostic assay is 1 minus the false positive rate, where the "false positive” rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • diagnosis refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
  • detecting may also optionally encompass any of the above.
  • Diagnosis of a disease according to the present invention can be affected by dete ⁇ iiguing a level of a polynucleotide or a polypeptide of the present invention in a biological sample obtained from the subject, wherein the level determined can be correlated with predisposition to, or presence or absence of the disease.
  • a biological sample obtained from the subject may also optionally comprise a sample that has not been physically removed from the subject, as described in greater detail below.
  • the term “level” refers to expression levels of RNA and/or protein or to
  • DNA copy number of a marker of the present invention is preferably the level of the marker in a biological sample obtained from the subject is different (i.e., increased or decreased) from the level of the same variant in a similar sample obtained from a healthy individual (examples of biological samples are described herein).
  • tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of DNA, RNA and/or polypeptide of the variant of interest in the subject.
  • Examples include, but are not limited to, fine needle biopsy, needle biopsy, core needle biopsy and surgical biopsy (e.g., brain biopsy), and lavage. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.
  • Determining the level of the same variant in normal tissues of the same origin is preferably effected along- side to detect an elevated expression and/or amplification and/or a decreased expression, of the variant as opposed to the normal tissues.
  • test amount of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of prostate cancer.
  • a test amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
  • a "control amount" of a marker can be any amount or a range of amounts to be compared against a test amount of a marker.
  • a control amount of a marker can be the amount of a marker in a patient with prostate cancer or a person without prostate cancer.
  • a control amount can be either in absolute amount (e.g., microgram/ml) or a relative amount (e.g., relative intensity of signals).
  • Detect refers to identifying the presence, absence or amount of the object to be detected.
  • label includes any moiety or item detectable by spectroscopic, photo chemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavadin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target.
  • the label often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound label in a sample.
  • a measurable signal such as a radioactive, chromogenic, or fluorescent signal
  • the label can be incorporated in or attached to a primer or probe either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., incorporation of radioactive nucleotides, or biotinylated nucleotides that are recognized by streptavadin.
  • the label may be directly or indirectly detectable. Indirect detection can involve the binding of a second label to the first label, directly or indirectly.
  • the label can be the ligand of a binding partner, such as biotin, which is a binding partner for streptavadin, or a nucleotide sequence, which is the binding partner for a complementary sequence, to which it can specifically hybridize.
  • the binding partner may itself be directly detectable, for example, an antibody may be itself labeled with a fluorescent molecule.
  • the binding partner also may be indirectly detectable, for example, a nucleic acid having a complementary nucleotide sequence can be a part of a branched DNA molecule that is in turn detectable through hybridization with other labeled nucleic acid molecules (see, e.g., P. D. Fahrlander and A. Klausner, Bio/Technology 6:1165 (1988)). Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.
  • Exemplary detectable labels include but are not limited to magnetic beads, fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic beads.
  • the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker- specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
  • Immunoassay is an assay that uses an antibody to specifically bind an antigen.
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
  • the specified antibodies bind to a particular protein at least two times greater than the background (non-specific signal) and do not substantially bind in a significant amount to other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • polyclonal antibodies raised to seminal basic protein from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with seminal basic protein and not with other proteins, except for polymorphic variants and alleles of seminal basic protein.
  • This selection may be achieved by subtracting out antibodies that cross-react with seminal basic protein molecules from other species.
  • a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
  • solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 1,2, 3 and 4.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 and 89.
  • an isolated polypeptide comprising SEQ ID NOs: 327, 328, 329, 330.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: SEQ ID NOs. 5, 6, 7, 8, 9 and 10.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114 and 115.
  • an isolated polypeptide comprising SEQ ID NOs: 331, 332, 333, 334 and 335.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 11.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 and 129.
  • an isolated polypeptide comprising SEQ ID NOs: 336.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 12. According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a segment SEQ ID NOs: 130, 131, 132, 133, 134 and 135.
  • an isolated polypeptide comprising SEQ ID NOs: 337.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 34, 35, 36, 37, 38 and 39.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236 and 237.
  • an isolated polypeptide comprising SEQ ID Nos: 359, 360, 361, 362 and 363.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and 33.
  • an isolated polypeptide comprising SEQ ID NOs: 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220 and 221.
  • an isolated polypeptide comprising SEQ ID NOs: 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357 and 358.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 13 and 14. According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising SEQ ID NOs: 136. 137, 138, 139, 140, 141 and 142. According to preferred embodiments of the present invention, there is provided an isolated polypeptide comprising SEQ ID NOs: 338 and 339.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 40, 41 and 42.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 238, 239, 240. 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269 and 270.
  • an isolated polypeptide comprising SEQ ID NOs: 364, 365 and 366.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 43.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283 and 284.
  • an isolated polypeptide comprising SEQ ID NOs: 367.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 44 and 45. According to preferred embodiments of the present invention, there is provided an isolated polynucleotide comprising a segment SEQ ID NOs: 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303 and 304.
  • an isolated polypeptide comprising SEQ ID NOs: 368 and 369.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 and 59.
  • an isolated polynucleotide comprising a segment SEQ ID NOs: 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325 and 326.
  • an isolated polypeptide comprising SEQ ID NOs: 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 and 383.
  • amino acids 1 - 45 of SEQ ID NO. 383 corresponds to amino acids 1 - 45 of SEQ ID NO. 383, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 46 - 85 of SEQ ID NO. 383, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated chimeric polypeptide encoding for SEQ ID NOs. 359 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 163 of SEQ ID NOs. 391 , which also corresponds to amino acids 1 - 163 of SEQ ID NOs. 359 , a bridging amino acid H corresponding to amino acid 164 of SEQ ID NOs. 359 , a second amino acid sequence being at least 90 % homologous to corresponding to amino acids 165 - 445 of SEQ ID NOs. 391 , which also corresponds to amino acids 165 - 445 of SEQ ID NO.
  • a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 446 - 496 of SEQ ID NO. 359 , wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 359 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 446 - 496 in SEQ
  • an isolated chimeric polypeptide encoding for SEQ ID NOs.360 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 163 of SEQ ID NOs. 391 , which also corresponds to amino acids 1 - 163 of SEQ ID NO.360 , a bridging amino acid H corresponding to amino acid 164 of SEQ ID NO. 360 , a second amino acid sequence being at least 90 % homologous to corresponding to amino acids 165 - 358 of SEQ ID NOs. 391 , which also corresponds to amino acids 165 - 358 of SEQ ID NO.
  • a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 359 - 382 of SEQ ID NO. 360 , wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO.
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 361 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 163 of SEQ ID NOs. 391 , which also corresponds to amino acids 1 - 163 of SEQ ID NO. 361 , a bridging amino acid H corresponding to amino acid 164 of SEQ ID NO.
  • a second amino acid sequence being at least 90 % homologous to corresponding to amino acids 165 - 359 of SEQ ID NOs. 391 , which also corresponds to amino acids 165 - 359 of SEQ ID NO. 361
  • a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 360 - 370 of SEQ ID NO. 361 , wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 361 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 360 - 370 in SEQ ID NO. 361 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 362 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 163 of SEQ ID NOs. SEQ ID NOs. 391 , which also corresponds to amino acids 1 - 163 of SEQ ID NO. 362 , a bridging amino acid H corresponding to amino acid 164 of SEQ ID NO. 362 , a second amino acid sequence being at least 90 % homologous to corresponding to amino acids 165 - 286 of SEQ ID NOs. 391 , which also corresponds to amino acids 165 - 286 of SEQ ID NO.
  • a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 287 - 301 of SEQ ID NO. 362 , wherein said first amino acid sequence, bridging amino acid, second amino acid sequence and third amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 362 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 287 - 301 in SEQ ID NO. 362 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 363 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 96 of SEQ ID NOs. 391 , which also corresponds to amino acids 1 - 96 of SEQ ID NO. 363 , a second amino acid sequence being at least 90 % homologous to corresponding to amino acids 113 - 163 of SEQ ID NOs. 391 , which also corresponds to amino acids 97 - 147 of SEQ ID NO. 363 , a bridging amino acid H corresponding to amino acid 148 of SEQ ID NO.
  • a third amino acid sequence being at least 90 % homologous to corresponding to amino acids 165 - 359 of SEQ ID NOs. 391 , which also corresponds to amino acids 149 - 343 of SEQ ID NO. 363
  • a fourth amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 344 - 354 of SEQ ID NO. 363 , wherein said first amino acid sequence, second amino acid sequence, bridging amino acid, third amino acid sequence and fourth amino acid sequence are contiguous and in a sequential order.
  • an isolated chimeric polypeptide encoding for an edge portion of SEQ ID NO. 363 comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise KR, having a structure as follows: a sequence starting from any of amino acid numbers 96-x to 96; and ending at any of amino acid numbers 97+ ((n-2) - x), in which x varies from 0 to n-2.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 363 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence in SEQ ID NO. 363 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 340 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 865 of CO4_HUMAN, which also corresponds to amino acids 1 - 865 of SEQ ID NO. 340 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 866 - 887 of SEQ ID NO. 340 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID 'NO. 340 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 866 - 887 in SEQ ID NO. 340 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 341 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 818 of CO4_HUMAN, which also corresponds to amino acids 1 - 818 of SEQ ID NO.
  • a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 819 - 843 of SEQ ID NO. 341 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 341 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 819 - 843 in
  • an isolated chimeric polypeptide encoding for SEQ SEQ ID NO. 342 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1052 of
  • CO4_HUMAN which also corresponds to amino acids 1 - 1052 of SEQ ID NO. 342 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1053 - 1084 of SEQ ID NO. 342 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 342 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence in SEQ ID NO.
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 343, comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1380 of SEQ ID NO. 389 , which also corresponds to amino acids 1 - 1380 of SEQ ID NO. 343 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1381 - 1397 of SEQ ID NO. 343 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ SEQ ID NO. 343 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence in SEQ
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 344 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1359 of SEQ ID NO. 389 , which also corresponds to amino acids 1 - 1359 of SEQ ID NO. 344 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1360 - 1415 of SEQ ID NO. 344 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 344 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1360 - 1415 in SEQ ID NO. 344 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 345 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1457 of SEQ ID NO. 389 , which also corresponds to amino acids 1 - 1457 of SEQ ID NO. 345 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 1458 - 1483 of SEQ ID NO. 345 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 345 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1458 - 1483 in SEQ ID NO. 345 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 346 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1303 of SEQ ID NO. 389 , which also corresponds to amino acids 1 - 1303 of SEQ ID NO. 346 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1304 - 1349 of SEQ ID NO. 346 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 346 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence in SEQ ID NO. 346 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 347 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1529 of SEQ ID NO. 389 , which also corresponds to amino acids 1 - 1529 of SEQ ID NO. 347 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1530 - 1533 of SEQ ID NO. 347 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 347 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1530 - 1533 in SEQ ID NO. 347 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 348 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1653 of SEQ ID NO. 389 , which also corresponds to amino acids 1 - 1653 of SEQ ID NO.
  • a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1654 - 1670 of SEQ ID NO. 348 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 348 comprising a polypeptide being at least 70%, optionally at least about 80%,. preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1654 - 1670 in SEQ ID NO. 348 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 349 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1626 of SEQ ID NO. 389 , which also corresponds to amino acids 1 - 1626 of SEQ ID NO. 349 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1627 - 1685 of SEQ ID NO. 349 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 349 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1627 - 1685 in SEQ ID NO. 349 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 350 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1528 of SEQ ID NO. 389 , which also corresponds to amino acids 1 - 1528 of SEQ ID NO.
  • a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1529 - 1579 of SEQ ID NO. 350 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 350 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1529 - 1579 in
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 351 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1593 of SEQ ID NO.
  • amino acids 1 - 1593 of SEQ ID NO. 351 which also corresponds to amino acids 1 - 1593 of SEQ ID NO. 351 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1594 - 1657 of SEQ ID NO. 351 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 351 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1594 - 1657 in SEQ ID NO. 351 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 352 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1593 of SEQ ID NO. 389 , which also corresponds to amino acids 1 - 1593 of SEQ ID NO. 352 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1594 - 1691 of SEQ ID NO. 352 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 352 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1594 - 1691 in SEQ ID NO. 352 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 353 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1232 of SEQ ID NO.
  • amino acids 1 - 1232 of SEQ ID NO. 353 which also corresponds to amino acids 1 - 1232 of SEQ ID NO. 353 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1233 - 1253 of SEQ ID NO. 353 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 353 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1233 - 1253 in SEQ ID NO. 353 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 354 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 818 of CO4_HUMAN, which also corresponds to amino acids 1 - 818 of SEQ ID NO. 354 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 819 - 843 of SEQ ID NO. 354, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 354 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to to amino acids 819 - 843 in
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 355 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 387 of CO4_HUMAN, which also corresponds to amino acids 1 - 387 of SEQ ID NO. 355 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 388 - 394 of SEQ SEQ ID NO. 355 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 355 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 388 - 394 in SEQ ID NO. 355 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 356 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 236 of CO4_HUMAN, which also corresponds to amino acids 1 - 236 of SEQ ID NO. 356 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 237 - 263 of SEQ ID NO. 356 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 356 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 237 - 263 in SEQ ID NO. 356.
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 357 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1529 of SEQ ID NO. 389 , which also corresponds to amino acids 1 - 1529 of SEQ ID NO.
  • a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1530 - 1533 of SEQ ID NO. 357 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 357 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence SGER in SEQ
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 358 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1473 of SEQ ID NO. 389 , which also corresponds to amino acids 1 - 1473 of SEQ ID NO. 358 , a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1474 - 1511 of SEQ ID NO.
  • a third amino acid sequence being at least 90 % homologous to corresponding to amino acids 1474 - 1503 of SEQ ID NO. 389 , which also corresponds to amino acids 1512 - 1541 of SEQ ID NO. 358
  • a fourth amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 1542 - 1555 of SEQ ID NO. 358 , wherein said first amino acid sequence, second amino acid sequence, third amino acid sequence and fourth amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for an edge portion of SEQ ID NO. 358 comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1474 - 1511, corresponding to SEQ ID NO. 358 .
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 358 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1542 - 1555 in SEQ ID NO. 358 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 339 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 27 of SEQ ID NO. 387 , which also corresponds to amino acids 1 - 27 of SEQ ID NO.
  • a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 28 - 41 of SEQ ID NO. 339 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 339 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 28 - 41 in SEQ
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 364 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 1617 of SEQ ID NO.
  • amino acids 1 - 1617 of SEQ ID NO. 364 corresponds to amino acids 1 - 1617 of SEQ ID NO. 364 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 1618 - 1645 of SEQ ID NO. 364 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 364 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1618 - 1645 in
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 365 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 2062 of SEQ ID NO. 393 , which also corresponds to amino acids 1 - 2062 of SEQ ID NO. 365, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 2063 - 2074 of SEQ ID NO. 365 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 365 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 2063 - 2074 in
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 366 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 587 of SEQ ID NO. 393 , which also corresponds to amino acids 1 - 587 of SEQ ID NO. 366 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 588 - 603 of SEQ ID NO. 366 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 366 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 588 - 603 in SEQ ID NO. 366 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 367 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 238 of SEQ ID NOs. 396 , which also corresponds to amino acids 1 - 238 of SEQ ID NO. 367 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 239 - 310 of SEQ ID NO. 367 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 367 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 239 - 310 in SEQ ED NO. 367 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 367 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 257 of SEQ ID NO. 395 , which also corresponds to amino acids 1 - 257 of SEQ ID NO. 367 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 258 - 310 of SEQ ID NO. 367 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 367 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 258 - 310 in SEQ ID NO. 367 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 367 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 257 of SEQ ID NO. 397 , which also corresponds to amino acids 1 - 257 of SEQ ID NO. 367 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 258 - 310 of SEQ ID NO. 367 , wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 367 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 258 - 310 in SEQ ID NO. 367 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 368 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 357 of Q8N441, which also corresponds to amino acids 1 - 357 of SEQ DD NO.
  • second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 358 - 437 of SEQ ID NO. 368
  • a third amino acid sequence being at least 90 % homologous to corresponding to amino acids 358 - 504 of Q8N441, which also corresponds to amino acids 438 - 584 of SEQ ID NO. 368 , wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for an edge portion of SEQ ID NO. 368 comprising an amino acid sequence being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 358 - 437, corresponding to SEQ ID NO. 368 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 369 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 269 of Q9H4D7, which also corresponds to amino acids 1 - 269 of SEQ ID NO. 369 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 270 - 490 of SEQ ID NO. 369 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 369 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 270 - 490 in
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 369 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 269 of Q8N441, which also corresponds to amino acids 1 - 269 of SEQ ID NO. 369 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 270 - 490 of SEQ ID NO. 369 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 369 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 270 - 490 in SEQ IDNO. 369 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 327 comprising a first amino acid sequence being at least 90 % homologous to to amino acids 1 - 274 of SEQ ID NO. 384, which also corresponds to amino acids 1 - 274 of SEQ ID NO. 327 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 275 - 322 of SEQ ID NO. 327 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 327 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 275 - 322 in SEQ ID NO. 327 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 327 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 274 of Q9UII8, which also corresponds to amino acids 1 - 274 of SEQ ID NO. 327 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 275 - 322 of SEQ ID NO. 327 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ SEQ ID NO. 327 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 275 - 322 in SEQ ID NO. 327 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 327 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 274 of CAD1_HUMAN, which also corresponds to amino acids 1 - 274 of SEQ ID NO. 327 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 275 - 322 of SEQ ID NO. 327 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 327 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 275 - 322 in SEQ ID NO. 327 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 328 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 379 of SEQ ID NO. 384, which also corresponds to amino acids 1 - 379 of SEQ ID NO. 328 , and a second amino acid sequence VIL corresponding to amino acids 380 - 382 of SEQ ID NO. 328 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 328 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 379 of SEQ ID NO. 384, which also corresponds to amino acids 1 - 379 of SEQ ID NO. 328 , and a second amino acid sequence VIL corresponding to amino acids 380 - 382 of SEQ ID NO. 328 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 328 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 379 of SEQ ID NO.
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 329 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 336 of SEQ ID NO. 384, which also corresponds to amino acids 1 - 336 of SEQ ID NO. 329 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 337 - 373 of SEQ ID NO. 329 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 329 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 337 - 373 in SEQ ID NO. 329 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 329 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 336 of SEQ ID NO. 384, which also corresponds to amino acids 1 - 336 of SEQ ID NO.
  • a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 337 - 373 of SEQ ID NO. 329 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 329 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 337 - 373 in SEQ ID NO. 329 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 329 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 336 of SEQ ID NO.
  • amino acids 1 - 336 of SEQ ID NO. 329 which also corresponds to amino acids 1 - 336 of SEQ ID NO. 329 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence corresponding to amino acids 337 - 373 of SEQ ID NO. 329 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ DD NO. 329 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 337 - 373 in SEQ ID NO. 329 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 330 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 229 of SEQ ID NO. 384, which also corresponds to amino acids 1 - 229 of SEQ ID NO. 330 , and a second amino acid sequence VSIS corresponding to amino acids 230 - 233 of SEQ ID NO. 330 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 330 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 229 of SEQ ID NO. 384, which also corresponds to amino acids 1 - 229 of SEQ ID NO. 330 , and a second amino acid sequence VSIS corresponding to amino acids 230 - 233 of SEQ ID NO. 330 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 330 comprising a first amino acid sequence being at least 90 % homologous to corresponding to amino acids 1 - 229 of SEQ ID NO.
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 332 comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 110 of SEQ ID NO. 332 , and a second amino acid sequence being at least 90 % homologous to TQ corresponding to amino acids 1 - 112 of Q8IXM0, which also corresponds to amino acids 111 - 222 of SEQ ID NO. 332 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a head of SEQ ID NO. 332 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1 - 110 of SEQ ID NO. 332 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 332 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 83 of Q96AC2, which also corresponds to amino acids 1 - 83 of SEQ ID NO. 332 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 84 - 222 of SEQ ID NO. 332 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 332 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 84 - 222 in SEQ
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 332 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 83 of Q8N2G4, which also corresponds to amino acids 1 - 83 of SEQ ID NO. 332 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 84 - 222 of SEQ ID NO. 332 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 332 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 84 - 222 in SEQ ID NO. 332 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 332 comprising a first amino acid sequence being at least 90 % homologous to amino acids 24 - 106 of BAC85518, which also corresponds to amino acids 1 - 83 of SEQ ID NO. 332 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 84 - 222 of SEQ DD NO. 332 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 332 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 84 - 222 in SEQ ID NO. 332 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 333 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 64 of Q96AC2, which also corresponds to amino acids 1 - 64 of SEQ ID NO. 333 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 65 - 93 of SEQ ID NO. 333 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 333 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 65 - 93 in SEQ ID NO. 333 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 333 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 64 of Q8N2G4, which also corresponds to amino acids 1 - 64 of SEQ ID NO. 333 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 65 - 93 of SEQ ID NO. 333 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 333 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 65 - 93 in SEQ ID NO. 333 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 333 comprising a first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 5 of SEQ ID NO. 333 , second amino acid sequence being at least 90 % homologous to amino acids 22 - 80 of BAC85273, which also corresponds to amino acids 6 - 64 of SEQ ID NO.
  • an isolated polypeptide encoding for a head of SEQ ID NO. 333 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1 - 5 of SEQ ID NO. 333 .
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 333 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 65 - 93 in SEQ
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 333 comprising a first amino acid sequence being at least 90 % homologous to amino acids 24 - 87 of BAC85518, which also corresponds to amino acids 1 - 64 of SEQ ID NO. 333 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 65 - 93 of SEQ ID NO. 333 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 333 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 65 - 93 in SEQ ID NO. 333 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 334 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 63 of Q96AC2, which also corresponds to amino acids 1 - 63 of SEQ ID NO. 334 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 64 - 84 of SEQ ID NO. 334 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 334 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 64 - 84 in SEQ ID NO. 334 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 335 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 63 of SEQ ID NOs. Q96AC2, which also corresponds to amino acids 1 - 63 of SEQ ID NO. 335 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 64 - 90 of SEQ ID NO. 335 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 335 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 64 - 90 in SEQ ID NO. 335 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 335 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 63 of Q8N2G4, which also corresponds to amino acids 1 - 63 of SEQ ID NO. 335 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 64 - 90 of SEQ ID NO. 335 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 335 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 64 - 90 in SEQ ID NO. 335 .
  • first amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 1 - 5 of SEQ ID NO. 335
  • second amino acid sequence being at least 90 % homologous to amino acids 22 - 79 of BAC85273, which also corresponds to amino acids 6 - 63 of SEQ ID NO. 335
  • a third amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 64 - 90 of SEQ ID NO.
  • an isolated polypeptide encoding for a head of SEQ ID NO. 335 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 1 - 5 of SEQ ID NO. 335 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 335 comprising a first amino acid sequence being at least 90 % homobgous to amino acids 24 - 86 of BAC85518, which also corresponds to amino acids 1 - 63 of SEQ ID NO.
  • a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 64 - 90 of SEQ ID NO. 335 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 335 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at bast about 95% homologous to amino acids 64 - 90 in SEQ ID NO. 335 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 336 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 247 of SEQ ID NO. 385 , which also corresponds to amino acids 1 - 247 of SEQ ID NO. 336 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 336 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 248 - 255 in SEQ ID NO. 336 .
  • an isolated chimeric polypeptide encoding for SEQ ID NO. 337 comprising a first amino acid sequence being at least 90 % homologous to amino acids 1 - 66 of SEQ ID NO. 386, which also corresponds to amino acids 1 - 66 of SEQ ID NO. 337 , and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at bast 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide sequence corresponding to amino acids 67 - 80 of SEQ ID NO. 337 , wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • an isolated polypeptide encoding for a tail of SEQ ID NO. 337 comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to amino acids 67 - 80 in SEQ
  • amino acid sequence corresponds to a bridge, edge portion, tail, head or insertion.
  • the antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.
  • kits for detecting prostate cancer comprising a kit from cluster HSECADH, Rl 1723, S78694, HUMTREFAC, HSCOC4, HSSTROL3, HUMF5A, Z40511, H53626 and HSMUClA for detecting overexpression of a splice variant.
  • the kit comprises a NAT-based technology.
  • the kit further comprises at least one primer pair capable of selectively hybridizing to a nucleic acid sequence.
  • the kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence .
  • the kit comprises an antibody.
  • the kit fiirther comprises at least one reagent for performing an ELISA or a Western blot.
  • a method for detecting prostate cancer comprising detecting overexpression of a splice variant from cluster HSECADH 3 Rl 1723, S78694, HUMTREFAC, HSCOC4, HSSTROL3, HUMF5A, Z40511, H53626 and HSMUClA.
  • detecting overexpression is performed with a NAT-based technology.
  • detecting overexpression is performed with an immunoassay.
  • the immunoassay comprises an antibody.
  • a biomarker capable of detecting prostate cancer comprising nucleic acid sequences or a fragment thereof, or amino acid sequences or a fragment thereof from cluster HSECADH, Rl 1723, S78694, HUMTREFAC, HSCOC4, HSSTROL3, HUMF5A, Z40511, H53626 and HSMUClA,.
  • a method for screening for prostate cancer comprising detecting prostate cancer cells with a biomarker or an antibody or a method or assay from cluster HSECADH, Rl 1723, S78694, HUMTREFAC, HSCOC4, HSSTROL3, HUMF5A, Z40511, H53626 and HSMUClA .
  • a method for diagnosing prostate cancer comprising detecting prostate cancer cells with a biomarker or an antibody or a method or assay from cluster HSECADH, Rl 1723, S78694, HUMTREFAC, HSCOC4, HSSTROL3, HUMF5A, Z40511, H53626 and HSMUClA .
  • a method for monitoring disease progression, treatment efficacy, relapse of prostate cancer comprising detecting prostate cancer cells with a biomarker or an antibody or a method or assay from cluster HSECADH, Rl 1723, S78694, HUMTREFAC, HSCOC4, HSSTROL3, HUMF5A,
  • a method of selecting a therapy for prostate cancer comprising detecting prostate cancer cells with a biomarker or an antibody or a method or assay from cluster HSECADH, Rl 1723, S78694, HUMTREFAC, HSCOC4, HSSTROL3, HUMF5A, Z40511, H53626 and HSMUClA .
  • any of the above nucleic acid and/or amino acid sequences further comprises any sequence having at least about 70%, preferably at least about 80%, more preferably at least about 90%, most preferably at least about 95% homology thereto.
  • All nucleic acid sequences and/or amino acid sequences shown herein as embodiments of the present invention relate to their isolated form, as isolated polynucleotides (including for all transcripts), oligonucleotides (including for all segments, amplicons and primers), peptides (including for all tails, bridges, insertions or heads, optionally including other antibody epitopes as described herein) and/or polypeptides (including for all proteins). It should be noted that oligonucleotide and polynucleotide, or peptide and polypeptide, may optionally be used interchangeably.
  • Figure 1 is a schematic description of the cancer biomarker selection engine.
  • Figure 2 is a schematic illustration, depicting grouping of transcripts of a given cluster based on presence or absence of unique sequence regions.
  • Figure 3 is a schematic summary of quantitative real-time PCR analysis.
  • Figure 4 is a schematic presentation of the oligonucleotide based microarray fabrication.
  • Figure 5 is a schematic summary of the oligonucleotide based microarray experimental flow.
  • Figure 6 is a histogram is a histogram showing Cancer and cell- line vs. normal tissue expression for Cluster HSECADH, demonstrating overexpression in a mixture of malignant tumors from different tissues and ovarian carcinoma.
  • Figure 7 is a histogram showing Cancer and cell- line vs. normal tissue expression for Cluster Rl 1723, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues and kidney malignant tumors.
  • Figure 8 is a histogram showing over expression of the Rl 1723 transcripts which are detectable by amplicon as depicted in sequence name Rl 1723 segl3 in cancerous prostate samples relative to the normal samples.
  • Figure 9 is a histogram showing expression of Rl 1723 transcripts, which are detectable by amplicon as depicted in sequence name R11723segl3, in different normal tissues.
  • Figures 1OA are histograms showing over expression of the Rl 1723 transcripts, which are detectable by amplicon as depicted in sequence name Rl 1723 juncll-18 in cancerous prostate samples relative to the normal samples ( Figure 10A) or expression in normal tissues ( Figure 10B).
  • Figure 11 is a histogram showing Cancer and cell- line vs. normal tissue expression for Cluster HUMTREFAC, demonstrating overexpression in a mixture of malignant tumors from different tissues, breast malignant tumors, pancreas carcinoma and prostate cancer.
  • Figure 12 is a histogram showing Cancer and cell-line vs. normal tissue expression for Cluster HSCOC4, demonstrating overexpression in brain malignant tumors, a mixture of malignant tumors from different tissues, breast malignant tumors, pancreas carcinoma and prostate cancer.
  • Figure 13 is a histogram showing Cancer and cell- line vs. normal tissue expression for Cluster HSSTROL3, demonstrating overexpression in transitional cell carcinoma, epithelial malignant tumors, a mixture of malignant tumors from different tissues and pancreas carcinoma.
  • Figure 14 is a histogram showing the over expression of the Stromelysin-3 precursor transcripts, which are detectable by amplicon as depicted in sequence name HSSTROL3 seg24, in cancerous Prostate samples relative to the normal samples.
  • Figure 15 is a histogram demonstrating the expression of Stromelysin-3 transcripts which are detectable by amplicon as depicted in sequence name HSSTROL3 seg24 in different normal tissues.
  • Figure 16 is a histogram showing Cancer and cell- line vs. normal tissue expression for Cluster H53626, demonstrating overexpression in epithelial malignant tumors, a mixture of malignant tumors from different tissues and myosarcoma.
  • Figure 17 is a histogram showing Cancer and cell- line vs. normal tissue expression for Cluster HSMUClA, demonstrating overexpression in a mixture of malignant tumors from different tissues, breast malignant tumors, pancreas carcinoma and prostate cancer.
  • FIG. 18A-B is a histogram showing the relative expression of AA315457 variants in normal, benign and tumor derived prostate samples as determined by real time PCR using primers for SEQ ID NO: 413.
  • Figure 18B is a duplicate experiment.
  • FIG. 19 is a histogram showing the relative expression of Thrombospondin 1 (THBSl) variants in normal, benign and tumor derived prostate samples as determined by real time PCR using primers for SEQ ID NO: 421.
  • THBSl Thrombospondin 1
  • FIG. 20 is a histogram showing the relative expression of Thrombospondin 1 (THBSl) variants in normal, benign and tumor derived prostate samples as determined by real time PCR using primers for SEQ ID NO: 418.
  • THBSl Thrombospondin 1
  • FIG. 21 is a histogram showing the relative expression of transcripts detectable by SEQ ID NOs: 413, 418 and 421 in normal, benign and tumor derived prostate samples as determined by real time PCR.
  • FIG. 22 is a histogram showing the relative expression of DD3/PCA3 variants in normal, benign and tumor derived prostate samples as determined by real time PCR using primers for SEQ ID NO: 475.
  • FIG. 23 is a histogram showing the relative expression of Thrombospondin 1 (THBSl) variants (e.g., variants no. 4, 6, 8, 11, 14, 15, 26, 27, 28, 30) in normal, benign and tumor derived prostate samples as determined by oligonucleotide-based micro-array experiments with SEQ ID NOs: 477, 478, 479, 480, 481, 482. For every oligonucleotide (SEQ ID NOs: 477, 478, 479, 480, 481, 482) the averaged intensity determined for every sample was divided by the averaged intensity of all the normal samples.
  • THBSl Thrombospondin 1
  • the present invention is of novel markers for prostate cancer that are both sensitive and accurate.
  • Biomolecular sequences amino acid and/or nucleic acid sequences
  • uncovered using the methodology of the present invention and described herein can be efficiently utilized as tissue or pathological markers and/or as drugs or drug targets for treating or preventing a disease.
  • These markers are specifically released to the bloodstream under conditions of prostate cancer and/or other prostate pathology, and/or are otherwise expressed at a much higher level and/or specifically expressed in prostate cancer tissue or cells.
  • the measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a probable diagnosis of prostate cancer and/or pathology.
  • the present invention therefore also relates to diagnostic assays for prostate cancer and/or prostate pathology, and methods of use of such markers for detection of prostate cancer and/or prostate pathology, optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
  • the markers of the present invention can be used for prognosis, prediction, screening, early diagnosis, staging, therapy selection and treatment monitoring of prostate cancer.
  • these markers maybe used for staging prostate cancer and/or monitoring the progression of the disease.
  • the markers of the present invention alone or in combination, can be used for detection of the source of metastasis found in anatomical places other then prostate.
  • one or more of the markers may optionally be used in combination with one or more other prostate cancer markers (other than those described herein).
  • Biomolecular sequences (amino acid and/or nucleic acid sequences) uncovered using the methodology of the present invention and described herein can be efficiently utilized as tissue or pathological markers and/or as drugs or drug targets for treating or preventing a disease.
  • These markers are specifically released to the bloodstream under conditions of prostate cancer (or one of the above indicative conditions), and/or are otherwise expressed at a much higher level and/or specifically expressed in prostate cancer tissue or cells, and/or tissue or cells under one of the above indicative conditions.
  • the measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can correlate with a probable diagnosis of prostate cancer and/or a condition that it is indicative of a higher risk for prostate cancer.
  • the present invention therefore also relates to diagnostic assays for prostate cancer and/or an indicative condition, and methods of use of such markers for detection of prostate cancer and/or an indicative condition, optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
  • use of the marker optionally and preferably permits a non-cancerous prostate disease state to be distinguished from prostate cancer and/or an indicative condition.
  • a non limiting example of a non-cancerous prostate disease state includes BPH.
  • use of the marker optionally and preferably permits an indicative condition to be distinguished from prostate cancer.
  • the present invention relates to bridges, tails, heads and/or insertions, and/or analogs, homologs and derivatives of such peptides.
  • bridges, tails, heads and/or insertions are described in greater detail below with regard to the Examples.
  • a "tail” refers to a peptide sequence at the end of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a tail may optionally be considered as a chimera, in that at least a first portion of the splice variant is typically highly homologous (often 100% identical) to a portion of the corresponding known protein, while at least a second portion of the variant comprises the tail.
  • a "head” refers to a peptide sequence at the beginning of an amino acid sequence that is unique to a splice variant according to the present invention. Therefore, a splice variant having such a head may optionally be considered as a chimera, in that at least a first portion of the splice variant comprises the head, while at least a second portion is typically highly homologous (often 100% identical) to a portion of the corresponding known protein.
  • an edge portion refers to a connection between two portions of a splice variant according to the present invention that were not joined in the wild type or known protein.
  • An edge may optionally arise due to a join between the above "known protein” portion of a variant and the tail, for example, and/or may occur if an internal portion of the wild type sequence is no longer present, such that two portions of the sequence are now contiguous in the splice variant that were not contiguous in the known protein.
  • a “bridge” may optionally be an edge portion as described above, but may also include a join between a head and a "known protein” portion of a variant, or a join between a tail and a "known protein” portion of a variant, or a join between an insertion and a "known protein” portion of a variant.
  • a bridge between a tail or a head or a unique insertion, and a "known protein" portion of a variant comprises at least about 10 amino acids, more preferably at least about 20 amino acids, most preferably at least about 30 amino acids, and even more preferably at least about 40 amino acids, in which at least one amino acid is from the tail/head/insertion and at least one amino acid is from the "known protein" portion of a variant.
  • the bridge may comprise any number of amino acids from about 10 to about 40 amino acids (for example, 10, 11, 12, 13...37, 38, 39, 40 amino acids in length, or any number in between). It should be noted that a bridge cannot be extended beyond the length of the sequence in either direction, and it should be assumed that every bridge description is to be read in such manner that the bridge length does not extend beyond the sequence itself.
  • bridges are described with regard to a sliding window in certain contexts below.
  • a bridge portion of CONTIG-N AME_P1 (representing the name of the protein), comprising a polypeptide having a length "n", wherein n is at least about 10 amino acids in length, optionally at least about 20 amino acids in length, preferably at least about 30 amino acids in length, more preferably at least about 40 amino acids in length and most preferably at least about 50 amino acids in length, wherein at least two amino acids comprise XX (2 amino acids in the center of the bridge, one from each end of the edge), having a structure as follows (numbering according to the sequence of CONTIG-N AME_P1): a sequence starting from any of amino acid numbers 49- x to 49 (for example); and ending at any of amino acid numbers 50 + ((n-2) -
  • n is any number of amino acids between 10-50 amino acids in length.
  • the bridge polypeptide cannot extend beyond the sequence, so it should be read such that 49-x (for example) is not less than 1, nor 50 + ((n-2) - x) (for example) greater than the total sequence length.
  • this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention.
  • antibodies differentially recognize splice variants of the present invention but do not recognize a corresponding known protein (such known proteins are discussed with regard to their splice variants in the Examples below).
  • this invention provides an isolated nucleic acid molecule encoding for a splice variant according to the present invention, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto.
  • this invention provides an isolated nucleic acid molecule, having a nucleotide sequence as set forth in any one of the sequences listed herein, or a sequence complementary thereto.
  • this invention provides an oligonucleotide of at least about 12 nucleotides, specifically hybridizable with the nucleic acid molecules of this invention.
  • this invention provides vectors, cells, liposomes and compositions comprising the isolated nucleic acids of this invention.
  • this invention provides a method for detecting a splice variant according to the present invention in a biological sample, comprising: contacting a biological sample with an antibody specifically recognizing a splice variant according to the present invention under conditions whereby the antibody specifically interacts with the splice variant in the biological sample but do not recognize known corresponding proteins (wherein the known protein is discussed with regard to its splice variant(s) in the Examples below), and detecting said interaction; wherein the presence of an interaction correlates with the presence of a splice variant in the biological sample.
  • this invention provides a method for detecting a splice variant nucleic acid sequences in a biological sample, comprising: hybridizing the isolated nucleic acid molecules or oligonucleotide fragments of at least about a minimum length to a nucleic acid material of a biological sample and detecting a hybridization complex; wherein the presence of a hybridization complex correlates with the presence of a splice variant nucleic acid sequence in the biological sample.
  • the splice variants described herein are non-limiting examples of markers for diagnosing prostate cancer and/or prostate pathology. Each splice variant marker of the present invention can be used alone or in combination, for various uses, including but not limited to, prognosis, prediction, screening, early diagnosis, determination of progression, therapy selection and treatment monitoring of prostate cancer and/or prostate pathology.
  • any marker according to the present invention may optionally be used alone or combination.
  • Such a combination may optionally comprise a plurality of markers described herein, optionally including any subcombination of markers, and/or a combination featuring at least one other marker, for example a known marker.
  • such a combination may optionally and preferably be used as described above with regard to determining a ratio between a quantitative or semi-quantitative measurement of any marker described herein to any other marker described herein, and/or any other known marker, and/or any other marker.
  • the known marker comprises the "known protein" as described in greater detail below with regard to each cluster or gene.
  • a splice variant protein or a fragment thereof, or a splice variant nucleic acid sequence or a fragment thereof may be featured as a biomarker for detecting prostate cancer and/or prostate pathology, such that a biomarker may optionally comprise any of the above.
  • the present invention optionally and preferably encompasses any amino acid sequence or fragment thereof encoded by a nucleic acid sequence corresponding to a splice variant protein as described herein.
  • Any oligopeptide or peptide relating to such an amino acid sequence or fragment thereof may optionally also (additionally or alternatively) be used as a biomarker, including but not limited to the unique amino acid sequences of these proteins that are depicted as tails, heads, insertions, edges or bridges.
  • the present invention also optionally encompasses antibodies capable of recognizing, and/or being elicited by, such oligopeptides or peptides.
  • the present invention also optionally and preferably encompasses any nucleic acid sequence or fragment thereof, or amino acid sequence or fragment thereof, corresponding to a splice variant of the present invention as described above, optionally for any application.
  • Non- limiting examples of methods or assays are described below.
  • the present invention also relates to kits based upon such diagnostic methods or assays.
  • Various embodiments of the present invention encompass nucleic acid sequences described hereinabove; fragments thereof, sequences hybridizable therewith, sequences homologous thereto, sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or artificially induced, either randomly or in a targeted fashion.
  • the present invention encompasses nucleic acid sequences described herein; fragments thereof, sequences hybridizable therewith, sequences homologous thereto [e.g., at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 % identical to the nucleic acid sequences set forth below], sequences encoding similar polypeptides with different codon usage, altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion.
  • the present invention also encompasses homologous nucleic acid sequences (i.e., which form a part of a polynucleotide sequence of the present invention) which include sequence regions unique to the polynucleotides of the present invention.
  • the present invention also encompasses novel polypeptides or portions thereof, which are encoded by the isolated polynucleotide and respective nucleic acid fragments thereof described hereinabove.
  • a “nucleic acid fragment” or an “oligonucleotide” or a “polynucleotide” are used herein interchangeably to refer to a polymer of nucleic acids.
  • a polynucleotide sequence of the present invention refers to a single or double stranded nucleic acid sequences which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).
  • complementary polynucleotide sequence refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. Such a sequence can be subsequently amplified in vivo or in vitro using a DNA dependent DNA polymerase.
  • genomic polynucleotide sequence refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.
  • composite polynucleotide sequence refers to a sequence, which is composed of genomic and cDNA sequences.
  • a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing therebetween.
  • the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. Such intronic sequences may further include cis acting expression regulatory elements.
  • Preferred embodiments of the present invention encompass oligonucleotide probes.
  • An example of an oligonucleotide probe which can be utilized by the present invention is a single stranded polynucleotide which includes a sequence complementary to the unique sequence region of any variant according to the present invention, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
  • an oligonucleotide probe of the present invention can be designed to hybridize with a nucleic acid sequence encompassed by any of the above nucleic acid sequences, particularly the portions specified above, including but not limited to a nucleotide sequence coding for an amino sequence of a bridge, tail, head and/or insertion according to the present invention, and/or the equivalent portions of any nucleotide sequence given herein (including but not limited to a nucleotide sequence of a node, segment or amplicon described herein).
  • Oligonucleotides designed according to the teachings of the present invention can be generated according to any oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis.
  • Oligonucleotides used according to this aspect of the present invention are those having a length selected from a range of about 10 to about 200 bases preferably about 15 to about 150 bases, more preferably about 20 to about 100 bases, most preferably about 20 to about 50 bases.
  • the oligonucleotide of the present invention features at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40, bases specifically hybridizable with the biomarkers of the present invention.
  • oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
  • Preferably used oligonucleotides are those modified at one or more of the backbone, internucleoside linkages or bases, as is broadly described hereinunder.
  • oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat.
  • Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and ammoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms can also be used.
  • modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts, as disclosed in U.S. Pat. Nos.
  • oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, Le., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target.
  • An example for such an oligonucleotide mimetic includes peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference.
  • Other backbone modifications, which can be used in the present invention are disclosed in U.S. Pat. No: 6,303,374.
  • Oligonucleotides of the present invention may also include base modifications or substitutions.
  • "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified bases include but are not limited to other synthetic and natural bases such as 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5- halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and
  • Further bases particularly useful for increasing the binding affinity of the oligomeric compounds of the invention include 5-substituted pyrirnidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2 0 C and are presently preferred base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.
  • oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates, which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S- tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium 1,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmity
  • oligonucleotides of the present invention may include further modifications for more efficient use as diagnostic agents and/or to increase bioavailability, therapeutic efficacy and reduce cytotoxicity.
  • a nucleic acid construct according to the present invention may be used, which includes at least a coding region of one of the above nucleic acid sequences, and further includes at least one cis acting regulatory element.
  • cis acting regulatory element refers to a polynucleotide sequence, preferably a promoter, which binds a trans acting regulator and regulates the transcription of a coding sequence located downstream thereto. Any suitable promoter sequence can be used by the nucleic acid construct of the present invention.
  • the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transformed.
  • cell type- specific and/or tissue - specific promoters include promoters such as albumin that is liver specific, lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740], neuron- specific promoters such as the neurofilament promoter [Byrne et al. (1989) Proc. Natl.
  • the nucleic acid construct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom.
  • the nucleic acid construct of the present invention preferably further includes an appropriate selectable marker and/or an origin of replication.
  • the nucleic acid construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in a gene and a tissue of choice.
  • the construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
  • suitable constructs include, but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com).
  • retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif., includingRetro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and the trasgene is transcribed from CMV promoter.
  • Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.
  • nucleic acid transfer techniques include transfection with viral or non- viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno- associated virus (AAV) and lipid-based systems.
  • viral or non- viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno- associated virus (AAV) and lipid-based systems.
  • Useful lipids for lipid- mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Choi [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus -defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • LTRs long terminal repeats
  • such a construct typically includes a signal sequence for secretion of the peptide from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence or the signal sequence of the polypeptide variants of the present invention.
  • the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • a signal that directs polyadenylation will typically include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second- strand DNA synthesis, and a 3' LTR or a portion thereof.
  • Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • Hybridization assays Detection of a nucleic acid of interest in a biological sample may optionally be effected by hybridization-based assays using an oligonucleotide probe (non- limiting examples of probes according to the present invention were previously described).
  • RNA detection Traditional hybridization assays include PCR, RT-PCR, Real-time PCR, RNase protection, in-situ hybridization, primer extension, Southern blots (DNA detection), dot or slot blots (DNA, RNA), and Northern blots (RNA detection) (NAT type assays are described in greater detail below). More recently, PNAs have been described (Nielsen et al. 1999, Current Opin. Biotechnol. 10:71-75). Other detection methods include kits containing probes on a dipstick setup and the like.
  • Hybridization based assays which allow the detection of a variant of interest (i.e., DNA or RNA) in a biological sample rely on the use of oligonucleotides which can be 10, 15, 20, or 30 to 100 nucleotides long preferably from 10 to 50, more preferably from 40 to 50 nucleotides long.
  • the isolated polynucleotides (oligonucleotides) of the present invention are preferably hybridizable with any of the herein described nucleic acid sequences under moderate to stringent hybridization conditions.
  • Moderate to stringent hybridization conditions are characterized by a hybridization solution such as containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 10 ⁇ cpm 32 P labeled probe, at 65 0 C, with a final wash solution of 0.2 x SSC and 0.1 % SDS and final wash at 65 0 C and whereas moderate hybridization is effected using a hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 10 6 cpm 32 P labeled probe, at 65 0 C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 0 C.
  • hybridization of short nucleic acids can be effected using the following exemplary hybridization protocols which can be modified according to the desired stringency;
  • hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected.
  • labels refer to radioactive, fluorescent, biological or enzymatic tags or labels of standard use in the art.
  • a label can be conjugated to either the oligonucleotide probes or the nucleic acids derived from the biological sample.
  • Probes can be labeled according to numerous well known methods.
  • Non- limiting examples of radioactive labels include 3H, 14C, 32P, and 35S.
  • Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies.
  • Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radio- nucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
  • oligonucleotides of the present invention can be labeled subsequent to synthesis, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo- cross- linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin- conjugated streptavidin) or the equivalent.
  • biotinylated dNTPs or rNTP or some similar means (e.g., photo- cross- linking a psoralen derivative of biotin to RNAs)
  • streptavidin e.g., phycoerythrin- conjugated streptavidin
  • fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and others [e.g., Kricka et al. (1992), Academic Press San Diego, Calif] can be attached to the oligonucleotides.
  • wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.
  • standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes. It will be appreciated that a variety of controls may be usefully employed to improve accuracy of hybridization assays. For instance, samples may be hybridized to an irrelevant probe and treated with RNAse A prior to hybridization, to assess false hybridization.
  • Probes can be labeled according to numerous well known methods.
  • radioactive nucleotides can be incorporated into probes of the invention by several methods.
  • Non- limiting examples of radioactive labels include 3 H, 14 C, 32 P, and 35 S.
  • wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate.
  • standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
  • Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and a-nucleotides and the like. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • Detection of a nucleic acid of interest in a biological sample may also optionally be effected by NAT-based assays, which involve nucleic acid amplification technology, such as PCR for example (or variations thereof such as real-time PCR for example).
  • nucleic acid amplification technology such as PCR for example (or variations thereof such as real-time PCR for example).
  • a "primer” defines an oligonucleotide which is capable of annealing to (hybridizing with) a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.
  • Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14 Numerous amplification techniques have been described and can be readily adapted to suit particular needs of a person of ordinary skill. Non- limiting examples of amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the q3 replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci.
  • amplification pair refers herein to a pair of oligonucleotides (oligos) of the present invention, which are selected to be used together in amplifying a selected nucleic acid sequence by one of a number of types of amplification processes, preferably a polymerase chain reaction.
  • amplification processes include ligase chain reaction, strand displacement amplification, or nucleic acid sequence-based amplification, as explained in greater detail below.
  • oligos are designed to bind to a complementary sequence under selected conditions.
  • amplification of a nucleic acid sample from a patient is amplified under conditions which favor the amplification of the most abundant differentially expressed nucleic acid.
  • RT-PCR is carried out on an mRNA sample from a patient under conditions which favor the amplification of the most abundant mRNA.
  • the amplification of the differentially expressed nucleic acids is carried out simultaneously. It will be realized by a person skilled in the art that such methods could be adapted for the detection of differentially expressed proteins instead of differentially expressed nucleic acid sequences.
  • the nucleic acid i.e. DNA or RNA
  • the nucleic acid may be obtained according to well known methods.
  • Oligonucleotide primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed.
  • the oligonucleotide primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system.
  • the oligonucleotide primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (Sambrook et al., 1989, Molecular Cloning -A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology,
  • antisense oligonucleotides may be employed to quantify expression of a splice isoform of interest. Such detection is effected at the pre-mRNA level.
  • oligonucleotides may compete with splicing factors for the splice site sequences. Thus, low activity of the antisense oligonucleotide is indicative of splicing activity.
  • the polymerase chain reaction and other nucleic acid amplification reactions are well known in the art (various non- limiting examples of these reactions are described in greater detail below).
  • the pair of oligonucleotides according to this aspect of the present invention are preferably selected to have compatible melting temperatures (Tm), e.g., melting temperatures which differ by less than that 7 0 C, preferably fess than 5 0 C, more preferably less than 4 0 C, most preferably less than 3 0 C, ideally between 3 0 C and 0 0 C.
  • Tm melting temperatures
  • PCR Polymerase Chain Reaction
  • PCR The polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis and Mullis et ah, is a method of increasing the concentration of a segment of target sequence in a mixture of genomic DNA without cloning or purification.
  • This technology provides one approach to the problems of low target sequence concentration.
  • PCR can be used to directly increase the concentration of the target to an easily detectable level.
  • This process for amplifying the target sequence involves the introduction of a molar excess of two oligonucleotide primers which are complementary to their respective strands of the double- stranded target sequence to the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize.
  • the primers are extended with polymerase so as to form complementary strands.
  • the steps of denaturation, hybridization (annealing), and polymerase extension (elongation) can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence.
  • the length of the segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and, therefore, this length is a controllable parameter. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are said to be "PCR-amplified.”
  • LCR Ligase Chain Reaction
  • LAR Ligase Amplification Reaction
  • LCR 5 two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, and ligation amplify a short segment of DNA. LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes: see for example Segev, PCT Publication No. W09001069 Al (1990). However, because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target- independent background signal. The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
  • the self- sustained sequence replication reaction (3SR) is a transcription-based in vitro amplification system that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection. In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5 1 end of the sequence of interest.
  • the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second- strand synthesis to amplify the area of interest.
  • 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs).
  • Q-Beta (Q ⁇ ) Replicase In this method, a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Q ⁇ replicase.
  • a previously identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence- specific ligation step.
  • available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37 degrees C). This prevents the use of high temperature as a means of achieving specificity as in the LCR, the ligation event can be used to detect a mutation at the junction site, but not elsewhere.
  • a successful diagnostic method must be very specific.
  • a straight-forward method of controlling the specificity of nucleic acid hybridization is by controlling the temperature of the reaction. While the 3SR/NASBA, and Q ⁇ systems are all able to generate a large quantity of signal, one or more of the enzymes involved in each cannot be used at high temperature (i.e., > 55 degrees C). Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes. If probes are shortened in order to make them melt more easily at low temperatures, the likelihood of having more than one perfect match in a complex genome increases. For these reasons, PCR and LCR currently dominate the research field in detection technologies.
  • PCR has yet to penetrate the clinical market in a significant way.
  • LCR LCR must also be optimized to use different oligonucleotide sequences for each target sequence.
  • both methods require expensive equipment, capable of precise temperature cycling.
  • nucleic acid detection technologies such as in studies of allelic variation, involve not only detection of a specific sequence in a complex background, but also the discrimination between sequences with few, or single, nucleotide differences.
  • One method of the detection of allele-specific variants by PCR is based upon the fact that it is difficult for Taq polymerase to synthesize a DNA strand when there is a mismatch between the template strand and the 3' end of the primer.
  • An allele-specific variant may be detected by the use of a primer that is perfectly matched with only one of the possible alleles; the mismatch to the other allele acts to prevent the extension of the primer, thereby preventing the amplification of that sequence.
  • This method has a substantial limitation in that the base composition of the mismatch influences the ability to prevent extension across the mismatch, and certain mismatches do not prevent extension or have only a minimal effect.
  • thermostable ligase A similar 3'-mismatch strategy is used with greater effect to prevent ligation in the LCR. Any mismatch effectively blocks the action of the thermostable ligase, but LCR still has the drawback of target-independent background ligation products initiating the amplification. Moreover, the combination of PCR with subsequent LCR to identify the nucleotides at individual positions is also a clearly cumbersome proposition for the clinical laboratory.
  • the direct detection method may be, for example a cycling probe reaction (CPR) or a branched DNA analysis.
  • CPR cycling probe reaction
  • CPR Cycling probe reaction
  • the cycling probe reaction (CPR) uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini are made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate. While the repeating process increases the signal, the RNA portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.
  • Branched DNA involves oligonucleotides with branched structures that allow each individual oligonucleotide to carry 35 to 40 labels (e.g., alkaline phosphatase enzymes). While this enhances the signal from a hybridization event, signal from non-specific binding is similarly increased.
  • labels e.g., alkaline phosphatase enzymes
  • the detection of at least one sequence change may be accomplished by, for example restriction fragment length polymorphism (RFLP analysis), allele specific oligonucleotide (ASO) analysis,
  • RFLP analysis restriction fragment length polymorphism
  • ASO allele specific oligonucleotide
  • SSCP Conformation Polymorphism
  • ddF Dideoxy fingerprinting
  • nucleic acid segments for mutations.
  • One option is to determine the entire gene sequence of each test sample (e.g., a bacterial isolate). For sequences under approximately 600 nucleotides, this may be accomplished using amplified material (e.g., PCR reaction products). This avoids the time and expense associated with cloning the segment of interest.
  • amplified material e.g., PCR reaction products
  • a given segment of nucleic acid may be characterized on several other levels.
  • the size of the molecule can be determined by electrophoresis by comparison to a known standard run on the same gel.
  • a more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis, to allow construction of an ordered map.
  • the presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain- terminating nucleotide analogs.
  • Restriction fragment length polymorphism For detection of single-base differences between like sequences, the requirements of the analysis are often at the highest level of resolution. For cases in which the position of the nucleotide in question is known in advance, several methods have been developed for examining single base changes without direct sequencing. For example, if a mutation of interest happens to fall within a restriction recognition sequence, a change in the pattern of digestion can be used as a diagnostic tool (e.g., restriction fragment length polymorphism [RFLP] analysis).
  • RFLP restriction fragment length polymorphism
  • MCC Mismatch Chemical Cleavage
  • RFLP analysis is used for tfie detection of point mutations, it is, by its nature, limited to the detection of only those single base changes which fall within a restriction sequence of a known restriction endonuclease. Moreover, the majority of the available enzymes have 4 to 6 base-pair recognition sequences, and cleave too frequently for many large-scale DNA manipulations. Thus, it is applicable only in a small fraction of cases, as most mutations do not fall within such sites.
  • Allele specific oligonucleotide can be designed to hybridize in proximity to the mutated nucleotide, such that a primer extension or ligation event can bused as the indicator of a match or a mis- match.
  • Hybridization with radioactively labeled allelic specific oligonucleotides also has been applied to the detection of specific point mutations. The method is based on the differences in the melting temperature of short DNA fragments differing by a single nucleotide. Stringent hybridization and washing conditions can differentiate between mutant and wild-type alleles.
  • the ASO approach applied to PCR products also has been extensively utilized by various researchers to detect and characterize point mutations in ras genes and gsp/gip oncogenes. Because of the presence of various nucleotide changes in multiple positions, the ASO method requires the use of many oligonucleotides to cover all possible oncogenic mutations.
  • the precise location of the suspected mutation must be known in advance of the test. That is to say, they are inapplicable when one needs to detect the presence of a mutation within a gene or sequence of interest.
  • DGGE/TGGE Denaturing/Temperature Gradient Gel Electrophoresis
  • the fragments to be analyzed are "clamped" at one end by a long stretch of GC base pairs (30-80) to allow complete denaturation of the sequence of interest without complete dissociation of the strands.
  • the attachment of a GC "clamp" to the DNA fragments increases the fraction of mutations that can be recognized by DGGE. Attaching a GC clamp to one primer B critical to ensure that the amplified sequence has a low dissociation temperature. Modifications of the technique have been developed, using temperature gradients, and the method can be also applied to RNA-.RNA duplexes. Limitations on the utility of DGGE include the requirement that the denaturing conditions must be optimized for each type of DNA to be tested.
  • DGGE constant denaturant gel electrophoresis
  • TGGE uses a thermal gradient rather than a chemical denaturant gradient.
  • TGGE requires the use of specialized equipment which can generate a temperature gradient perpendicularly oriented relative to the electrical field.
  • TGGE can detect mutations in relatively small fragments of DNA therefore scanning of large gene segments requires the use of multiple PCR products prior to running the gel.
  • Single-Strand Conformation Polymorphism (SSCP): Another common method, called “Single-Strand Conformation Polymorphism” (SSCP) was developed by Hayashi, Sekya and colleagues and is based on the observation that single strands of nucleic acid can take on characteristic conformations in non-denaturing conditions, and these conformations influence electrophoretic mobility. The complementary strands assume sufficiently different structures that one strand may be resolved from the other. Changes in sequences within the fragment will also change the conformation, consequently altering the mobility and allowing this to be used as an assay for sequence variations.
  • SSCP Single-Strand Conformation Polymorphism
  • the SSCP process involves denaturing a DNA segment (e.g., a PCR product) that is labeled on both strands, followed by slow electrophoretic separation on a non-denaturing polyacrylamide gel, so that intra- molecular interactions can form and not be disturbed during the run.
  • This technique is extremely sensitive to variations in gel composition and temperature. A serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.
  • Dideoxy fingerprinting (ddF) The dideoxy fmge ⁇ rinting (ddF) is another technique developed to scan genes for the presence of mutations. The ddF technique combines components of Sanger dideoxy sequencing with SSCP.
  • a dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction products are electrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis. While ddF is an improvement over SSCP in terms of increased sensitivity, ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of the size suitable for SSCP (i.e., fragments of 200-300 bases for optimal detection of mutations).
  • the step of searching for any of the nucleic acid sequences described here, in tumor cells or in cells derived from a cancer patient is effected by any suitable technique, including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self- sustained synthetic reaction, Q ⁇ -Replicase, cycling probe reaction, branched DNA, restriction fragment length polymorphism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonucleotides, denaturing gradient gel electrophoresis, constant denaturant gel electrophoresis, temperature gradient gel electrophoresis and dideoxy finge ⁇ rinting.
  • any suitable technique including, but not limited to, nucleic acid sequencing, polymerase chain reaction, ligase chain reaction, self- sustained synthetic reaction, Q ⁇ -Replicase, cycling probe reaction, branched DNA, restriction fragment length polymorphism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonucleotides, denaturing
  • Detection may also optionally be performed with a chip or other such device.
  • the nucleic acid sample which includes the candidate region to be analyzed is preferably isolated, amplified and labeled with a reporter group.
  • This reporter group can be a fluorescent group such as phycoerythrin.
  • the labeled nucleic acid is then incubated with the probes immobilized on the chip using a fluidics station, describe the fabrication of fluidics devices and particularly microcapillary devices, in silicon and glass substrates.
  • the chip is inserted into a scanner and patterns of hybridization are detected.
  • the hybridization data is collected, as a signal emitted from the reporter groups already incorporated into the nucleic acid, which is now bound to the probes attached to the chip. Since the sequence and position of each probe immobilized on the chip is known, the identity of the nucleic acid hybridized to a given probe can be determined.
  • polypeptide refers to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
  • polypeptide include glycoproteins, as well as non-glycoproteins.
  • Polypeptide products can be biochemically synthesized such as by employing standard solid phase techniques. Such methods include but are not limited to exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry. Solid phase polypeptide synthesis procedures are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).
  • Synthetic polypeptides can optionally be purified by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. WH Freeman and Co. N.Y.], after which their composition can be confirmed via amino acid sequencing.
  • a polypeptide can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in En2ymol. 153:516- 544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511- 514, Takamatsu et al. (1987) EMBO J.
  • the present invention also encompasses polypeptides encoded by the polynucleotide sequences of the present invention, as well as polypeptides according to the amino acid sequences described herein.
  • the present invention also encompasses homologues of these polypeptides, such homologues can be at least 50 %, at least 55 %, at least 60%, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 95 % or more say 100 % homologous to the amino acid sequences set forth below, as can be determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters, optionally and preferably including the following: filtering on (this option filters repetitive or low- complexity sequences from the query using the Seg (protein) program), scoring matrix is BLOSUM62 for proteins, word size is 3, E value is 10, gap costs are 11, 1 (initialization and extension), and number of alignments shown is 50.
  • NCBI National Center of Biotechnology Information
  • nucleic acid sequence identity/homology is determined with BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters, which preferably include using the DUST filter program, and also preferably include having an E value of 10, filtering low complexity sequences and a word size of 11.
  • NCBI National Center of Biotechnology Information
  • the present invention also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or artificially induced, either randomly or in a targeted fashion.
  • peptides identified according the present invention may be degradation products, synthetic peptides or recombinant peptides as well as peptidomimetics, typically, synthetic peptides and peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
  • Natural aromatic amino acids, Trp, Tyr and Phe may be substituted for synthetic non- natural acid such as Phenylglycine, TIC, naphthylelanine (NoI), ring- methylated derivatives of Phe, halogenated derivatives of Phe or o- methyl- Tyr.
  • synthetic non- natural acid such as Phenylglycine, TIC, naphthylelanine (NoI), ring- methylated derivatives of Phe, halogenated derivatives of Phe or o- methyl- Tyr.
  • the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids. Table 1 non-conventional or modified amino acids which can be used with the present invention. Table 1
  • the peptides of the present invention are preferably utilized in diagnostics which require the peptides to be in soluble form, the peptides of the present invention preferably include one or more non-natural or ratural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl- containing side chain.
  • the peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclicization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
  • the peptides of present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis well known in the art, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. These methods are preferably used when the peptide is relatively short (i.e., 10 kDa) and/or when it cannot be produced by recombinant techniques (i.e., not encoded by a nucleic acid sequence) and therefore involves different chemistry.
  • Synthetic peptides can be purified by preparative high performance liquid chromatography and the composition of which can be confirmed via amino acid sequencing.
  • the peptides of the present invention can be generated using recombinant techniques such as described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990)
  • Antibodies refers to a polypeptide ligand that is preferably substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen).
  • the recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad- immunoglobulin variable region genes.
  • Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab' and F(ab)' 2 fragments.
  • antibody also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CHl, CH2 and CH3, but does not include the heavy chain variable region.
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule
  • Fab' the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain
  • two Fab 1 fragments are obtained per antibody molecule
  • (Fab')2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • SCA Single chain antibody
  • Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (1972O].
  • the variable chains can be linked by an intermolecular disulfide bond or cross- linked by chemicals such as glutaraldehyde.
  • the Fv fragments comprise VH and VL chains connected by a peptide linker.
  • These single- chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • CDR peptides ('minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].
  • 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') 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 from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of 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); andPresta, 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 Whiter 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. Pat. 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. MoI. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boemer et al.
  • human antibodies can be made by introduction 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. Pat. Nos.
  • the antibody of this aspect of the present invention specifically binds at least one epitope of the polypeptide variants of the present invention.
  • epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • a unique epitope may be created in a variant due to a change in one or more post-translational modifications, including but not limited to glycosylation and/or phosphorylation, as described below. Such a change may also cause a new epitope to be created, for example through removal of glycosylation at a particular site.
  • An epitope according to the present invention may also optionally comprise part or all of a unique sequence portion of a variant according to the present invention in combination with at least one other portion of the variant which is not contiguous to the unique sequence portion in the linear polypeptide itself, yet which are able to form an epitope in combination.
  • One or more unique sequence portions may optionally combine with one or more other non-contiguous portions of the variant (including a portion which may have high homology to a portion of the known protein) to form an epitope.
  • an immunoassay can be used to qualitatively or quantitatively detect and analyze markers in a sample.
  • This method comprises: providing an antibody that specifically binds to a marker; contacting a sample with the antibody; and detecting the presence of a complex of the antibody bound to the marker in the sample.
  • Antibodies that specifically bind to a protein marker can be prepared using any suitable methods known in the art. After the antibody is provided, a marker can be detected and/or quantified using any of a number of well recognized immunological binding assays.
  • Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme- linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).
  • EIA enzyme immune assay
  • ELISA enzyme- linked immunosorbent assay
  • RIA radioimmune assay
  • Western blot assay e.g., Western blot assay
  • slot blot assay see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,5
  • the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample.
  • solid supports include but are not limited to glass or plastic in the form of, e.g., a microliter plate, a stick, a bead, or a microbead.
  • Antibodies can also be attached to a solid support. After incubating the sample with antibodies, the mixture is washed and the antibody- marker complex formed can be detected. This can be accomplished by incubating the washed mixture with a detection reagent.
  • the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
  • an indirect assay wherein, for example, a second, labeled antibody is used to detect bound marker-specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker are incubated simultaneously with the mixture.
  • incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, marker, volume of solution, concentrations and the like. Usually the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10 0 C to 40 0 C.
  • the immunoassay can be used to determine a test amount of a marker in a sample from a subject.
  • a test amount of a marker in a sample can be detected using the immunoassay methods described above. If a marker is present in the sample, it will form an antibody- marker complex with an antibody that specifically binds the marker under suitable incubation conditions described above.
  • the amount of an antibody- marker complex can optionally be determined by comparing to a standard.
  • the test amount of marker need not be measured in absolute units, as long as the unit of measurement can be compared to a control amount and/or signal.
  • antibodies which specifically interact with the polypeptides of the present invention and not with wild type proteins or other isoforms thereof, for example.
  • Such antibodies are directed, for example, to the unique sequence portions of the polypeptide variants of the present invention, including but not limited to bridges, heads, tails and insertions described in greater detail below.
  • Preferred embodiments of antibodies according to the present invention are described in greater detail with regard to the section entitled "Antibodies”.
  • Radioimmunoassay In one version, this method involves precipitation of the desired substrate and in the methods detailed hereinbelow, with a specific antibody and
  • J25 radiolabeled antibody binding protein e.g., protein A labeled with 1
  • a precipitable carrier such as agarose beads.
  • the number of counts in the precipitated pellet is proportional to the amount of substrate.
  • a labeled substrate and an unlabelled antibody binding protein are employed.
  • a sample containing an unknown amount of substrate is added in varying amounts.
  • the decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
  • Enzyme linked immunosorbent assay This method involves fixation of a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate to a surface such as a well of a microtiter plate. A substrate specific antibody coupled to an enzyme is applied and allowed to bind to the substrate. Presence of the antibody is then detected and quantitated by a colorimetric reaction employing the enzyme coupled to the antibody. Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy.
  • Western blot This method involves separation of a substrate from other protein by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nylon or PVDF).
  • a membrane e.g., nylon or PVDF
  • Antibody binding reagents may be, for example, protein
  • Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
  • Immunohistochemical analysis This method involves detection of a substrate in situ in fixed cells by substrate specific antibodies.
  • the substrate specific antibodies may be enzyme linked or linked to fluorophores. Detection is by r ⁇ croscopy and subjective evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required.
  • Fluorescence activated cell sorting This method involves detection of a substrate in situ in cells by substrate specific antibodies.
  • the substrate specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • 6,696,686 describes the use of SPECT for detection of breast cancer, and is hereby incorporated by reference as if fully set forth herein.
  • Display Libraries According to still another aspect of the present invention there is provided a display library comprising a plurality of display vehicles (such as phages, viruses or bacteria) each displaying at least 6, at least 7, at least 8, at least 9, at least 10, 10-15, 12-17, 15-20, 15-30 or 20- 50 consecutive amino acids derived from the polypeptide sequences of the present invention.
  • display vehicles such as phages, viruses or bacteria
  • This Section relates to Examples of sequences according to the present invention, including illustrative methods of selection thereof.
  • GenBank sequences the human EST sequences from the EST (GBEST) section and the human mRNA sequences from the primate (GBPRI) section were used; also the human nucleotide RefSeq mRNA sequences were used (see for example www.ncbi.nlm.nih.gov/Genbank/GenbankOverview.html and for a reference to the EST section, see www.ncbi.nhn.nih.gov/dbEST/; a general reference to dbEST, the EST database in
  • GenBank may be found in Boguski et al, Nat Genet. 1993 Aug;4(4):332-3; all of which are hereby incorporated by reference as if fully set forth herein).
  • Novel splice variants were predicted using the LEADS clustering and assembly system as described in Sorek, R., Ast, G. & Graur, D. Alu-containing exons are alternatively spliced. Genome Res 12, 1060-7 (2002); US patent No: 6,625,545; and U.S. Pat. Appl. No. 10/426,002, published as US20040101876 on May 27 2004; all of which are hereby incorporated by reference as if fully set forth herein. Briefly, the software cleans the expressed sequences from repeats, vectors and immunoglobulins. It then aligns the expressed sequences to the genome taking alternatively splicing into account and clusters overlapping expressed sequences into "clusters" that represent genes or partial genes.
  • the GeneCarta platform includes a rich pool of annotations, sequence information (particularly of spliced sequences), chromosomal information, alignments, and additional information such as SNPs, gene ontology terms, expression profiles, functional analyses, detailed domain structures, known and predicted proteins and detailed homology reports.
  • Biological source examples include cancer cell- lines; normal tissues; cancer tissues; fetal tissues; and others such as normal cell lines and pools of normal cell- lines, cancer cell- lines and combinations thereof. A specific description of abbreviations used below with regard to these tissues/cell lines etc is given above.
  • Protocol of library construction various methods are known in the art for library construction including normalized library construction; non-normalized library construction; subtracted libraries; ORESTES and others. It will be appreciated that at times the protocol of library construction is not indicated in GenBank and/or other library annotaion. The following rules are followed:
  • Clusters having at least five sequences including at least two sequences from the tissue of interest were analyzed. Splice variants were identified by using the LEADS software package as described above.
  • the basic algorithm - for each cluster the number of cancer and normal libraries contributing sequences to the cluster was counted. Fisher exact test was used to check if cancer libraries are significantly over-represented in the cluster as compared to the total number of cancer and normal libraries.
  • Clones no. score - Generally, when the number of ESTs is much higher in the cancer libraries relative to the normal libraries it might indicate actual over-expression.
  • Clones number score The total weighted number of EST clones from cancer libraries was compared to the EST clones from normal libraries. To avoid cases where one library contributes to the majority of the score, the contribution of the library that gives most clones for a given cluster was limited to 2 clones. The score was computed as
  • Clones number score significance - Fisher exact test was used to check if EST clones from cancer libraries are significantly over-represented in the cluster as compared to the total number of EST clones from cancer and normal libraries. Two search approaches were used to find either general cancer- specific candidates or tumor specific candidates.
  • tissue libraries/sequences were compared to the total number of libraries/sequences in cluster. Similar statistical tools to those described in above were employed to identify tissue specific genes. Tissue abbreviations are the same as for cancerous tissues, but are indicated with the header "normal tissue”.
  • Each cluster includes at least 2 libraries from the tissue T. At least 3 clones (weighed - as described above) from tissue T in the cluster; and
  • Clones from the tissue T are at least 40 % from all the clones participating in the tested cluster Fisher exact test P- values were computed both for library and weighted clone counts to check that the counts are statistically significant.
  • Cancer-specific splice variants containing a unique region were identified.
  • a Region is defined as a group of adjacent exons that always appear or do not appear together in each splice variant.
  • a “segment” (sometimes referred also as “seg” or “node”) is defined as the shortest contiguous transcribed region without known splicing inside.
  • Each unique sequence region divides the set of transcripts into 2 groups: (i) Transcripts containing this region (group TA).
  • Region 1 common to all transcripts, thus it is not considered; Region 2: specific to Transcript 1: T_l unique regions (2+6) against T_2+3 unique regions (3+4); Region 3: specific to Transcripts 2+3: T_2+3 unique regions (3+4) against Tl unique regions (2+6); Region 4: specific to Transcript 3: T_3 unique regions (4) against Tl+2 unique regions (2+5+6); Region 5: specific to Transcript 1+2: T_l+2 unique regions (2+5+6) against T3 unique regions (4); Region 6: specific to Transcript 1: same as region 2.
  • Reliable EST supported-regions were defined as supported by minimum of one of the following:
  • This Section relates to Examples describing experiments involving these sequences, and illustrative, non- limiting examples of methods, assays and uses thereof. The materials and experimental procedures are explained first, as all experiments used them as a basis for the work that was performed.
  • the markers of the present invention were tested with regard to their expression in various cancerous and non-cancerous tissue samples.
  • a description of the samples used in the panel is provided in Table 2 below.
  • a description of the samples used in the normal tissue panel is provided in Table 3 below. Tests were then performed as described in the "Materials and
  • RNA preparation - RNA was obtained from Clontech (Franklin Lakes, NJ USA 07417, www.clontech.com), BioChain Inst. Inc. (Hayward, CA 94545 USA www.biochain.com), ABS (Wilmington, DE 19801, USA, http://www.absbioreagents.com) or Ambion (Austin, TX 78744 USA, http://www.ambion.com).
  • RNA was generated from tissue samples using TRI-Reagent (Molecular Research Center), according to Manufacturer's instructions. Tissue and RNA samples were obtained from patients or from postmortem. Total RNA samples were treated with DNaseI (Ambion) and purified using RNeasy columns (Qiagen).
  • RT PCR - Purified RNA (1 ⁇ g) was mixed with 150 ng Random Hexamer primers (Invitrogen) and 500 ⁇ M dNTP in a total volume of 15.6 ⁇ l. The mixture was incubated for 5 min at 65 °C and then quickly chilled on ice. Thereafter, 5 ⁇ l of 5X Superscript!! first strand buffer (Invitrogen), 2.4 ⁇ l 0.1M DTT and 40 units RNasin (Promega) were added, and the mixture was incubated for 10 min at 25 0 C, followed by further incubation at 42 0 C for 2 min. Then, 1 ⁇ l (200units) of Superscript]!
  • Real-Time RT-PCR analysis- cDNA (5 ⁇ l), prepared as described above, was used as a template in Real- Time PCR reactions using the SYBR Green I assay (PE Applied Biosystem) with specific primers and UNG Enzyme (Eurogentech or ABI or Roche).
  • the amplification was effected as follows: 50 0 C for 2 min, 95 0 C for 10 min, and then 40 cycles of 95 0 C for 15sec, followed by 60 0 C for 1 min.
  • Detection was performed by using the PE Applied Biosystem SDS 7000. The cycle in which the reactions achieved a threshold level (Ct) of fluorescence was registered and was used to calculate the relative transcript quantity in the RT reactions.
  • Ct threshold level
  • the efficiency of the PCR reaction was calculated from a standard curve, created by using serial dilutions of several reverse transcription (RT) reactions. To minimize inherent differences in the RT reaction, the resulting relative quantities were normalized to the geometric mean of the relative quantities of several housekeeping (HSKP) genes.
  • RT reverse transcription
  • HSKP housekeeping
  • SDHA GenBank Accession No. NM_004168
  • SDHA Forward primer TGGGAACAAGAGGGCATCTG
  • SDHA Reverse primer CCACCACTGCATCAAATTCATG SDHA-amplicon :
  • PBGD GenBank Accession No. BC019323
  • PBGD Forward primer TGAGAGTGATTCGCGTGGG
  • HPRTl -amplicon TGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAA
  • RPLl 9 (GenBank Accession No. NM_000981
  • RPL19Forward primer TGGCAAGAAGAAGGTCTGGTTAG
  • RPL19Reverse primer TGATCAGCCCATCTTTGATGAG
  • RPL19 Forward primer TGGCAAGAAGAAGGTCTGGTTAG
  • RPLl 9 Reverse primer: TGATCAGCCCATCTTTGATGAG RPLl 9 -amplicon:
  • SDHA (GenBank Accession No. NM_004168) SDHA Forward primer:
  • SDHA Reverse primer CCACCACTGCATCAAATTCATG SDHA-amplicon :
  • the designed oligonucleotides were synthesized and purified by desalting with the Sigma-Genosys system (The Woodlands, TX, US) and all of the oligonucleotides were joined to a C6 amino- modified linker at the 5' end, or being attached directly to CodeLmk slides (Cat #25-6700-01. Amersham Bioscience, Piscataway, NJ, US).
  • the 50-mer oligonucleotides, forming the target sequences were first suspended in Ultra-pure DDW (Cat # 01-866- IA Kibbutz Beit-Haemek, Israel) to a concentration of 50 ⁇ M. Before printing the slides, the oligonucleotides were resuspended in 30OmM sodium phosphate (pH 8.5) to final concentration of 15OmM and printed at 35-40% relative humidity at 21 0 C.
  • Each slide contained a total of 9792 features in 32 subarrays. Of these features, 4224 features were sequences of interest according to the present invention and negative controls that were printed in duplicate. An additional 288 features (96 target sequences printed in triplicate) contained housekeeping genes from Human Evaluation Library2, Compugen Ltd, Israel. Another 384 features are E.coli spikes 1-6, which are oligos to E-CoIi genes which are commercially available in the Array Control product (Array control- sense oligo spots, Ambion Inc. Austin, TX. Cat #1781, Lot #112K06).
  • Slides were treated for blocking of the residual reactive groups by incubating them in blocking solution at 5O 0 C for 15 minutes (lOml/slide of buffer containing 0.1M Tris, 5OmM ethanolamine, 0.1% SDS). The slides were then rinsed twice with Ultra-pure DDW (double distilled water). The slides were then washed with wash solution (10ml/slide. 4X SSC, 0.1% SDS)) at 50 0 C for 30 minutes on the shaker. The slides were then rinsed twice with Ultra-pure DDW, followed by drying by centrifugation for 3 minutes at 800 rpm. Next, in order to assist in automatic operation of the hybridization protocol, the slides were treated with Ventana Discovery hybridization station barcode adhesives.
  • the printed slides were loaded on a Bio-Optica (Milan, Italy) hematology staining device and were incubated for 10 minutes in 50ml of 3-Aminopropyl Triethoxysilane (Sigma A3648 lot #122K589). Excess fluid was dried and slides were then incubated for three hours in 20 rnm/Hg in a dark vacuum desiccator (Pelco 2251, Ted Pella, Inc. Redding CA).
  • the following protocol was then followed with the Genisphere 900-RP (random primer), with mini elute columns on the Ventana Discovery HybStationTM, to perform the microarray experiments. Briefly, the protocol was performed as described with regard to the instructions and information provided with the device itself. The protocol included cDNA synthesis and labeling. cDNA concentration was measured with the TBS-380 (Turner Biosystems. Sunnyvale, CA.) PicoFlour, which is used with the OliGreen ssDNA Quantitation reagent and kit.
  • Hybridization was performed with the Ventana Hybridization device, according to the provided protocols (Discovery Hybridization Station Tuscon AZ).
  • DNA oligonucleotides at 25uM were deposited (printed) onto Amersham 'CodeLink' glass slides generating a well defined 'spot'. These slides are covered with a long-chain, hydrophilic polymer chemistry that creates an active 3-D surface that covalently binds the DNA oligonucleotides 5 '-end via the
  • FIG. 5 shows a schematic method for performing the microarray experiments. It should be noted that stages on the left-hand or right-hand side may optionally be performed in any order, including in parallel, until stage 4 (hybridization). Briefly, on the left-hand side, the target oligonucleotides are being spotted on a glass microscope slide (although optionally other materials could be used) to form a spotted slide (stage 1). On the right hand side, control sample RNA and cancer sample RNA are Cy3 and Cy5 labeled, respectively (stage 2), to form labeled probes. It should be noted that the control and cancer samples come from corresponding tissues (for example, normal prostate tissue and cancerous prostate tissue).
  • the tissue from which the RNA was taken is indicated below in the specific examples of data for particular clusters, with regard to overexpression of an oligonucleotide from a "chip” (microarray), as for example "prostate” for chips in which prostate cancerous tissue and normal tissue were tested as described above.
  • the probes are mixed.
  • hybridization is performed to form a processed slide.
  • stage 5 the slide is washed and scanned to form an image file, followed by data analysis in stage 6.
  • Cluster HSECADH features 4 transcript(s) and 30 segment(s) of interest, the names for which are given in Tables 4 and 5, respectively, the sequences themselves are given at the end of the application.
  • the selected protein variants are given in table 6.
  • sequences are variants of the known protein Epithelial- cadherin precursor (SwissProt accession identifier CAD1_HUMAN; known also according to the synonyms E- cadherin; Uvomorulin; Cadherin- 1; CAM 120/80), SEQ ID NO: 384, referred to herein as the previously known protein.
  • the variant proteins according to the present invention are variants of a known diagnostic marker, called E- Cadherin.
  • Cadherins are calcium dependent cell adhesion proteins. They preferentially interact with themselves in a homophilic manner in connecting cells; cadherins may thus contribute to the sorting of heterogeneous cell types.
  • E-cadherin has a potent invasive suppressor role. It is also a ligand for integrin alpha- E/beta-7.
  • the sequence for protein Epithelial-cadherin precursor is given at the end of the application, as "Epithelial-cadherin precursor amino acid sequence". Known polymorphisms for this sequence are as shown in Table 7.
  • Protein Epithelial- cadherin localization is believed to be Type I membrane protein.
  • the GO assignment relies on information from one or more of the SwissProt/TremBl Protein knowledgebase, available from ⁇ http://www.expasy.ch/sprot/>; or Locuslink, available from ⁇ http://www.ncbi.nhn.nih.gov/projects/LocusLink/>.
  • Cluster HSECADH can be used as a diagnostic marker according to overexpression of transcripts of this cluster in cancer. Expression of such transcripts in normal tissues is also given according to the previously described methods.
  • the term "number" in the right hand column of the table and the numbers on the y-axis of Figure 6 refer to weighted expression of ESTs in each category, as "parts per million” (ratio of the expression of ESTs for a particular cluster to the expression of all ESTs in that category, according to parts per million).
  • cluster HSECADH features 4 transcript(s), which were listed in Table 4 above. These transcript(s) encode for protein(s) which are variant(s) of protein Epithelial- cadherin precursor. A description of each variant protein according to the present invention is now provided.
  • Variant protein HSECADHJP9 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HSECADH_T11. An alignment is given to the known protein (Epithelial-cadherin precursor) at the end of the application. One or more alignments to one or more previously published protein sequences are given at the end of the application. A brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • Comparison report between HSECADH_P9 and Q9UII7 (SEQ ID NO:483): 1.An isolated chimeric polypeptide encoding for HSECADH_P9, comprising a first amino acid sequence being at least 90 % homologous to
  • polypeptide encoding for a tail of HSECADH P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence TACRSRIANSCHSGDSWRNSCFANSDSAALAVSSEESGGQRALTAPRG in
  • GNAVEDPMEILITVTDQNDNKPEFTQEVFKGSVMEG corresponding to amino acids 1 - 274 of Q9UII8, which also corresponds to amino acids 1 - 274 of HSECADH P9, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having
  • polypeptide encoding for a tail of HSECADH_P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably
  • GNAVEDPMEILITVTDQNDNKPEFTQEVFKGSVMEG corresponding to amino acids 1 - 274 of CAD IJHUMAN, which also corresponds to amino acids 1 - 274 of HSECADH_P9, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably at least 90% and most preferably at least 95% homologous to a polypeptide having the sequence
  • polypeptide encoding for a tail of HSECADH P9, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence TACRSRIANSCHSGDSWRNSCFANSDSAALAVSSEESGGQRALTAPRG in
  • the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
  • the variant protein is believed to be located as follows with regard to the cell: secreted.
  • the protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans -membrane region prediction program predicts that this protein has a trans -membrane region..
  • Variant protein HSECADH_P9 also has the following non- silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 10, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HSECADH_P9 sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • SNPs Single Nucleotide Polymorphisms
  • Variant protein HSECADH_P9 is encoded by the following transcript(s): HSECADH Tll, for which the sequence(s) is/are given at the end of the application.
  • the coding portion of transcript HSECADH Tl 1 is shown in bold; this coding portion starts at position 125 and ends at position 1090.
  • the transcript also has the following SNPs as listed in Table 11 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HSECADH_P9 sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • Variant protein HSECADH_P13 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HSECADH_T18.
  • An alignment is given to the known protein (Epithelialrcadherin precursor) at the end of the application.
  • One or more alignments to one or more previously published protein sequences are given at the end of the application.
  • a brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • STTATAVITVTDTNDNPPIFNPTT corresponding to amino acids 1 - 379 of Q9UII7, which also corresponds to amino acids 1 - 379 of HSECADH P13, and a second amino acid sequence VTL corresponding to amino acids 380 - 382 of HSECADH P 13, wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • STTATAVITVTDTNDNPPIFNPTT corresponding to amino acids 1 - 379 of Q9UII8, which also corresponds to amino acids 1 - 379 of HSECADH_P13, and a second amino acid sequence VIL corresponding to amino acids 380 - 382 of HSECADH P 13, wherein said first and second amino acid sequences are contiguous and in a sequential order.
  • Comparison report between HSECADH_P13 and CAD1_HUMAN 1.An isolated chimeric polypeptide encoding for HSECADH_P13, comprising a first amino acid sequence being at least 90 % homologous to MGPWSRSLSALLLLLQVSSWLCQEPEPCHPGFDAESYTFTVPRRHLERGRVLGRVNFED CTGRQRTAYFSLDTRFKVGTDGVITVKRPLRPHNPQIHFLVYAWDSTYRKFSTKVTLNT VGHHHRPPPHQASVSGIQAELLTFPNSSPGLRRQKRDWVIPPISCPENEKGPFPKNLVQI KSNKDKEGKVFYSITGQGADTPPVGVFIIERETGWLKVTEPLDRERIATYTLFSHAVSSN GNAVEDPMEILITVTDQNDNKPEFTQEVFKGSVMEGALPGTSVMEVTATDADDDVNT YNAAIAYTILSQDPELPDKNMFTINRNTGVISWTTGLDRESFPTYTLWQAADL
  • the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
  • the variant protein is believed to be located as follows with regard to the cell: secreted.
  • the protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans- membrane region prediction program predicts that this protein has a trans -membrane region..
  • Variant protein HSECADH_P13 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 12, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HSECADH_P13 sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • SNPs Single Nucleotide Polymorphisms
  • Variant protein HSECADH_P13 is encoded by the following transcript(s): HSECADH-Tl 8, for which the sequence(s) is/are given at the end of the application.
  • the coding portion of transcript HSECADHJT 18 is shown in bold; this coding portion starts at position 125 and ends at position 1270.
  • the transcript also has the following SNPs as listed in Table 13 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HSECADH_P13 sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • Variant protein HSECADH-P 14 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HSECADH_T19.
  • An alignment is given to the known protein (Epithelialrcadherin precursor) at the end of the application.
  • One or more alignments to one or more previously published protein sequences are given at the end of the application.
  • a brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • polypeptide encoding for a tail of HSECADH_P14, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRGQEDPEGVEDKCVLAQSRGQSKILLGQLSVNTVMV in HSECADH_P14.
  • chimeric polypeptide encoding for HSECADHJP 14 comprising a first amino acid sequence being at least 90 % homologous to MGPWSRSLSALLLLLQVSSWLCQEPEPCHPGFDAESYTFTVPRRHLERGRVLGRVNFED CTGRQRTAYFSLDTRFKVGTDGVITVKRPLRFHNPQIHFLVYAWDSTYRKFSTKVTLNT VGHHHRPPPHQASVSGIQAELLTFPNSSPGLRRQKRDWVIPPISCPENEKGPFPKNLVQI KSNKDKEGKVFYSITGQGADTPPVGVFIIERETGWLKVTEPLDRERIATYTLFSHAVSSN GNAVEDPMEILITVTDQNDNKPEFTQEVFKGSVMEGALPGTSVMEVTATDADDDVNT YNAAIAYTILSQDPELPDKNMFTINRNTGVISVVTTGLDRE corresponding to amino acids 1 - 336 of Q9UII8, which also corresponds to amino acids 1 - 336
  • polypeptide encoding for a tail of HSECADH_P14, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRGQEDPEGVEDKCVLAQSRGQSKILLGQLSVNTVMV in HSECADH_P14.
  • HSECADHJP14 Comparison report between HSECADHJP14 and CADIJTUMAN: 1.An isolated chimeric polypeptide encoding for HSECADH P14, comprising a first amino acid sequence being at least 90 % homologous to MGPWSRSLSALLLLLQVSSWLCQEPEPCHPGFDAESYTFTWRRHLERGRVLGRVNFED CTGRQRTAYFSLDTRFKVGTDGVITVKRPLRFHNPQIHFLVYAWDSTYRKFSTKVTLNT VGHHHRPPPHQASVSGIQAELLTFPNSSPGLRRQKRDWVIPPISCPENEKGPFPKNLVQI KSNKDKEGKVFYSITGQGADTPPVGVFIIERETGWLKVTEPLDRERIATYTLFSHAVSSN GNAVEDPMEILITVTDQNDNKPEFTQEVFKGSVMEGALPGTSVMEVTATDADDDVNT YNAAIAYTILSQDPELPDKNMFTINRNTGVISVVTTGLDRE
  • polypeptide encoding for a tail of HSECADH-P 14, comprising a polypeptide being at least 70%, optionally at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95% homologous to the sequence VRGQEDPEGVEDKCVLAQSRGQSKILLGQLSVNTVMV in HSECADH_P14.
  • the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
  • the variant protein is believed to be located as follows with regard to the cell: secreted.
  • the protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans -membrane region prediction program predicts that this protein has a trans -membrane region..
  • Variant protein HSECADH_P14 also has the following non- silent SNPs (Single
  • Nucleotide Polymorphisms as listed in Table 14, (given according to tieir position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HSECADHJP 14 sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • Variant protein HSECADH_P14 is encoded by the following transcript(s): HSECADH Tl 9, for which the sequence(s) is/are given at the end of the application.
  • the coding portion of transcript HSECADHJT 19 is shown in bold; this coding portion starts at position 125 and ends at position 1243.
  • the transcript also has the following SNPs as listed in Table 15 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HSECADH_P14 sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • Variant protein HSECADHJP 15 has an amino acid sequence as given at the end of the application; it is encoded by transcript(s) HSECADHJT20.
  • One or more alignments to one or more previously published protein sequences are given at the end of the application.
  • a brief description of the relationship of the variant protein according to the present invention to each such aligned protein is as follows:
  • Comparison report between HSECADH_P15 and Q9UII7 1.An isolated chimeric polypeptide encoding for HSECADH_P15, comprising a first amino acid sequence being at least 90 % homologous to MGPWSRSLSALLLLLQVSSWLCQEPEPCHPGFDAESYTFTVPRRHLERGRVLGRVNFED CTGRQRTAYFSLDTRFKVGTDGVITVKRPLRFHNPQIHFLVYAWDSTYRKFSTKVTLNT VGHHHRPPPHQASVSGIQAELLTFPNSSPGLRRQKRDWVIPPISCPENEKGPFPKNLVQI KSNKDKEGKVFYSITGQGADTPPVGVFIIERETGWLKVTEPLDRERIATYT corresponding to amino acids 1 - 229 of Q9UII7, which also corresponds to amino acids 1 - 229 of HSECADH_P15, and a second amino acid sequence VSIS corresponding to amino acids 230 - 233 of HSECADH P 15,
  • Comparison report between HSECADHJP 15 and CAD1_HUMAN 1.An isolated chimeric polypeptide encoding for HSECADH P 15, comprising a first amino acid sequence being at least 90 % homologous to MGPWSRSLSALLLLLQVSSWLCQEPEPCHPGFDAESYTFTVPRRHLERGRVLGRVNFED CTGRQRTAYFSLDTRFKVGTDGVITVKRPLRFHNPQIHFLVYAWDSTYRKFSTKVTLNT VGHHHRPPPHQASVSGIQAELLTFPNSSPGLRRQKRDWVIPPISCPENEKGPFPKNLVQI KSNKDKEGKVFYSITGQGADTPPVGVFIIERETGWLKVTEPLDRERIATYT corresponding to amino acids 1 - 229 of CAD1_HUMAN, which also corresponds to amino acids 1 - 229 of HSECADH_P15, and a second amino acid sequence VSIS corresponding to amino acids 230 - 233 of HSECADH_P
  • the location of the variant protein was determined according to results from a number of different software programs and analyses, including analyses from SignalP and other specialized programs.
  • the variant protein is believed to be located as follows with regard to the cell: secreted.
  • the protein localization is believed to be secreted because both signal-peptide prediction programs predict that this protein has a signal peptide, and neither trans- membrane region prediction program predicts that this protein has a trans -membrane region..
  • Variant protein HSECADH_P15 also has the following non-silent SNPs (Single Nucleotide Polymorphisms) as listed in Table 16, (given according to their position(s) on the amino acid sequence, with the alternative amino acid(s) listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HSECADH_P15 sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • SNPs Single Nucleotide Polymorphisms
  • Variant protein HSECADH P 15 is encoded by the following transcript(s): HSECADH_T20, for which the sequence(s) is/are given at the end of the application.
  • the coding portion of transcript HSECADH_T20 is shown in bold; this coding portion starts at position 125 and ends at position 823.
  • the transcript also has the following SNPs as listed in Table 17 (given according to their position on the nucleotide sequence, with the alternative nucleic acid listed; the last column indicates whether the SNP is known or not; the presence of known SNPs in variant protein HSECADH_P15 sequence provides support for the deduced sequence of this variant protein according to the present invention).
  • cluster HSECADH features 30 segment(s), which were listed in Table 2 above and for which the sequence(s) are given at the end of the application. These segment(s) are portions of nucleic acid sequence(s) which are described herein separately because they are of particular interest. A description of each segment according to the present invention is now provided.
  • Segment cluster HSECADH_node_0 is supported by 17 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADHJTll, HSECADH_T18, HSECADH_T19 and HSECADH_T20. Table 18 below describes the starting and ending position of this segment on each transcript. Table 18 - Segment location on transcripts
  • Segment cluster HSECADH_node_14 is supported by 40 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADHJT11, HSECADH_T18, HSECADHJT19 and HSECADH_T20. Table 19 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_15 is supported by 1 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T20. Table 20 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_21 is supported by 40 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADHJT18 and HSECADH_T19. Table 21 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_22 is supported by 1 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T19. Table 22 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_25 is supported by 34 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T18. Table 23 below describes the starting and ending position of this segment on each transcript. Table 23 - Segment location on transcripts
  • Segment cluster HSECADH_node_26 is supported by 1 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T18. Table 24 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_48 is supported by 44 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T11. Table 25 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_52 is supported by 39 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T11. Table 26 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_53 is supported by 59 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADHJT11. Table 27 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_54 is supported by 44 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADHJTll. Table 28 below describes the starting and ending position of this segment on each transcript. Table 28 - Segment location on transcripts
  • Segment cluster HSECADH_node_57 is supported by 67 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADHJTll. Table 29 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_60 is supported by
  • Segment cluster HSECADH_node_62 is supported by 173 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T11. Table 31 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_63 is supported by 162 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T11. Table 32 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_7 according to the present invention is supported by
  • short segments related to the above cluster are also provided. These segments are up to about 120 bp in length, and so are included in a separate description.
  • Segment cluster HSECADH_node_l can be found in the following transcript(s): HSECADH_T11, HSECADH_T18, HSECADH_T19 and HSECADH T20. Table 34 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_l 1 is supported by
  • Segment cluster HSECADH_node_12 is supported by 26 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADHJTl l, HSECADHJT18, HSECADH_T19 and HSECADH_T20. Table 36 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_17 can be found in the following transcript(s): HSECADH T11, HSECADHJT18 and HSECADH_T19. Table 37 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_18 is supported by 41 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T11, HSECADHJT18 and HSECADH_T19. Table 38 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_19 according to the present invention can be found in the following transcript(s): HSECADH_T18 and HSECADH_T19. Table 39 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_3 is supported by 18 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADHJTll, HSECADH_T18, HSECADH_T19 and HSECADH_T20. Table 40 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_42 is supported by 43 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T11. Table 41 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_45 is supported by 39 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T11. Table 42 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_46 is supported by 40 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T11. Table 43 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_55 is supported by 36 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T11. Table 44 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_56 is supported by 42 libraries. The number of libraries was determined as previously described. This segment can be found in the fellowing transcript(s): HSECADH T11. Table 45 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_58 is supported by 61 libraries. The number of libraries was determined as previously described. This segment can be found in the following transcript(s): HSECADH_T11. Table 46 below describes the starting and ending position of this segment on each transcript.
  • Segment cluster HSECADH_node_59 according to the present invention can be found in the following transcript(s): HSECADH T11. Table 47 below describes the starting and ending position of this segment on each transcript.
  • Matching Percent Similarity 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent
  • Gaps 0
  • Matching length 379 Total length: 379 Matching Percent Similarity: 100.00 Matching Percent
  • Gaps 0
  • Gaps 0
  • Alignment segment 1/1 Quality: 3313.00
  • Matching length 336 Total length: 336 Matching Percent Similarity: 100.00 Matching Percent Identity: 100.00
  • Gaps 0
  • Matching Percent Similarity 100.00 Matching Percent Identity: 100.00 Total Percent Similarity: 100.00 Total Percent Identity: 100.00
  • Gaps 0
  • Gaps 0

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  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne des nouveaux marqueurs pour le cancer de la prostate qui sont à la fois sensibles et précis. De plus, ces marqueurs sont capables de distinguer un cancer de la prostate d'une hyperplasie prostatique bénigne. Lesdits marqueurs sont surexprimés spécifiquement dans le cancer de la prostates par opposition à des tissus prostatiques normaux et/ou une hyperplasie prostatique bénigne. La mesure de ces marqueurs, seuls ou en combinaison, dans des échantillons prélevés sur un patient donne des informations que le diagnosticien peut corréler avec un diagnostic probable de cancer de la prostate. Les marqueurs de la présente invention, seuls ou en combinaison, autorisent une détection différentielle poussée entre le cancer de la prostate et des états non cancéreux.
PCT/IB2005/002560 2004-01-27 2005-01-27 Nouvelles sequences de nucleotides et d'acides amines; essais et methodes d'utilisation pour le diagnostic du cancer de a la prostate WO2006021874A2 (fr)

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CA002554707A CA2554707A1 (fr) 2004-01-27 2005-01-27 Nouvelles sequences de nucleotides et d'acides amines; essais et methodes d'utilisation pour le diagnostic du cancer de a la prostate
EP05805032A EP1735468A2 (fr) 2004-01-27 2005-01-27 Nouvelles sequences de nucleotides et d'acides amines; essais et methodes d'utilisation pour le diagnostic du cancer de a la prostate

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US53912804P 2004-01-27 2004-01-27
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US60/539,128 2004-01-27
US58416604P 2004-07-01 2004-07-01
US60/584,166 2004-07-01
US60725904P 2004-09-07 2004-09-07
US60/607,259 2004-09-07
US62065604P 2004-10-22 2004-10-22
US62067704P 2004-10-22 2004-10-22
US62091604P 2004-10-22 2004-10-22
US62091804P 2004-10-22 2004-10-22
US62085304P 2004-10-22 2004-10-22
US62087404P 2004-10-22 2004-10-22
US60/620,918 2004-10-22
US60/620,656 2004-10-22
US60/620,853 2004-10-22
US60/620,677 2004-10-22
US60/620,916 2004-10-22
US60/620,874 2004-10-22
US62113104P 2004-10-25 2004-10-25
US60/621,131 2004-10-25
US62825104P 2004-11-17 2004-11-17
US62811204P 2004-11-17 2004-11-17
US62815604P 2004-11-17 2004-11-17
US62814504P 2004-11-17 2004-11-17
US62816704P 2004-11-17 2004-11-17
US62813404P 2004-11-17 2004-11-17
US62810104P 2004-11-17 2004-11-17
US62811104P 2004-11-17 2004-11-17
US62823104P 2004-11-17 2004-11-17
US62817804P 2004-11-17 2004-11-17
US62812304P 2004-11-17 2004-11-17
US60/628,123 2004-11-17
US60/628,134 2004-11-17
US60/628,112 2004-11-17
US60/628,145 2004-11-17
US60/628,231 2004-11-17
US60/628,178 2004-11-17
US60/628,167 2004-11-17
US60/628,251 2004-11-17
US60/628,101 2004-11-17
US60/628,156 2004-11-17
US11/043,806 2005-01-27
US11/043,806 US7368548B2 (en) 2004-01-27 2005-01-27 Nucleotide and amino acid sequences, and assays and methods of use thereof for diagnosis of prostate cancer

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US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture

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WO2003105758A2 (fr) * 2002-06-12 2003-12-24 Avalon Pharmaceuticals, Inc. Gene lie au cancer utilise comme cible pour la chimiotherapie

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Publication number Priority date Publication date Assignee Title
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture

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WO2006021874A3 (fr) 2009-04-09

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