WO2005107364A2 - Polynucleotide, polypeptides, and diagnostic methods - Google Patents

Polynucleotide, polypeptides, and diagnostic methods Download PDF

Info

Publication number: WO2005107364A2
Authority: WO; WIPO (PCT)
Prior art keywords: pea; node; hscreact; amino acids; amino acid
Prior art date: 2004-01-27

Application number

PCT/IB2005/002407

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English (en)

French (fr)

Other versions

WO2005107364A9 (en

WO2005107364A3 (en

Inventor

Amir Toporik

Sarah Pollock

Zurit Levine

Michal Ayalon-Soffer

Gad S. Cojocaru

Alexander Diber

Amit Novik

Dvir Dahary

Pinchas Akiva

Rotem Sorek

Ronen Shemesh

Original Assignee

Compugen Usa, Inc.

Cohen, Yossi

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2004-01-27

Filing date

2005-01-27

Publication date

2005-11-17

2005-01-27 Application filed by Compugen Usa, Inc., Cohen, Yossi filed Critical Compugen Usa, Inc.

2005-01-27 Priority to EP05767217A priority Critical patent/EP1735342A2/en

2005-01-27 Priority claimed from US11/043,806 external-priority patent/US7368548B2/en

2005-11-17 Publication of WO2005107364A2 publication Critical patent/WO2005107364A2/en

2006-05-11 Publication of WO2005107364A9 publication Critical patent/WO2005107364A9/en

2009-04-16 Publication of WO2005107364A3 publication Critical patent/WO2005107364A3/en

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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

Definitions

the present invention is related to novel nucleotide and protein sequences, and assays and methods of use thereof.
IHC Immunohistochemistry
IHC is therefore particularly suitable for antibody- antigen reactions that are not disturbed or destroyed by the process of fixing the tissue slices.
IHC permits determining the localization of binding, and hence mapping of the presence of the antigen within the tissue and even within different compartments in the cell. Such mapping can provide useful diagnostic information, including:
IHC information is valuable for more than diagnosis. It can also be used to determine prognosis and therapy treatment (as in the case of HER- 2 in breast cancer) and monitor disease.
IHC protein markers could be from any cellular location. Most often these markers are membrane proteins but secreted proteins or intracellular proteins (including intranuclear) can be used as an IHC marker too.
IHC has at least two major disadvantages. It is performed on tissue samples and therefore a tissue sample has to be collected from the patient, which most often requires invasive procedures like biopsy associated with pain, discomfort, hospitalization and risk of infection. In addition, the interpretation of the result is observer dependant and therefore subjective. There is no measured value but rather an estimation (on a scale of 1-4) of how prevalent the antigen on target is.
the present invention provides, in different embodiments, many novel amino acid and nucleic acid sequences, which may optionally be used as diagnostic markers.
the present invention provides a number of different variants of known serum proteins, which may optionally be used as diagnostic markers, preferably as serum markers, or optionally as IHC markers.
the present invention therefore overcomes the many deficiencies of the background art with regard to the need to obtain tissue samples and subjective interpretations of results.
serum markers require only a simple blood test and their result is typically a scientifically measured number.
the variants of the present invention may also provide different and/or better measurement parameters for various diseases and/or pathological conditions.
the present invention also provides a number of different variants of known IHC proteins, which may optionally be used as diagnostic markers, preferably as serum markers, or optionally as IHC markers.
the present invention therefore overcomes the many deficiencies of the background art with regard to the need to obtain tissue samples and subjective interpretations of results.
serum markers require only a simple blood test and their result is typically a scientifically measured number.
the variants of the present invention may also provide different and/or better measurement parameters for various diseases and/or pathological conditions.
Other variants are also provided by the present invention as described in greater detail below. The diseases for which such variants may be useful diagnostic markers are described in greater detail below for each of the variants.
a “marker- detectable disease” refers to a disease that may be detected by a particular marker, with regard to the description of such diseases below.
the markers of the present invention alone or in combination, show a high degree of differential detection between disease and non-disease states.
the present invention therefore also relates to diagnostic assays for disease detection optionally and preferably in a biological sample taken from a subject (patient), which is more preferably some type of body fluid or secretion including but not limited to seminal plasma, blood, serum, urine, prostatic fluid, seminal fluid, semen, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, cerebrospinal fluid, sputum, saliva, milk, peritoneal fluid, pleural fluid, cyst fluid, broncho alveolar lavage, lavage of the reproductive system and/or lavage of any other part of the body or system in the body, and stool or a tissue sample.
the term may also optionally encompass samples of in vivo cell culture constituents.
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 for example, 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.
a key to the p values with regard to the analysis of such overexpression is as follows: - library- based statistics: P- value without including the level of expression in cell- lines (PI) - library based statistics: P- value including the of expression in cell- lines (P2) - EST clone statistics: P- value without including the level of expression in cell- lines (SP1) - EST clone statistics: predicted overexpression ratio without including the level of expression in cell- lines (R3) - EST cbne statistics: P- value including the level of expression in cell- lines (SP2) - 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
the probe names follow the
ADP adipocyte
BM bone marrow
BRS mammary gland
CAR cartilage
CNS central nervous system
E-ADR endocrine_adrenal_gland
E-PAN endocrine_pancreas
E-PT endocrine_parathyroid_thyroid
EPID epididymis
GI gastrointestinal tract
lymph node MUS muscle
PNS peripheral nervous system
TCELL immune T cells
THYM thymus
TST testes
UTER cervix- uterus
VAS vascular endothelial artery
adipocyte "Al”; “adrenalcortex”, “A2”; “adrenalgland”, “A3”; “amygdala”, “A4"; “appendix”, “A5"; “atrioventricularnode”, “A6”; “bm_cdl05_endothelial”, “El”; “bm_cd33_myeloid”, “Ml”; “bm_cd34_", “Bl”; “bm_cd7 l_earlyerythroid", "El”; "bonemarrow”, “B2”; “bronchialepithelialcells", “B3”;
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 bebw.
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.
the phrase "disease” includes any type of pathology and/or damage, including both chronic and acute damage, as well as a progress from acute to chronic damage.
the term "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 patients (subjects) having one of the herein-described diseases or conditions, as compared to a comparable sample taken from subjects who do not have one the above- described diseases or conditions.
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.
the marker is detectable in one sample and not detectable in the other, then such a marker can be considered to be differentially present.
a relatively low amount of up- regulation may serve as the marker, as described herein.
diagnostic means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
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.
Diagnosing 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.
the term “detecting” may also optionally encompass any of the above. Diagnosis of a disease according to the present invention can be effected by determining a level of a polynucleotide ⁇ 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.
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).
Numerous well known 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.
test amount refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of a particular disease or condition.
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 a particular disease or condition or a person without such a disease or condition.
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.
a “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.
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.
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 nucleic acid sequence in the table below and/or:
nucleic acid sequence comprising a sequence in the table below:
an isolated chimeric polypeptide encoding for HSAPHOL_P2 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 having the sequence PHSGPAAAFIRRRGWWPGPRCA corresponding to amino acids 1 - 22 of HSAPHOL_P2, second amino acid sequence being at least 90 % homologous to
PATPRPLSWLRAPTRLCLDGPSPVLCA corresponding to amino acids 1 - 27 of AAH21289, which also corresponds to amino acids 23 - 49 of HSAPHOL_P2, and a third amino acid sequence being at least 90 % homologous to EKEKDPKYWRDQ AQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQL HHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVGVSAAT ERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNE MPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKTDVEYESDEKARGTRLD GLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNNVT DPSLSEMVV VAIQILRKNPKGFFLLVEGGRIDHGHHEG
an isolated polypeptide encoding for a head of HSAPHOL_P2 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 PHSGPAAAFIRRRGWWPGPRCA of HSAPHOL_P2.
an isolated chimeric polypeptide encoding for an edge portion of HS APHOL_P2 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 AE, having a structure as follows : a sequence starting from any of amino acid numbers 49-x to 50; and ending at any of amino acid numbers 50+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSAPHOL_P2 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 having the sequence PHSGPAAAFIRRRGWWPGPRCAPATPRPLSWLRAPTRLCLDGPSPVLCA corresponding to amino acids 1 - 49 of HSAPHOL_P2, second amino acid sequence being at least 90 % homologous to
HSAPHOL_P2 wherein said first, second and third amino acid sequences are contiguous and in a sequential order.
an isolated polypeptide encoding for a head of HSAPHOL_P2 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 PHSGPAAAFIRRRGWWPGPRCAPATPRPLSWLRAPTRLCLDGPSPVLCA of HSAPHOL_P2.
an isolated chimeric polypeptide encoding for an edge portion of HSAPHOL_P2 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 AE, having a structure as follows: a sequence starting from any of amino acid numbers 49-x to 50; and ending at any of amino acid numbers 50+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSAPHOL_P3, comprising a first amino acid sequence being at least 90 % homologous to MISPFLVLAIGTCLTNSLVP corresponding to amino acids 63 - 82 of AAH21289, which also corresponds to amino acids 1 - 20 of HSAPHOL_P3, and a second amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of HSAPHOL_P3, 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 PG, having a structure as follows: a sequence starting from any of amino acid numbers 20-x to 20; and ending at any of amino acid numbers 21+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSAPHOL_P3, comprising a first amino acid sequence being at least 90 % homologous to MISPFLVLAIGTCLTNSLVP corresponding to amino acids 1 - 20 of PPBT_HUMAN, which also corresponds to amino acids 1 - 20 of HSAPHOL_P3, and a second amino acid sequence being at least 90 % homologous to GMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYL CGVKANEGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSA AYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKTD VEYESDEK ⁇ RGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFE PGDMQYELNRNN
an isolated chimeric polypeptide encoding for HSAPHOL_P4 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of HSAPHOL_P5 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 MD, having a structure as follows: a sequence starting from any of amino acid numbers 355-x to 355; and ending at any of amino acid numbers 356+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSAPHOL_P5 comprising a first amino acid sequence being at least 90 % homologous to MISPFLVLAIGTCLTNSLVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFL GDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTAT AYLCGVKANEGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHA TPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPK NKTDVEYESDEKARGTRLDGLDLVDTWKSFK RYKHSHFIWNRTELLTLDPHNVDYLL GLFEPGDMQYELNRNNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKA KQALHEAVEM
an isolated chimeric polypeptide encoding for an edge portion of HSAPHOL_P5 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 MD, having a structure as follows: a sequence starting from any of amino acid numbers 355-x to 355; and ending at any of amino acid numbers 356+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSAPHOL_P6 comprising a first amino acid sequence being at least 90 % homologous to MISPFLVLAIGTCLTNSLVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFL GDGMGVSTVTAARTLKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTAT AYLCGVKANEGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHA TPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPK NKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVDYLL corresponding to amino acids 63 - 349 of AAH21289, which also corresponds to amino acids 1 - 287 of HS APHOL_P6, and
an isolated chimeric polypeptide encoding for an edge portion of HSAPHOL_P6, 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 LG, having a structure as follows: a sequence starting from any of amino acid numbers 287-x to 287; and ending at any of amino acid numbers 288+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSAPHOL_P6, comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of HSAPHOL_P6 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 LG, having a structure as follows: a sequence starting from any of amino acid numbers 287-x to 287; and ending at any of amino acid numbers 288+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSAPHOL_P7 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSAPHOL_P7 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
an isolated chimeric polypeptide encoding for HSAPHOL_P7 comprising a first amino acid sequence being at least 90 % homologous to MISPFLVLAIGTCLTNSLVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFL GDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTAT AYLCGVKANEGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHA TPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPK NKTDVEYESDEKARGTRLDGLDLVDTWKSFKPR corresponding to amino acids 1 - 262 of PPBT_HUMAN,
an isolated polypeptide encoding for a tail of HSAPHOL_P7 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 YKLPPRCPLANRVDFSWAGREYRLQTFSKPLIFLANVFLQTQRP in HSAPHOL_P7.
an isolated chimeric polypeptide encoding for HSAPHOL_P7 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSAPHOL_P7 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 LPPRCPLANRVDFSWAGREYRLQTFSKPLIFLANVFLQTQRP in HSAPHOL_P7.
an isolated chimeric polypeptide encoding for HSAPHOL_P8 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSAPHOL_P8 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 KWRGWRGGCMARSLVAGAACGQHLGTRP in HSAPHOL_P8.
an isolated chimeric polypeptide encoding for HSAPHOL_P8 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSAPHOL_P8, 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 KWRGWRGGCMARSLVAGAACGQHLGTRP in HSAPHOL P8.
an isolated chimeric polypeptide encoding for HSAPHOL_P8 comprising a first amino acid sequence being at least 90 % homologous to MISPFLVLAIGTCLTNSLVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFL GDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTAT AYLCGVKANEGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKSVG ⁇ VTTTRVNHA TPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPK NKTDVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFI NRTELLTLDPHNVDYLL G corresponding to amino acids 1 - 288 of 075090, which also corresponds to amino acids 1 - 288 of HSAPHOL_P8, and
an isolated polypeptide encoding for a tail of HSAPHOLJP8 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 KWRGWRGGCMARSLVAGAACGQHLGTRP in HSAPHOL_P8.
the Alkaline phosphatase vanant- detectable disease comprises one or more of the following: liver diseases including but not limited to infectious, malignant, degenerating, cholestatic and autoimmune diseases, bone conditions including but not limited to Paget's disease, Osteomalacia, Rickets, bone tumors, osteoporosis, bone changes occu ⁇ ing due to parathyroid disorders, tumors (either benign, malignant or metastatic) in general, and more specific diseases including but not limited to Hodgkin's disease, diabetes, hyperthyroidism and congestive heart failure.
liver diseases including but not limited to infectious, malignant, degenerating, cholestatic and autoimmune diseases
bone conditions including but not limited to Paget's disease, Osteomalacia, Rickets, bone tumors, osteoporosis, bone changes occu ⁇ ing due to parathyroid disorders, tumors (either benign, malignant or metastatic) in general, and more specific diseases including but not limited to Hodgkin's disease, diabetes,
HSKITCR T2 HSKITCR_T4 HSKITCR T5 HSKITCR T6 a nucleic acid sequence comprising a sequence in the table below: gggmentNam ⁇ T HSKITCR node 0 HSKITCR node 11 HSKITCR_node_17 HSKITCR node 2 HSKITCR node 21 HSKITCR node 27 HSKITCR node_3 HSKITCR node 31 HSKITCR node_33 HSKITCR node 34 HSKITCR node 36 HSKITCR node 44 HSKITCR node_46 HSKITCR node 5 HSKITCR node_50 HSKITCR node 7 HSKITCR node_9 HSKITCR node 13 HSKITCR node_15 HSKITCR node 19 HSKITCR node_23 HSKITCR node 25 HSKITCR_node_29 HSKITCR node 37 HSKITCR node 39 HSKITCR node 41 HSK
an isolated chimeric polypeptide encoding for HSKITCR_P2 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for HSKITCR_P4 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSKITCR_P4 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 VSTHSLLDSPAKDF in HSKITCR_P4.
an isolated chimeric polypeptide encoding for HSKITCR_P5 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSKITCR_P5 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 GKCLAFCSAVLSRI in HSKITCR_P5.
CD117-detectable cancers comprise gastrointestinal stromal tumors, mast cell tumors, and/or seminomatous germ cell tumors.
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
HSCREACT PEA 1 T39 a nucleic acid sequence comprising a sequence in the table below:
an isolated polypeptide comprising an amino acid sequence in the table below:
an isolated chimeric polypeptide encoding for HSCREACT_PEA_1_P9 comprising a first amino acid sequence being at least 90 % homologous to
MEKLLCFLVLTSLSHAFGQTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYT ELSST corresponding to amino acids 1 - 64 of CRP_HUMAN, which also corresponds to amino acids 1 - 64 of HSCREACT_PEA_1_P9, second (bridging) amino acid sequence comprising H, and a third amino acid sequence being at least 90 % homologous to EINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP conesponding to amino acids 188 - 224 of CRP_HUMAN, which also corresponds to amino acids 66 - 102 of HSCREACT_PEA_1_P9, 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 HSCREACT_PEA_1_P9 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 THE having a structure as follows
an isolated chimeric polypeptide encoding for HSCREACT_PEA_1_P10 comprising a first amino acid sequence being at least 90 % homologous to MEKLLCFLVLTSLSHAFGQTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYT
ELSSTRG corresponding to amino acids 1 - 66 of CRP_HUMAN, which also corresponds to amino acids 1 - 66 of HSCREACT_PEA_1_P10.
an isolated chimeric polypeptide encoding for HSCREACT_PEA_1_P12 comprising a first amino acid sequence being at least 90 % homologous to
ELSSTRG corresponding to amino acids 1 - 66 of CRP_HUMAN, which also corresponds to amino acids 1 - 66 of HSCREACT_PEA_1_P12, and a second amino acid sequence being at least 90 % homologous to PNVLNWRALKYEVQGEVFTKPQLWP corresponding to amino acids 200 - 224 of CRP_HUMAN, which also corresponds to amino acids 67 - 91 of
HSCREACT_PEA_1_P12 wherein said first and second amino acid sequences are contiguous and in a sequential order.
an isolated chimeric polypeptide encoding for an edge portion of HSCREACT_PEA_1_P12 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 GP, having a structure as follows: a sequence starting from any of amino acid numbers 66-x to 66; and ending at any of amino acid numbers 67 + ((n-2) - x), in which x varies from 0 to n-2.
an isolated polypeptide encoding for a tail of HSCREACT_PEA_1_P16 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 VSESGHWPGVWFGSRVLIIMS in HSCREACT_PEA_1_P16.
an isolated chimeric polypeptide encoding for HSCREACT_PEA_1_P22 comprising a first amino acid sequence being at least 90 % homologous to MEKLLCFLVLTSLSHAFGQTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYT ELSSTRG conesponding to amino acids 1 - 66 of CRP_HUMAN, which also conesponds to amino acids 1 - 66 of HSCREACT_PEA_1_P22, 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 AFLILWLFWETPPLFHTNLVGL conesponding to amino acids 67 - 88 of HSCREACT_PEA_1_P22, wherein said first and second amino acid sequences are contiguous and in a sequential order.
an isolated polypeptide encoding for a tail of HSCREACT_PEA_1_P22 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 AFLILWLFWETPPLFHTNLVGL in HSCREACT_PEA_1_P22.
an isolated chimeric polypeptide encoding for HSCREACT_PEA_1_P28 comprising a first amino acid sequence being at least 90 % homologous to
the CRP variant- detectable disease may comprise one or more of the following: Assessment of disease activity in inflammatory conditions (Juvenile rheumatoid arthritis, Rheumatoid arthritis, Ankylosing spondylitis, Reiter disease, Psoriatic arthropathy, Vasculitides Behcet syndrome, Wegener granulomatosis, Polyarteritis nodosa, Polymyalgia rheumatica, Crohn's disease, Rheumatic fever, Familial fevers including familial Mediterranean fever, Acute pancreatitis); Diagnosis and management of infection (Bacterial endocarditis, Neonatal septicemia and meningitis, Intercunent infection in systemic lupus erythematosus, Intercunent infection in leukemia and its treatment, Postoperative complications including infection, and thromboembolism); Differential diagnosis/classification of inflammatory disease (Systemic lupus erythematosus vs.
rheumatoid arthritis Crohn disease vs. ulcerative colitis
Tissue necrosis after myocardial infarction and/or outcome of such an infarction 1. Coronary artery disease, non- fatal and fatal 2. Stroke 3. Progression of peripheral vascular disease 4. Development of Congestive Heart Failure 5. Sudden Cardiac Death. 6. Poor prognosis in severe unstable angina 7. Poor prognosis after angioplasty.
low-grade upregulation of CRP vanant production may be detected for predicting coronary Events, stroke and cerebrovascular events, and other cardiovascular diseases such as unstable angina.
a diagnostic method or assay according to measurement of upregulated, slightly upregulated and/or baseline levels of a CRP variant according to the present invention.
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
Z25227 T19 a nucleic acid sequence comprising a sequence in the table below:
an isolated polypeptide comprising an amino acid sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
an isolated polypeptide comprising an amino acid sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
an isolated polypeptide comprising an amino acid sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table bebw and/or:
HS ⁇ R PEA 1 T2 HSTIR_PEA_1 T3 a nucleic acid sequence comprising a sequence in the table below:
HSTIR PEA 1 P4 HSTIR PEA 1 P6 According to prefened embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or: a nucleic acid sequence comprising a sequence in the table below:
an isolated polypeptide comprising an amino acid sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
HSCDIA_PEA 1 Ti l a nucleic acid sequence comprising a sequence in the table below:
an isolated polypeptide comprising an amino acid sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
S69686 T36 S69686 T48 a nucleic acid sequence comprising a sequence in the table below:
S69686_node__54 S69686_node_ _55 S69686_node. _56 S69686_node . . 57 S69686_node. .58 S69686_node_59 S69686_node_ . 60
S69686_node_61 S69686_node . . 62
S69686_node . .63 S69686_node . .64 S69686_node_ . 65 S69686_node_ .66 S69686_node .
an isolated polypeptide comprising an amino acid sequence in the table below: S69686 P2 S69686 P6 S69686 P7 S69686 P13
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
HUMTCXAAA PEA_1_T12 a nucleic acid sequence comprising a sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or: jfr anscripfNa e : W " W-d HSPPI PEA 1 T3 HSPPI PEA 1 T5 HSPPI PEA 1_T6 HSPPI PEA 1 T12 HSPPI_PEA_1_T13 HSPPI PEA_1_T17 HSPPI PEA 1 T18 a nucleic acid sequence comprising a sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or: ifBg geifcii D11581 PEA 1 T4 D11581 PEA 1 T5 D11581 PEA 1 T12 D11581 PEA 1 T14 a nucleic acid sequence comprising a sequence in the table below:
an isolated polypeptide comprising an amino acid sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
nucleic acid sequence comprising a sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
nucleic acid sequence comprising a sequence in the table below:
an isolated polypeptide comprising an amino acid sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or: l TranscriptNarne,. HSPRO204 PEA 1_T3
nucleic acid sequence comprising a sequence in the table below:
HSPRO204 PEA 1 P21 According to prefened embodiments ofthe present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
nucleic acid sequence comprising a sequence in the table below:
an isolated polypeptide comprising an amino acid sequence in the table below:
an isolated chimeric polypeptide encoding for HSMUCl A_PEA_1_P63 comprising a first amino acid sequence being at least 90 % homologous to MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETS ATQRSS V conesponding to amino acids 1 - 45 of MUC1_HUMAN, which also conesponds to amino acids 1 - 45 of HSMUC 1A_PEA_1_P63, 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 EEEVSADQVSVGASGVLGSFKEARNAPSFLSWSFSMGPSK conesponding to amino acids 46 - 85 of HSMUC1A_PEA_1_P63, 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 HSMUC1A_PEA_1_P63 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 EEEVSADQVSVGASGVLGSFKEARNAPSFLSWSFSMGPSK in HSMUC 1A_PEA_1_P63.
an isolated chimeric polypeptide encoding for S42303_PEA_1_P2 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 having the sequence SRRNYGKWKLDGMFLLRRYVCIFTEKLKNQAELYVFLS conesponding to amino acids 1 - 38 of S42303_PEA_1_P2, and a second amino acid sequence being at least 90 % homologous to VKFSNCNGKRKVQYESSEPADFKVDEDGMVYAVRSFPLSSEHAKFLIYAQDKETQEK WQVAVKLSLKPTLTEESVKESAEVEEIVFPRQFSKHSGHLQRQKRDWVIPPINLPENSRG PFPQELVRIRSDRDKNLSLRYSVTGPGADQPPTGIFIINPISGQLSVTKPLDR
an isolated polypeptide encoding for a head of S42303_PEA_1_P2 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 SRRNYGKWKLDGMFLLRRYVCIFTEKLKNQAELYVFLS of S42303_PEA_1_P2.
an isolated polypeptide encoding for a head of S42303_PEA_1_P3 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 MCNTQRM of S42303_PEA_1_P3.
an isolated chimeric polypeptide encoding for S42303_PEA_1_P4 comprising a first amino acid sequence being at least 90 % homologous to
S42303_PEA_1_P4 According to prefened embodiments ofthe present invention, there is provided an isolated chimeric polypeptide encoding for S42303_PEA_1_P5, comprising a first amino acid sequence being at least 90 % homologous to MCRIAGALRTLLPLLAALLQASVEASGEIALCKTGFPEDVYSAVLSKDVHEGQPLLNVK
an isolated chimeric polypeptide encoding for S42303_PEA_1_P6 comprising a first amino acid sequence being at least 90 % homologous to MCRIAGALRTLLPLLAALLQASVEASGEIALCKTGFPEDVYSAVLSKDVHEGQPLLNVK FSNCNGKRKVQYESSEPADFKVDEDGMVYAVRSFPLSSEHAKFLIYAQDKETQEKWQ VAVKLSLKPTLTEESVKESAEVEEIVFPRQFSKHSGHLQRQKRDWVIPPINLPENSRGPFP QELVRIRSDRDKNLSLRYSVTGPGADQPPTGIFIINPISGQLSVTKPLDREQIARFHLRAH A ⁇ GNQVENPIDIVINVIDMNDNRPEFLHQVWNGTVPEGSKPGTYVMTVTAIDADDP NALNGMLRYRIVSQAPSTPSPNMFTINNETGDIITVAAGLDREKVQY
an isolated polypeptide encoding for a tail of S42303_PEA_1_P6 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 KTWPIESLHL in S42303_PEA_1_P6.
an isolated chimeric polypeptide encoding for S42303_PEA_1_P7 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of S42303_PEA_1_P7 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 VRFQPADN in S42303_PEA_1_P7.
an isolated chimeric polypeptide encoding for T87096_PEA_1_P11 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of T87096_PEA_1_P11 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 VSAGGWGWGWGWQGEPQGHHYHPDTAVTPLST in T87096_PEA_1_P11.
an isolated chimeric polypeptide encoding for T87096_PEA_1_P27 comprising a first amino acid sequence being at least 90 % homologous to MQPSSLLPLALCLLAAPASALVRIPLHKFTS1RRTMSEVGGSVEDLIAKGPVSKYSQAVP AVTEGPIPEVLKNYMDAQYYGEIGIGTPPQCFTWFDTGSSNLWVPSIHCKLLDIACWIH HKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSQDTVSVPCQSASSASALGGVKVERQVF GEATKQPGITFIAAKFDGILGMAYPRISVNNVLPVFDNLMQQKLVDQNIFSFYLSRDPD AQPGGELMLGGTDSKYYKGSLSYLNVTRKAYWQVHLDQV conesponding to amino acids 1 - 277 of CATD_HUMAN, which also conesponds to amino acids 1 - 277 of CATD_HUMAN, which also conespond
an isolated polypeptide encoding for a tail of T87096_PEA_1_P27 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 WAAVG in T87096_PEA_1_P27.
an isolated chimeric polypeptide encoding for T87096_PEA_1_P39 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of T87096_PEA_1_P39 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 CESRTLAPSPRSCPSGMSLQGCLRNHLGNAILLPLGPVSQASPPPCSSH in T87096_PEA_1_P39.
an isolated chimeric polypeptide encoding for HSPRO204_PEA_l_P3, comprising a first amino acid sequence being at least 90 % homologous to MNIKGSPWKGSLLLLLVSNLLLCQSVAPLPICPGGAARCQVTLRDLFDRAVVLSHYIHN LSSEMFSEFDKRYTHGRGFITKAINSCHTSSLATPEDKEQAQQMN corresponding to amino acids 1 - 104 of PRL_HUMAN, which also conesponds to amino acids 1 - 104 of HSPRO204_PEA_l_P3, 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 KTF conesponding to amino acids 105 - 107 of HSPRO204_PEA_l_P3, wherein said first amino acid sequence and second amino acid sequence are contig
an isolated chimeric polypeptide encoding for HSPRO204_PEA_l_P4 comprising a first amino acid sequence being at least 90 % homologous to MNIKGSPWKGSLLLLLVSNLLLCQSVAPLPICPGGAARCQ VTLRDLFDRAVVLSHYIHN LSSEMFSEFDKRYTHGRGFITKAINSCHTSSLATPEDKEQAQQMNQKDFLSLIVSILRSW NEPLYHLVTEVRGMQEAPEAILSKAVEIEEQTKRLLEGMELIVSQ conesponding to amino acids 1 - 164 of PRL_HUMAN, which also conesponds to amino acids 1 - 164 of HSPRO204_PEA_l_P4, 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 LERTRTYKY cones
an isolated polypeptide encoding for a tail of HSPRO204_PEA_l_P4 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 LERTRTYKY in HSPRO204_PEA_l_P4.
an isolated chimeric polypeptide encoding for HSPRO204_PEA_l_P5 comprising a first amino acid sequence being at least 90 % homologous to MNIKGSPWKGSLLLLLVSNLLLCQSVAPLPICPGGAARCQVTLRDLFDRAVVLSHYfflN LSSEMFSEFDKRYTHGRGFITKALNSCHTSSLATPEDKEQAQQMNQKDFLSLIVSILRSW NEPLYHLVTEVRGMQEAPEAILSKAVEIEEQTKRLLEGMELTVSQ conesponding to amino acids 1 - 164 of PRL_HUMAN, which also conesponds to amino acids 1 - 164 of HSPRO204_PEA_l_P5, 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
an isolated polypeptide encoding for a tail of HSPRO204_PEA_l_P5 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 SLFFCVMRFILKPKKMRSTLSGRDFHPCRWLMKSLAFLLIITCSTAYAGIHIKSTIISSS in HSPRO204_PEA_l_P5.
an isolated polypeptide encoding for a tail of HSPRO204_PEA_l_P6 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 SSLLVLLCFSH in HSPRO204_PEA_l_P6.
an isolated chimeric polypeptide encoding for HSPRO204_PEA_l_Pl 1 comprising a first amino acid sequence being at least 90 % homologous to
LSSEMFSEFDKRYTHGRGFITKAINSCHTSSLATPEDKEQAQQMN conesponding to amino acids 1 - 104 of PRL_HUMAN, which also conesponds to amino acids 1 - 104 of HSPRO204_PEA_l_Pl 1, and a second amino acid sequence being at least 90 % homologous to VHPETKENEIYPVWSGLPSLQM ADEESRLS A YYNLLHCLRRDSHKIDNYLKLLKCRIIH NNNC corresponding to amino acids 165 - 227 of PRL_HUMAN, which also conesponds to amino acids 105 - 167 of HSPRO204_PEA_l_Pl 1, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
an isolated chimeric polypeptide encoding for HSPRO204_PEA_l_P12 comprising a first amino acid sequence being at least 90 % homologous to MNIKGSPWKGSLLLLLVSNLLLCQSVAPLPICPGGAARCQVTLRDLFDRAVVLSHYIHN LSSEMFSEFDKRYTHGRGFITKAINSCHTSSLATPEDKEQAQQMN conesponding to amino acids 1 - 104 of PRL_HUMAN, which also conesponds to amino acids 1 - 104 of HSPRO204_PEA_l_P12, 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 AKRTDCSASSMGQAVV corresponding to amino acids 105 - 120 of HSPRO204_PEA_l_P12, wherein said
an isolated polypeptide encoding for a tail of HSPRO204_PEA_l_P12 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 AKRTDCSASSMGQAVV in HSPRO204_PEA_l_P12.
an isolated chimeric polypeptide encoding for HSPRO204_PEA_l_P21 comprising a first amino acid sequence being at least 90 % homologous to
LPHFFPCHPRRQGASPTDESKRLSEPDSQHIAILE corresponding to amino acids 41 - 75 of HSPRO204_PEA_l_P21, 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 HSPRO204_PEA_l_P21 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 LPHFFPCHPRRQGASPTDESKRLSEPDSQHIAILE in HSPRO204_PEA_l_P21.
an isolated chimeric polypeptide encoding for S69686_P2 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 having the sequence MLRLPYPLTWRQRPKQLEALCVGAATGPRA conesponding to amino acids 1 - 30 of S69686_P2, and a second amino acid sequence being at least 90 % homologous to MWLCPLALNLILMAASGAVCEVKDVCVGSPGIPGTPGSHGLPGRDGRDGLKGDPGPPG PMGPPGEMPCPPGNDGLPGAPGIPGECGEKGEPGERGPPGLPAHLDEELQATLHDFRHQ ILQTRGALSLQGSIMTVGEKVFSSNGQSITFDAIQEACARAGGRIAVPRNPEENEAIASFV KKYNTYAYVGLTEGPSPGDFRYSDGTPVNY
an isolated polypeptide encoding for a head of S69686_P2 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 MLRLPYPLTWRQRPKQLEALCVGAATGPRA of S69686_P2.
an isolated chimeric polypeptide encoding for S69686_P6, 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 having the sequence MLRLPYPLTWRQRPKQLEALCVATGPRA conesponding to amino acids 1 - 28 of S69686_P6, and a second amino acid sequence being at least 90 % homologous to MWLCPLALNLILMAASGAVCEVKDVCVGSPGIPGTPGSHGLPGRDGRDGLKGDPGPPG PMGPPGEMPCPPGNDGLPGAPGIPGECGEKGEPGERGPPGLPAHLDEELQATLHDFRHQ ILQTRGALSLQGSIMTVGEKVFSSNGQSITFDAIQEACARAGGRIAVPRNPEENEAIASFV KKYNTYAYVGLTEGPSPGDFRYSDGTPVNYTN
an isolated polypeptide encoding for a head of S69686_P6 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 MLRLPYPLTWRQRPKQLEALCVATGPRA of S69686_P6.
an isolated chimeric polypeptide encoding for S69686_P7 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 having the sequence MLRLPYPLTWRQRPKQLEALCVGAATGPRA conesponding to amino acids 1 - 30 of S69686_P7, a second amino acid sequence being at least 90 % homologous to MWLCPLALNLILMAASGAVCEVKDVCVGSPGIPGTPGSHGLPGRDGRDGLKGDPGPPG PMGPPGEMPCPPGNDGLPGAPGIPGECGEKGEPGERGPP conesponding to amino acids 1 - 97 of PSPA_HUMAN_V1, which also conesponds to amino acids 31 - 127 of S69686_P7, and a third amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a head of S69686_P7 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 MLRLPYPLTWRQRPKQLEALCVGAATGPRA of S69686_P7.
an isolated chimeric polypeptide encoding for an edge portion of S69686_P7 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 PA, having a structure as follows: a sequence starting from any of amino acid numbers 127-x to 127; and ending at any of amino acid numbers 128+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for S69686_P13 comprising a first amino acid sequence being at least 90 % homologous to MWLCPLALNLILMAASGAVCEVKDVCVGSP conesponding to amino acids 1 - 30 ofPSPA_HUMAN_Vl, which also conesponds to amino acids 1 - 30 of S69686_P13, and a second amino acid sequence being at least 90 % homologous to GRGKEQCVEMYTDGQWNDRNCLYSRLTICEF conesponding to amino acids 218 - 248 of PSPA_HUMAN_V1, which also conesponds to amino acids 31 - 61 of S69686_P13, wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
an isolated chimeric polypeptide encoding for an edge portion of S69686_P13 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 PG, having a structure as follows: a sequence starting from any of amino acid numbers 30-x to 30; and ending at any of amino acid numbers 31+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HUMTCXAAA_PEA_1_P6 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 having the sequence MRNQAPGRPKGATFPPRRPTGSRAPPLAPELRAKQRPGERV conesponding to amino acids 1 - 41 of HUMTCXAAA_PEA_1_P6, a second amino acid sequence being at least 90 % homologous to MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQ PRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSAL SNSIMYFSHFVPVFLP conesponding to amino acids 1 - 134 of CD8A_HUMAN, which also cone
an isolated polypeptide encoding for a head of HUMTCXAAA_PEA_1_P6 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 MRNQAPGRPKGATFPPRRPTGSRAPPLAPELRAKQRPGERV of HUMTCXAAA_PEA_1_P6.
an isolated polypeptide encoding for an edge portion of HUMTCXAAA_PEA_1_P6 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 PGN having a stmcture as follows (numbering according to HUMTCXAAA_PEA_1_P6): a sequence starting from any of amino acid numbers 175-x to 175; and ending at any of amino acid numbers 177 + ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HUMTCXAAA_PEA_1_P12 comprising
an isolated polypeptide encoding for an edge portion of HUMTCXAAA_PEA_1_P12 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 AGN having a structure as follows (numbering according to HUMTCXAAA_PEA_1_P12): a sequence starting from any of amino acid numbers 171-x to 171; and ending at any of amino acid numbers 173 + ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HUMTCXAAA_PEA_1_P13 comprising a first
HUMTCXAAA_PEA_1_P13 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 SKSRGIAAGRSRPRSCPWLC conesponding to amino acids 209 - 228 of HUMTCXAAA_PEA_1_P13, 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 HUMTCXAAA_PEA_1_P13 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 SKSRGIAAGRSRPRSCPWLC in HUMTCXAAA_PEA_1_P13.
an isolated chimeric polypeptide encoding for HUMTCXAAA_PEA_1_P14 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of HUMTCXAAA_PEA_1_P14 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 HF, having a structure as follows: a sequence starting from any of amino acid numbers 127-x to 127; and ending at any of amino acid numbers 128+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HUMTCXAAA_PEA_1_P15 comprising a first amino acid sequence being at least 90 % homologous to MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCS WLFQ PRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSAL SNSIMYFSHFVPVFLP corresponding to amino acids 1 - 134 of CD8 A_HUMAN, which also corresponds to amino acids 1 - 134 of HUMTCXAAA_PEA_1_P15, a second amino acid sequence bridging amino acid sequence comprising of G, and a third amino acid sequence being at least 90 % homologous to NRRRVCKCPRPWKSGDKPSLSARYV conesponding to amino acids 210 - 235 of CD8A_HUMAN, which also cones
an isolated polypeptide encoding for an edge portion of HUMTCXAAA_PEA_1_P15 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 PGN having a structure as follows (numbering according to HUMTCXAAA_PEA_1_P15): a sequence starting from any of amino acid numbers 134-x to 134; and ending at any of amino acid numbers 136 + ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSPPI_PEA_1_P6 comprising a first amino acid sequence being
HSPPI_PEA_1_P6 wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
an isolated chimeric polypeptide encoding for HSPPI_PEA_1_P9 comprising a first amino acid sequence being at least 90 % homologous to MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHL VEAL YLVCGERGFFYTPKTRRE AEDLQ conesponding to amino acids 1 - 62 of INS_HUMAN, which also conesponds to amino acids 1 - 62 of HSPPI_PEA_1_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 GEPTAHCCPWPPPATPCSWRSHPAWAEGGRRLPPSRGSGALF conesponding to amino acids 63 - 104 of HSPPI_PEA_1_P9, wherein said first amino acid sequence and second amino acid sequence are
an isolated polypeptide encoding for a tail of HSPPI_PEA_1_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 GEPTAHCCPWPPPATPCSWRSHPAWAEGGRRLPPSRGSGALF in HSPPI_PEA_1_P9.
an isolated chimeric polypeptide encoding for HSPPI_PEA_1_P10 comprising a first amino acid sequence being at least 90 % homologous to MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRE AEDLQ conesponding to amino acids 1 - 62 of INS_HUMAN, which also conesponds to amino acids 1 - 62 of HSPPI_PEA_1_P10, 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 GGAGRGPWCRQPAALGPGGVPAEAWHCGTMLYQHLLPLPAGELLQLDAARRQPHTR RLLHRERWNKALEPA conesponding to amino acids 63 - 133 of HSPPI_PEA_1_P
an isolated polypeptide encoding for a tail of HSPPI_PEA_1_P10 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 GGAGRGPWCRQPAALGPGGVPAEAWHCGTMLYQHLLPLPAGELLQLDAARRQPHTR RLLHRERWNKALEPA in HSPPI_PEA_1_P10.
an isolated chimeric polypeptide encoding for HSPPI_PEA_1_P12 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSPPI_PEA_1_P12 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 AGELLQLDAARRQPHTRRLLHRERWNKALEPA in HSPPI_PEA_1_P12.
an isolated chimeric polypeptide encoding for HSPPI_PEA_1_P14 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSPPI_PEA_1_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 AGELLQLDAARRQPHTRRLLHRERWNKALEPALLCRLCVLGALGQAPLPGTWSPSQL SPRSLGAHRCQRRPGPACSGSPQSGHACRLPAAPTLWLRVQYGSCGGL in HSPPI_PEA_1_P14.
an isolated chimeric polypeptide encoding for HSPPI_PEA_1_P15 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSPPI_PEA_1_P15 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 GGAGRGPWCRQPAALGPGGVPAEAWHCGTMLYQHLLPLPAGELLQLDAARRQPHTR RLLHRERWNKALEPALLCRLCVLG ALGQAPLPGTWSPSQLSPRSLGAHRCQRRPGPA CSGSPQSGHACRLPAAPTLWLRVQYGSCGGL in HSPPI_PEA_1_P15.
an isolated chimeric polypeptide encoding for an edge portion of DI 1581_PEA_1_P6 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 QY, having a stmcture as follows: a sequence starting from any of amino acid numbers 90-x to 90; and ending at any of amino acid numbers 91+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for DI 1581_PEA_1_P10 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for DI 1581_PEA_1_P12 comprising a first amino acid sequence being at least 90 % homologous to MKWVESIFLIFLLNFTESRTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATYK EVSKMVKDALTAIEKPTGDEQSSGCLENQLPAFLEELCHEKEILEKYGHSDCCSQSEEG RHNCFLAHKKPTPASIPLFQVPEPVTSCEAYEEDRETFMNKFIYEIARRHPFLYAPTILLW AARYDKJIPSCCKAENAVECFQTKAATVTKELRESSLLNQHACAVMKNFGTRTFQAITV TKLSQKFTKVNFTEIQKL ⁇ DVAHVHEHCCRGDVLDCLQDGEKLMSYICSQQDTLSNKI TECCKLTTLERGQCIIHAENDEKPEGLSPNLNRFLGDRDFN
DI 1581_PEA_1_P12 wherein said first amino acid sequence and second amino acid sequence are contiguous and in a sequential order.
an isolated chimeric polypeptide encoding for an edge portion of DI 1581_PEA_1_P12 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 AS, having a stmcture as follows: a sequence starting from any of amino acid numbers 352-x to 352; and ending at any of amino acid numbers 353+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for DI 1581_PEA_1_P16 comprising a first amino acid sequence being at least 90 % homologous to MKWVESIFLIFLLNFTESRTLHRNEYGIASILDSYQCTAEISLADLATIFFAQFVQEATYK EVSKMVKDALTAIEKPTGDEQSSGCLENQ conesponding to amino acids 1 - 90 of
FETA_HUMAN which also conesponds to amino acids 1 - 90 of DI 1581_PEA_1_P16, 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 NFAMRKKFWRSTDIQTAAAKVKREDITVFLHTKSPLQHRSHFSKFQNLSQAVKHMKKT GRHS conesponding to amino acids 91 - 152 of DI 1581_PEA_1_P16, 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 D11581_PEA_1_P16 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 NFAMRKKTWRSTDIQTAAAKVKJ EDITVFLHTKSPLQHRSHFSKFQNLSQAVKHMKKT GRHS in D11581_PEA_1_P16.
an isolated chimeric polypeptide encoding for Z25227_P10 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for T87719_P2 comprising a first amino acid sequence being at least 90 % homologous to MRCALALSALLLLLSTPPLLPS conesponding to amino acids 1 - 22 of PODX_HUMAN_Vl, which also conesponds to amino acids 1 - 22 of T87719_P2, a second amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of T87719_P2 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 SS, having a stmcture as follows: a sequence starting from any of amino acid numbers 22-x to 23; and ending at any of amino acid numbers 23+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated polypeptide encoding for a tail of T87719_P2 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 VTPAGVGQVGEPRLG in T87719_P2.
an isolated chimeric polypeptide encoding for T87719_P8 comprising a first amino acid sequence being at least 90 % homologous to MRCALALSALLLLLSTPPLLPS conesponding to amino acids 1 - 22 of PODX_HUMAN, which also conesponds to amino acids 1 - 22 of T87719_P8, a second amino acid sequence being at least 90 % homologous to SPSPSPSPSQNATQTTTDSSNKTAPTPASSVTIMATDTAQQSTVPTSKANEILASVKATTL GVSSDSPGTTTLAQQVSGPVNTTVARGGGSGNPTTTIESPKSTKSADTTTVATSTATAKP NTTSSQNGAEDTTNSGGKSSHSVTTDLTSTKAE conesponding to amino acids 25 - 178 of PODX_HUMAN, which also conesponds to amino acids 23 - 176 of T87719_P8, and a third amino acid sequence being at least 70%, optionally at least
an isolated chimeric polypeptide encoding for an edge portion of T87719_P8, 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 SS, having a structure as follows: a sequence starting from any of amino acid numbers 22-x to 23; and ending at any of amino acid numbers 23+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated polypeptide encoding for a tail of T87719_P8 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 RARVKL in T87719_P8.
an isolated chimeric polypeptide encoding for HSTIR_PEA_1_P6 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of HSTIR_PEA_1_P6 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 TF, having a structure as follows: a sequence starting from any of amino acid numbers 366-x to 366; and ending at any of amino acid numbers 367+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSALK1A_PEA_1_P14 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSALK1A_PEA_1_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 GTSSCPSTPSPSSWPLPSLPSFPLMLWPIKGLGAGERVGRTLGSNWQSGLARGGGS in HSALK1A_PEA_1_P14.
an isolated chimeric polypeptide encoding for HSCDIA_PEA_1_P5 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 having the sequence LQWGRKNLGAMFAF conesponding to amino acids 1 - 14 of HSCDIA_PEA_1_P5, a bridging amino acid T conesponding to amino acid 18 of HSCDIA_PEA_1_P5, and a second amino acid sequence being at least 90 % homologous to GLKEPLSFHVTWIASFYNHSWKQNLVSGWLSDLQTHTWDSNSSTIVFLWPWSRGNFSN EEWKELETLFRIRTIRSFEGIRRYAHELQFEYPFEIQVTGGCELHSGKVSGSFLQLAYQGS DFVSFQNNSWLPYPVAGNMAKHFCKVLN
an isolated polypeptide encoding for a head of HSCDIA_PEA_1_P5 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 LQWGRKNLGAMFAF of HSCDIA_PEA_1_P5.
an isolated chimeric polypeptide encoding for HSCDIA_PEA_1_P6 comprising a first amino acid sequence being at least 90 % homologous to MLFLLLPLLA VLPGDGNADGLKEPLSFHVTWIASFYNHS WKQNLVSG WLSDLQTHTW DSNSST ⁇ VFLWPWSRGNFSNEEWKELETLFRIRTIRSFEGIRRYAHELQFEYPFEIQVTGG CELHSGKVSGSFLQLAYQGSDFVSFQNNSWLPYPVAGNMAKHFCKVLNQNQHENDIT HNLLSDTCPRFILGLLDAGKAHLQRQVKPEAWLSHGPSPGPGHLQLVCHVSGFYPKPV WVMWMRGEQEQQGTQRGDILPSADGTWYLRATLEVAAGEAADLSCRVKHSSLEGQD rVLYWEHHSSVGFIILAVrVPLLLLIGLALWFRKR conespond
an isolated chimeric polypeptide encoding for HSCDIA_PEA_1_P7 comprising a first amino acid sequence being at least 90 % homologous to MLFLLLPLLA VLPGDGNADGLKEPLSFHVTWIASFYNHSWKQNLVS GWLSDLQTHTW DSNSSTIVFLWPWSRGNFSNEEWKELETLFRIRTIRSFEGIRRYAHELQFEYPFEIQVTGG CELHSGKVSGSFLQLAYQGSDFVSFQNNSWLPYPVAGNMAKHFCKVLNQNQHENDIT HNLLSDTCPRFILGLLDAGKAHLQRQVKPEAWLSHGPSPGPGHLQLVCHVSGFYPKPV WVMWMRGEQEQQGTQRGDILPSADGTWYLRATLEVAAGEAADLSCRVKHSSLEGQD rVLYW conesponding to amino acids 1 - 294 of CD1A_HUMAN_V
an isolated polypeptide encoding for a tail of HSCDIA_PEA_1_P7 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 GEKKLRPRLEMPGSGPQA in HSCDIA_PEA_1_P7.
an isolated chimeric polypeptide encoding for HSCDIA_PEA_1_P8 comprising a first amino acid sequence being at least 90 % homologous to MLFLLLPLLA VLPGDGNADGLKEPLSFHVTWIASFYNHSWKQNLVSGWLSDLQTHTW DSNSSTIVFLWPWSRGNFSNEEWKELETLFRIRTIRSFEGIRRYAHELQFEYPFEIQVTGG CELHSGKVSGSFLQLAYQGSDFVSFQNNSWLPYPVAGNMAKHFCKVLNQNQHENDIT HNLLSDTCPRFILGLLDAGKAHLQRQVKPEAWLSHGPSPGPGHLQLVCHVSGFYPKPV WVMWMRGEQEQQGTQRGDILPSADGTWYLRATLEVAAGEAADLSCRVKHSSLEGQD IVLYW conesponding to amino acids 1 - 294 of CD1 A_HUMAN_V1, which also cone
an isolated chimeric polypeptide encoding for HSCDIA_PEA_1_P9 comprising a first amino acid sequence being at least 90 % homologous to MLFLLLPLLA VLPGDGNAD conesponding to amino acids 1 - 19 of CD1A_HUMAN, which also corresponds to amino acids 1 - 19 of
HSCDIA_PEA_1_P9 HSCDIA_PEA_1_P9, and a second amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of HSCDIA_PEA_1_P9 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 DG, having a structure as follows: a sequence starting from any of amino acid numbers 19-x to 19; and ending at any of amino acid numbers 20+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSCDIA_PEA_1_P11 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of HSCDIA_PEA_1_P11 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 RE, having a structure as follows: a sequence starting from any of amino acid numbers 239-x to 239; and ending at any of amino acid numbers 240+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
HUM1NHA PEA 1 T2 HUMINHA PEA 1 T4 HUMINHA_PEA_1_T5 HUMINHA PEA 1 T6 a nucleic acid sequence comprising a sequence in the table below:
HUMINHA PEA_l_node 2 HUMINHA PEA 1 node 3 .MINHA_PEA_l_node_4 HUMINHA PEA 1 node 7 HUMINHA PEA 1 node 9 HUMP HA PEA 1 node 10 HUMINHA PEA 1 node 16 HUMINHA PEA 1 node 5 HUMINHA PEA 1 node 6 HUMINHA PEA 1 node 8 HUMINHA PEA 1 node 11 HUMINHA_PEA_l_node_12 HUMINHA PEA_l_node_14
an isolated polypeptide comprising an amino acid sequence in the table below: PfoteirrNarne , ⁇ t J Stfi HUMINHA PEA 1 P4 HUMINHA PEA 1 P5
an isolated chimeric polypeptide encoding for HUMINHA_PEA_1_P5 comprising a first amino acid sequence being at least 90 % homologous to MVLHLLLFLLLTPQGGHSCQGLELARELVLAKVRALFLDALGPPAVTREGGDPGVRRL PRRHALGGFTHRGSEPEEEEDVSQAILFPAT conesponding to amino acids 1 - 89 of IHA_HUMAN, which also conesponds to amino acids 1 - 89 of HUMINHA_PEA_1_P5, 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
an isolated polypeptide encoding for a tail of HUMINHA_PEA_1_P5 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 GSAPQRPVAMTTAQRDSLLWKLAGLLRESGDWLSGCSTLSLLTPTLQQLNHVFELHL GPWGPGQTGFV in HUMINHA_PEA_1_P5.
an isolated chimeric polypeptide encoding for HUMINHA__PEA_1_P10 comprising a first amino acid sequence being at least 90 % homologous to MVLHLLLFLLLTPQGGHSCQGLELARELVLAKVRALFLDALGPPAVTREGGDPGVRRL PRRHALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFRP SQHTR conesponding to amino acids 1 - 122 of IHA_HUMAN, which also conesponds to amino acids 1 - 122 of HUMINHA_PEA_1_P10, 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 NHPVEGREPDAQLP conesponding to amino acids 123 - 136 of HUMINHA_PEA_
an isolated polypeptide encoding for a tail of HUMINHA_PEA_1_P10 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 NHPVEGREPDAQLP in HUMINHA_PEA_1_P10.
an isolated chimeric polypeptide encoding for HUMEDF_PEA_2_P5 comprising a first amino acid sequence being at least 90 % homologous to
EQTSEIITFAESGT conesponding to amino acids 1 - 131 of IHB A_HUMAN, which also conesponds to amino acids 1 - 131 of HUMEDF_PEA_2_P5, 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 VKS corresponding to amino acids 132 - 134 of HUMEDF_PEA_2_P5, 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 HUMEDF_PEA_2_P5 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 VKS in HUMEDF_PEA_2_P5.
an isolated chimeric polypeptide encoding for HUMEDF_PEA_2_P6 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HUMEDF_PEA_2_P6 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 HSEA in HUMEDF_PEA_2_P6.
an isolated chimeric polypeptide encoding for HUMEDF_PEA_2_P8 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HUMEDF_PEA_2_P8 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 VKS in HUMEDF_PEA_2_P8.
the antibody is capable of differentiating between a splice variant having said epitope and a corresponding known protein.
a kit for detecting an inhibin variant-detectable disease comprising a kit detecting specific expression of a splice variant as described herein.
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 as described herein.
the kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence as described herein.
the kit comprises an antibody as described herein.
the kit further comprises at least one reagent for performing an ELISA or a Western blot.
the method for detecting an inhibin variant-detectable disease comprising detecting specific expression of a splice variant as described herein.
said detecting specific expression is performed with a NAT-based technology.
detecting specific expression is performed with an immunoassay.
the immunoassay comprises an antibody as described herein.
a biomarker capable of detecting Inhibin variant- detectable disease comprising any of the above nucleic acid sequences or a fragment thereof, or any ofthe above amino acid sequences or a fragment thereof.
a method for screening for variant-detectable disease comprising detecting cells affected by an inhibin variant-detectable disease with a biomarker or an antibody or a method or assay as described herein.
a method for diagnosing an inhibin variant detectable disease comprising detecting cells affected by inhibin variant-detectable disease with a biomarker or an antibody or a method or assay as described herein.
a method for monitoring disease progression and/or treatment efficacy and/or relapse of Inhibin variant- detectable disease comprising detecting cells affected by Inhibin variant-detectable disease with a biomarker or an antibody or a method or assay as described herein.
a method of selecting a therapy for Inhibin variant- detectable disease comprising detecting cells affected by an inhibin variant-detectable disease with a biomarker or an antibody or a method or assay as described herein and selecting a therapy according to said detection.
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
HSGR0W1_PEA 1 PEA 1 T16 a nucleic acid sequence comprising a sequence in the table below:
HSGROW1 PEA 1 PEA 1 node 19 HSGROW1 PEA 1 PEA 1 node 20 According to prefened embodiments ofthe present invention, there is provided an isolated polypeptide comprising an amino acid sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
T05709 PEA 1 P14 According to prefened embodiments of the present invention, there is provided an isolated chimeric polypeptide encoding for T05709_PEA_1_P3, comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for T05709_PEA_1_P3 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of T05709_PEA_1_P3 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 MSSMLQAATTSMQGSHSQEFMMLCLILKAKWTLPRPGEK in T05709_PEA_1_P3.
an isolated chimeric polypeptide encoding for T05709_PEA_1_P8 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for T05709_PEA_1_P9 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for T05709_PEA_1_P13 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of T05709_PEA_1_P13 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 NMLIGVELQRLLVFQVFLFIQLDTMMHRSS in T05709_PEA_1_P13.
an isolated chimeric polypeptide encoding for T05709_PEA_1_P13 comprising a first amino acid sequence being at least 90 % homologous to MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNM KAFLDELKAENIKKFL YNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLL SYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDL VYVNY ARTEDFFKLERDMKINCSGKIVIARYGKVFRGNK conesponding to amino acids 1 - 213 of FOHl_HUMAN, which also conesponds to amino acids 1 - 213 of T05709_PEA_1_P13, and a second amino acid sequence being at least 70%, optionally at least 80%, preferably at least 85%, more preferably
an isolated polypeptide encoding for a tail of T05709_PEA_1_P13 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 NMLIGVELQRLLVFQVFLFIQLDTMMHRSS in T05709_PEA_1_P13.
an isolated chimeric polypeptide encoding for T05709_PEA_1_P14 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of T05709_PEA_1_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 VKVGKRN in T05709_PEA_1_P14.
an isolated chimeric polypeptide encoding for T05709_PEA_1_P14 comprising a first amino acid sequence being at least 90 % homologous to MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLF conesponding to amino acids 1 - 39 of FOHl_HUMAN, which also conesponds to amino acids 1 - 39 of T05709_PEA_1_P14, 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 VKVGKRN conesponding to amino acids 40 - 46 of T05709_PEA_1_P14, wherein said first and second amino acid sequences are contiguous and in a sequential order.
an isolated polypeptide encoding for a tail of T05709_PEA_1_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 VKVGKRN in T05709_PEA_1_P14.
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or: HSPROSAP PEA 1 T3 HSPROSAP_PEA 1_T15 HSPROSAP PEA 1 T19 HSPROSAP PEA 1 T20 HSPROSAP PEA 1 T23 HSPROSAP PEA 1 T24 HSPROSAP PEA 1 T25 a nucleic acid sequence comprising a sequence in the table below:
an isolated polypeptide comprising an amino acid sequence in the table below:
an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
HUMPGCA PEA 1 TO HUMPGCA PEA 1_T1 HUMPGCA PEA 1 T5 a nucleic acid sequence comprising a sequence in the table below:
an isolated polypeptide comprising an amino acid sequence in the table below:
HUMPGCA PEA 1 P12 HUMPGCA PEA 1 P14 HUMPGCA PEA 1 P15 According to prefened embodiments of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence in the table below and/or:
nucleic acid sequence comprising a sequence in the table below:
an isolated polypeptide comprising an amino acid sequence in the table below: IKgiMHamaS ⁇ *s HUMFBRB PEA_1_P4 HUMFBRB PEA 1 P9 HUMFBRB PEA 1 Pl l HUMFBRB PEA 1 P13 HUMFBRB PEA 1 P17 HUMFBRB_PEA 1 P26
HSMRACP5 PEA 1 Ti l HSMRACP5_PEA_1 T14 HSMRACP5 PEA 1 T20
nucleic acid sequence comprising a sequence in the table below:
HSMRACP5 PEA 1 Pl l HSMRACP5 PEA 1 P12 HSMRACP5 PEA 1 P14
an isolated chimeric polypeptide encoding for HSMRACP5_PEA_1_P11 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSMRACP5_PEA_1_P11 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 VCAQQSGAGGGGGQWGEAALPSDLPLVRAEGR in HSMRACP5_PEA_1_P 11.
an isolated chimeric polypeptide encoding for HSMRACP5_PEA_1_P11 comprising a first amino acid sequence being at least 90 % homologous to MDMWTALLILQALLLPSLADGATPALRFVAVGDWGGVPNAPFHTAREMANAKEIART VQILGADFILSLGDNFYFTGVQDINDKRFQ conesponding to amino acids 1 - 87 of
PPA5_HUMAN which also conesponds to amino acids 1 - 87 of HSMRACP5_PEA_1_P11, 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 VCAQQSGAGGGGGQWGEAALPSDLPLVRAEGR conesponding to amino acids 88 - 1 19 of HSMRACP5_PEA_1_P11, wherein said first and second amino acid sequences are contiguous and in a sequential order.
an isolated polypeptide encoding for a tail of HSMRACP5_PEA_1_P11 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 VCAQQSGAGGGGGQWGEAALPSDLPLVRAEGR in HSMRACP5_PEA_1_P11.
an isolated chimeric polypeptide encoding for HSMRACP5_PEA_1_P12 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of HSMRACP5_PEA_1_P12 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 PW, having a structure as follows: a sequence starting from any of amino acid numbers 103-x to 104; and ending at any of amino acid numbers 104+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSMRACP5_PEA_1_P14 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSMRACP5_PEA_1_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 EGETQLMNCGAT in HSMRACP5_PEA_1_P14.
an isolated chimeric polypeptide encoding for HSMRACP5_PEA_1_P14 comprising a first amino acid sequence being at least 90 % homologous to
HSMRACP5_PEA_1_P14 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 EGETQLMNCGAT conesponding to amino acids 102 - 113 of HSMRACP5_PEA_1_P14, wherein said first and second amino acid sequences are contiguous and in a sequential order.
an isolated polypeptide encoding for a tail of HSMRACP5_PEA_1_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 EGETQLMNCGAT in HSMRACP5_PEA_1_P14.
an isolated chimeric polypeptide encoding for HSMRACP5_PEA_1_P14 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSMRACP5_PEA_1_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 EGETQLMNCGAT in HSMRACP5_PEA_1_P14.
an isolated chimeric polypeptide encoding for HUMFBRB_PEA_1_P4 comprising a first amino acid sequence being at least 90 % homologous to MKRMVSWSFHKLKTMKHLLLLLLCVFLVKSQGVNDNEEGFFSARGHRPLDKKREEAP SLRPAPPPISGGGYRARPAKAAATQKKVERKAPDAGGCLHADPDLGVLCPTGCQLQEA LLQQERPIR SVDELNNNVEAVSQTSSSSFQYMYLLKDLWQKRQKQVKDNENVVNEY SSELEKHQLYIDETVNSNIPTNLRVLRSILENLRSKIQKLESDVSAQMEYCRTPCTVSCNI PWSGKECEEIIRKGGETSEMYLIQPDSSVKPYRVYCDMNTENGGWTVIQNRQDGSVDF GRKWDPYKQGFGNVATNTDGKNYCGLPGEYWLGND
an isolated polypeptide encoding for a tail of HUMFBRB_PEA_1_P4 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 YVWHSLLLL in HUMFBRB_PEA_1_P4.
an isolated chimeric polypeptide encoding for HUMFBRB_PEA_1_P9 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HUMFBRB_PEA_1_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 NEQACKIKSFYLKWDFF in HUMFBRB_PEA_1_P9.
an isolated chimeric polypeptide encoding for HUMFBRB_PEA_1_P11 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HUMFBRB_PEA_1_P11 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 KLSTWDLLICNYLDTVKCQETRPGWAHTCNSSTLGGQSGLIA in HUMFBRB_PEA_1_P11.
an isolated chimeric polypeptide encoding for HUMFBRB_PEA_1_P13 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of HUMFBRB_PEA_1_P17 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 GG, having a structure as follows: a sequence starting from any of amino acid numbers 277-x to 277; and ending at any of amino acid numbers 278+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HUMFBRB_PEA_1_P26 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of HUMFBRB_PEA_1_P26 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 EE, having a stmcture as follows: a sequence starting from any of amino acid numbers 54-x to 54; and ending at any of amino acid numbers 55+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HUMPGCA_PEA_1_P12 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HUMPGCA_PEA_1_P12 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
an isolated chimeric polypeptide encoding for HUMPGCA_PEA_1_P14 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for HUMPGCA_PEA_1_P15 comprising a first amino acid sequence being at least 90 % homologous to MKWMWVLVCLQLLEAAWKVPLKKFKSIRETMKEKGLLGEFLRTHKYDPAWKYRF GDLSVTYEPMAYMD conesponding to amino acids 1 - 70 of PEPC_HUMAN, which also conesponds to amino acids 1 - 70 of HUMPGCA_PEA_1_P15, and a second amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for an edge portion of HUMPGCA_PEA_1_P15 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 DV, having a structure as follows: a sequence starting from any of amino acid numbers 70-x to 70; and ending at any of amino acid numbers 71+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSPROSAP_PEA_l_P3 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for HSPROSAP_PEA_l_P9 comprising a first amino acid sequence being at least 90 % homologous to MRAAPLLLARAASLSLGFLFLLFFWLDRSVLAKELKFVTLVFRHGDRSPIDTFPTDPIKE SSWPQGFGQLTQLGMEQHYELGEYIRKRYRKFLNESYKHEQVYIRSTDVDRTLMSAMT NLAALFPPEGVSIWNPILLWQPIPVHTVPLSEDQLLYLPFRNCPRFQELESETLKSEEFQK RLHPYKDFIATLGKLSGLHGQDLFGIWSKVYDPLYCESVHNFTLPSWATEDTMTKLREL SELSLLSLYGIHKQKEKSRLQGGVLVNEILNHMKRATQIPSYKKLIMYSAHDTTVSGLQ MALDVYNGLLPPYASCHLTELYFEKGEYFVEMYYRNET
PPAP_HUMAN which also conesponds to amino acids 1 - 380 of HSPROSAP_PEA_l_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 PAETAHSARRNHDIALPCGRSTCLENTVLYYHYG conesponding to amino acids 381 - 414 of HSPROSAP_PEA_l_P9, 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 HSPROSAP_PEA_l_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 PAETAHSARRNHDIALPCGRSTCLENTVLYYHYG in HSPROSAP_PEA_l_P9.
an isolated polypeptide encoding for a tail of HSPROSAP_PEA_l_Pl 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 VKEKKITG in HSPR0SAP_PEA_1_P11.
an isolated chimeric polypeptide encoding for HSPROSAP_PEA_l_P12 comprising a first amino acid sequence being at least 90 % homologous to
an isolated chimeric polypeptide encoding for HSPROSAP_PEA_l_P13 comprising a first amino acid sequence being at least 90 % homologous to MRAAPLLLARAASLSLGFLFLLFFWLDRSVLAKELKFVTLVFRHGDRSPIDTFPTDPIKE SSWPQGFGQLTQLGMEQHYELGEYIRKRYRKFLNESYKHEQVYIRSTDVDRTLMSAMT NLAALFPPEGVSIWNPILLWQPIPVHTVPLSEDQ conesponding to amino acids 1 - 152 of PPAP_HUMAN, which also conesponds to amino acids 1 - 152 of HSPROSAP_PEA_l_P13, 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 VSILGKPGD
an isolated polypeptide encoding for a tail of HSPROSAP_PEA_l_P13 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 VSILGKPGDFRWT in HSPROSAP_PEA_l_P13.
an isolated chimeric polypeptide encoding for HSPROSAP_PEA_l_P14 comprising a first amino acid sequence being at least 90 % homologous to MRAAPLLLARAASLSLGFLFLLFFWLDRS VLAKELKFVTLVFRHGDRSPIDTFPTDPIKE SSWPQGFGQLTQLGMEQFTYELGEYIRKRYRKFLNESYKHEQVYIRSTDVDRTLMSAMT NLAALFPPEGVSIWNPILLWQPIPVHTVPLSEDQ conesponding to amino acids 1 - 152 of PPAP_HUMAN, which also conesponds to amino acids 1 - 152 of HSPROSAP_PEA_l_P14, 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
SHHRHDHRISLWLKLSLTAGPRLLPSDLWGRLLSSLSCQYP corresponding to amino acids 153 - 193 of HSPROSAP_PEA_l_P14, 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 HSPROSAP_PEA_l_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 SHHRHDHRISLWLKLSLTAGPRLLPSDLWGRLLSSLSCQYP in HSPROSAP_PEA_l_P14.
an isolated chimeric polypeptide encoding for HSPROSAP_PEA_l_P23 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSPROSAP_PEA_l_P23 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 SRLFSLLFP in HSPROSAP_PEA_l_P23.
an isolated chimeric polypeptide encoding for HSU13680_PEA_1_P18 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSU13680_PEA_1_P18 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 GIIWNKRRTLSQYPLCLGAEWCLRCCEN in HSU13680_PEA_1_P18.
an isolated chimeric polypeptide encoding for HSU13680_PEA_1_P19 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSU13680_PEA_1_P19 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 MKLSS in HSU13680_PEA_1_P19.
an isolated chimeric polypeptide encoding for HSU13680_PEA_1_P15 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSU13680_PEA_1_P15 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 NFCIF in HSU13680_PEA_1_P15.
an isolated chimeric polypeptide encoding for HSGROWl_PEA_l_PEA_l_P17 comprising a first amino acid sequence being at least 90 % homologous to
an isolated polypeptide encoding for a tail of HSGROWl_PEA_l_PEA_l_P17 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 VSSWGMGAHQGWQEGVTFPRWEIRGGD in HSGROWl_PEA_l_PEA_l_P17.
an isolated chimeric polypeptide encoding for HSGROWl_PEA_l_PEA_l_P18 comprising a first amino acid sequence being at least 90 % homologous to
HSGROWl_PEA_l_PEA_l_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 VRVAPGVPNPGAPLTLRAVLEKHCCPLFSSQALTQENSPYSSFPLVNPPGLSLHPEGEGG K conesponding to amino acids 153 - 213 of HSGROWl_PEA_l_PEA_l_P9, 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 HSGROWl_PEA_l_PEA_l_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
an isolated chimeric polypeptide encoding for HSGROW1_PEA_1_PEA_1_P10 comprising a first amino acid sequence being at least 90 % homologous to
HSGROW1_PEA_1_PEA_1_P10 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 FL, having a structure as follows: a sequence starting from any of amino acid numbers 57-x to 57; and ending at any of amino acid numbers 58+ ((n-2) - x), in which x varies from 0 to n-2.
an isolated chimeric polypeptide encoding for HSGROWl_PEA_l_PEA_l_P15 comprising a first amino acid sequence being at least 90 % homologous to
HSGROWl_PEA_l_PEA_l_P15 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 FR, having a stmcture as follows: a sequence starting from any of amino acid numbers 57-x to 57; and ending at any of amino acid numbers 58+ ((n-2) - x), in which x varies from 0 to n-2.
an antibody capable of specifically binding to an epitope of an amino acid sequence as described herein.
the amino acid sequence corresponds to a bridge, edge portion, tail, head or insertion as in any of the previous claims.
the antibody is capable of differentiating between a splice variant having said epitope and a conesponding known protein.
a kit for detecting a Marker- detectable disease comprising a kit detecting specific expression of a splice variant as described herein.
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 as described herein.
the kit further comprises at least one oligonucleotide capable of selectively hybridizing to a nucleic acid sequence as described herein.
the kit comprises an antibody as described herein.
the kit further comprises at least one reagent for performing an ELISA or a Western blot.
the immunoassay comprises an antibody as described herein.
a biomarker capable of detecting Marker- detectable disease comprising any nucleic acid sequence described herein or a fragment thereof, or any amino acid sequence described herein or a fragment thereof.
a method for screening for variant-detectable disease comprising detecting cells affected by a Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
a method for diagnosing a marker-detectable disease comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any ofthe above claims.
a method for monitoring disease progression and or treatment efficacy and/or relapse of Marker- detectable disease comprising detecting cells affected by Marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims.
a method of selecting a therapy for a marker- detectable disease comprising detecting cells affected by a marker-detectable disease with a biomarker or an antibody or a method or assay according to any of the above claims and selecting a therapy according to said detection.
cardiac disease and/or pathology and/or condition and/or disorder may comprise one or more of Myocardial infarct, acute coronary syndrome, angina pectoris (stable and unstable), cardiomyopathy, myocarditis, congestive heart failure or any type of heart failure, the detection of reinfarction, the detection of success of thrombolytic therapy after Myocardial infarct, Myocardial infarct after surgery, assessing the size of infarct in Myocardial infarct, the differential diagnosis of heart related conditions from lung related conditions (as pulmonary embolism), the differential diagnosis of Dyspnea, and cardiac valves related conditions.
Biomolecular sequences amino acid and/or nucleic acid sequences
Biomolecular 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 cardiac disease and/or cardiac pathology, including but not limited to cardiac damage, and/or are otherwise expressed at a much higher level and/or specifically expressed in heart.
the method of the present invention identifies clusters (genes) which are characterized in that the transcripts are differentially expressed in heart muscle tissue compared with other normal tissues, preferably in comparison to skeletal muscle tissue.
heart muscle tissue differentially expressed proteins are potential acute heart damage markers. Leakage of intracellular content can also occur in chronic damage to the heart muscle, therefore proteins selected according to this method are potential markers for chronic heart conditions.
proteins selected according to this method are potential markers for chronic heart conditions.
BNP brain natriuretic peptide
ANF atrial natriuretic factor
BNP and ANF are not only differentially expressed in heart tissue, they are also overexpressed dramatically (hundreds of times greater expression) when heart failure occurs.
Other heart specific secreted proteins might present similar overexpression in chronic damage.
the markers described herein are overexpressed in heart as opposed to muscle, as described in greater detail below.
the measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can conelate with a probable diagnosis of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage.
the present invention therefore also relates to diagnostic assays for cardiac disease and/or cardiac pathology, including but not limited to cardiac damage, and methods of use of such markers for detection of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage (alone or in combination), optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
the present invention therefore also relates to diagnostic assays for cardiac disease and/or cardiac pathology, including but not limited to cardiac damage, and methods of use of such markers for detection of cardiac disease and/or cardiac pathology, including but not limited to cardiac damage (alone or in combination), optionally and preferably in a sample taken from a subject (patient), which is more preferably some type of blood sample.
any ofthe 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.
nucleic acid sequences and/or amino acid sequences shown herein as embodiments ofthe 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 schematic summary of cancer biomarkers selection engine and the wet validation stages.
Figure 2. Schematic illustration, depicting grouping of transcripts of a given cluster based on presence or absence of unique sequence regions.
Figure 3 is schematic presentation of the oligonucleotide based microarray fabrication.
Figure 4 is schematic summary of the oligonucleotide based microanay experimental flow.
Figure 5 is a histogram showing Cancer and cell- line vs. normal tissue expression for Cluster HSPROSAP, demonstrating overexpression in a mixture of malignant tumors from different tissues.
Figure 6 is a histogram showing Cancer and cell- line vs.
FIG. 7 is a histogram showing Cancer and cell- line vs. normal tissue expression for Cluster T87096, demonstrating overexpression in kidney malignant tumors and pancreas carcinoma.
Figure 8 is a histogram showing selective expression of cluster S42303 in heart tissue, calculated based on the number of heart- specific clones in libraries/sequences.
Figures 9-10 are histograms, demonstrating the expression of cluster S42303, as measured by the actual expression of oligonucleotides in various tissues, including heart.
Figure 11 is a histogram showing Cancer and cell- line vs.
FIG. 12 is a schematic summary of quantitative real-time PCR analysis.
Figure 13 is a histogram showing down regulation of the Mast/stem cell growth factor receptor SCFR (HSKITCR) transcripts, which are detectable by amplicon as depicted in sequence name HSKITCR seg3F2R2, in cancerous ovary samples relative to the normal samples.
HSKITCR Mast/stem cell growth factor receptor SCFR
Figure 14 is a histogram showing down regulation of the Mast/stem cell growth factor receptor SCFR (HSKITCR) transcripts, which are detectable by amplicon as depicted in sequence name HSKITCR seg3F2R2, in cancerous colon samples relative to the normal samples.
Figures 15 is a histogram showing overexpression of the Mast/stem cell growth factor receptor SCFR (HSKITCR) transcripts, which are detectable by amplicon as depicted in sequence name HSKITCR seg3F2R2, in cancerous lung samples relative to the normal samples.
HSKITCR Mast/stem cell growth factor receptor SCFR
Figures 16 is a histogram showing down regulation of the Mast/stem cell growth factor receptor SCFR (HSKITCR) transcripts, which are detectable by amplicon as depicted in sequence name HSKITCR seg3F2R2, in cancerous lung samples relative to the normal samples.
Figures 17 is a histogram showing overexpression of the Mast/stem cell growth factor receptor SCFR (HSKITCR) transcripts, which are detectable by amplicon as depicted in sequence name HSKITCR seg3F2R2, in cancerous prostate samples relative to the normal samples.
HSKITCR Mast/stem cell growth factor receptor SCFR
Figures 18 is a histogram showing down regulation of the Mast stem cell growth factor receptor SCFR (HSKITCR) transcripts, which are detectable by amplicon as depicted in sequence name HSKITCR seg3F2R2, in cancerous breast samples relative to the normal samples.
Figure 19 is a histogram demonstrating the expression of Mast/stem cell growth factor receptor Kit HSKITCR transcripts which are detectable by amplicon as depicted in sequence name HSKITCR seg3F2R2 in different normal tissues.
HSKITCR Mast stem cell growth factor receptor SCFR
Figure 20 is a histogram showing down regulation of the Mast/stem cell growth factor receptor SCFR (HSKITCR) transcripts, which are detectable by amplicon as depicted in sequence name HSKITCR seg44F2R2, in cancerous colon samples relative to the normal samples.
Figure 21 is a histogram showing down regulation of the Mast/stem cell growth factor receptor SCFR (HSKITCR) transcripts, which are detectable by amplicon as depicted in sequence name HSKITCR seg44F2R2, in cancerous breast samples relative to the normal samples.
HSKITCR Mast/stem cell growth factor receptor SCFR
Figure 22 is a histogram showing down regulation of the Mast/stem cell growth factor receptor SCFR (HSKITCR) transcripts, which are detectable by amplicon as depicted in sequence name HSKITCR seg44F2R2, in cancerous lung samples relative to the normal samples.
Figure 23 is a histogram showing down regulation of the Mast/stem cell growth factor receptor SCFR (HSKITCR) transcripts, which are detectable by amplicon as depicted in sequence name HSKITCR seg44F2R2, in cancerous ovary samples relative to the normal samples.
HSKITCR Mast/stem cell growth factor receptor SCFR
Figure 24 is a histogram demonstrating the expression of Mast/stem cell growth factor receptor Kit HSKITCR transcripts which are detectable by amplicon as depicted in sequence name HSKITCR seg44F2R2 in different normal tissues.
Figure 25 is a histogram showing selective expression of cluster HUMCKMA in heart tissue, calculated based on the number of heart- specific clones in libraries/sequences.
Figure 26 is a histogram, demonstrating the expression of cluster HUMCKMA, as measured by the actual expression of oligonucleotides in various tissues, including heart.
Figure 27 is a schematic presentation of the wild type and new variants of Homo sapiens alkaline phosphatase, liver/bone/kidney (ALPL) HSAPHOL mRNA and protein structures.
Orange boxes indicate the regions, representing exons. Arrows represent the introns. Yellow boxes indicate the amino acid coding regions. Green boxes represent the unique amino acids, encoded by the new variants; the number of the unique amino acids in each variant is indicated within each box.
the known mRNA and protein is indicated by "WT”.
the new variants are marked as T10, T4, T6, T5, and T8, respectively.
the location of the GPI-anchor and the location of the CGEN-oligo are indicated.
Figures 28-29 are histograms showing on two different scales the expression of Homo sapiens alkaline phosphatase, liver/bone/kidney (ALPL) HSAPHOL transcripts, which are detectable by amplicon as depicted in sequence name HSAPHOL junc2- 13, in different normal tissues.
Figure 30 is a histogram showing the expression of the Homo sapiens alkaline phosphatase, liver/bone/kidney (ALPL) HSAPHOL transcripts which are detectable by amplicon as depicted in sequence name HSAPHOL seg26F2R2 in different normal tissues.
Figure 31 is a histogram showing the expression of the Homo sapiens alkaline phosphatase, liver/bone/kidney (ALPL) HSAPHOL transcripts which are detectable by amplicon as depicted in sequence name HSAPHOL seg38 in different normal tissues.
Figures 32-33 are histograms showing on two different scales the expression of Homo sapiens C- reactive protein, pentraxin- related (CRP) HSCREACT transcripts which are detectable by amplicon as depicted in sequence name HSCREACT juncl 1-53F2R2 in different normal tissues.
CRP pentraxin-related
Figure 34-35 are histograms showing on two different scales the expression of Homo sapiens C-reactive protein, pentraxin-related (CRP) HSCREACT transcripts which are detectable by amplicon as depicted in sequence name HSCREACT juncl2-30F2R2 in different normal tissues.
Figure 36-37 are histograms showing on two different scales the expression of Homo sapiens C-reactive protein, pentraxin-related (CRP) HSCREACT transcripts which are detectable by amplicon as depicted in sequence name HSCREACT juncl2-53F2R2 in different normal tissues.
Figure 38 is a histogram showing the expression of Homo sapiens C-reactive protein, pentraxin-related (CRP) HSCREACT transcripts which are detectable by amplicon as depicted in sequence name HSCREACT junc24-47F2R2 in different normal tissues.
Figure 39 is a histogram showing the expression of Homo sapiens C-reactive protein, pentraxin-related (CRP) HSCREACT transcripts which are detectable by amplicon as depicted in sequence name HSCREACT seg8-l l in different normal tissues.
Figure 40 is a histogram showing the expression of of the Mast/stem cell growth factor receptor SCFR (HSKITCR) transcripts, which are detectable by amplicon as depicted in sequence name HSKITCR seg44F2R2, in cancerous prostate samples relative to the normal samples.
HSKITCR Mast/stem cell growth factor receptor SCFR
the present invention provides variants, which may optionally be used as diagnostic markers.
these variants are useful as diagnostic markers for marker- detectable (also refened to herein as “variant-detectable") diseases as described herein.
Differential variant markers are collectively described as "variant disease markers”.
the markers of the present invention alone or in combination, can be used for prognosis, prediction, screening, early diagnosis, staging, therapy selection and treatment monitoring of a marker- detectable disease.
these markers may be used for staging the disease in patient (for example if the disease features cancer) and/or monitoring the progression of the disease.
the markers of the present invention can be used for detection of the source of metastasis found in anatomical places other than the originating tissue, again in the example of cancer.
one or more of the markers may optionally be used in combination with one or more other disease 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 a particular disease, and/or are otherwise expressed at a much higher level and/or specifically expressed in tissue or cells afflicted with or demonstrating the disease.
the measurement of these markers, alone or in combination, in patient samples provides information that the diagnostician can conelate with a probable diagnosis of a particular disease and/or a condition that is indicative of a higher risk for a particular disease.
the present invention therefore also relates to diagnostic assays for marker-detectable disease and/or an indicative condition, and methods of use of such markers for detection of marker-detectable disease 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.
the present invention relates to bridges, tails, heads and/or insertions, and/or analogs, homologs and derivatives of such peptides.
Such 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.
a splice variant having such a tail may optbnally 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 conesponding 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.
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 conesponding 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).
bridges 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. Furthermore, bridges are described with regard to a sliding window in certain contexts below.
a bridge between two edges may optionally be described as follows: a bridge portion of CONTIG-NAME_Pl (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-NAME_Pl): 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) - x) (for example), in which x varies from 0 to n-2.
this invention provides antibodies specifically recognizing the splice variants and polypeptide fragments thereof of this invention. Preferably such 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 conesponding 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 conelates 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 conelates 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 marker-detectable disease and/or an indicative condition.
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 marker- detectable disease and/or an indicative condition, including a transition from an indicative condition to marker- detectable disease.
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 fagment thereof, or a splice variant nucleic acid sequence or a fragment thereof may be featured as a biomarker for detecting marker- detectable disease and/or an indicative condition, 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 conesponding 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, conesponding 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.
Nucleic acid sequences and Oligonucleotides 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).
cDNA complementary polynucleotide sequence
genomic polynucleotide sequence e.g., a combination of the above.
composite polynucleotide sequences e.g., a combination of the above.
the phrase "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.
Prefened 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 inventbn 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.
the oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
oligonucleotides are those modified at one or more of the backbone, intemucleoside linkages or bases, as is broadly described hereinunder.
prefened oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural intemucleoside linkages.
Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat.
Prefened 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 aminoalkylphosphoramidates, 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'.
modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside 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
others having mixed N, O, S and CFfe 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 intemucleoside linkage, i.e., 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.
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 pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
5-methylcytosine substi tions have been shown to increase nucleic acid duplex stability by 0.6- 1.2 °C and are presently prefened base substitutions, even more particularly when combined with 2'-0-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 bast 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. Acad. Sci.
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.
the construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cos id, a phage, a vims 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.
Cunently preferred in vivo nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I vims, or adeno- associated vims (AAV) and lipid-based systems.
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 prefened constructs for use in gene therapy are viruses, most preferably adenovimses, AAV, lentiviruses, or refrovimses.
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 vims used, unless it is already present in the viral construct.
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.
such constructs 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).
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).
kits containing probes on a dipstick setup and the like 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 % dextrare sulfate, 1 M NaCl, 1 % SDS and 5 x 10 ⁇ cpm 32 P labeled probe, at 65 °C, with a final wash solution of 0.2 x SSC and 0.1 % SDS and final wash at 65°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 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 50 °C.
a hybridization solution such as containing 10 % dextrare sulfate, 1 M NaCl, 1 % SDS and 5 x 10 ⁇ cpm 32 P labeled probe, at 65 °C
moderate hybridization is effected using a
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.
detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies.
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.
oligonucleotide probes when fluorescent ly- labeled oligonucleotide probes are used, 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. Those skilled in the art will appreciate that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the oligonucleotide primers and probes.
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, l4 C, 32 P, and 35 S.
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
NAT-based assays 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).
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 canied out by a number of suitable methods. See generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8: 14
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. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 1989, supra).
PCR polymerase chain reaction
LCR ligase chain reaction
SDA strand displacement amplification
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.
the 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 canied 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
for practicing the present invention 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 Cunent Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.). It will be appreciated that antisense oligonucleotides may be employed to quantify expression of a splice isoform of interest. Such detection is effected at the pre- mRNA level. Essentially the ability to quantitate transcription from a splice site of interest can be effected based on splice site accessibility.
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 °C, preferably less than 5 °C, more preferably less than 4 °C, most preferably less than 3 °C, ideally between 3 °C and 0 °C.
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 Multis et ⁇ /., 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.
Ligase Chain Reaction (LCR or LAR): The ligase chain reaction [LCR; sometimes refened to as “Ligase Amplification Reaction” (LAR)] has developed into a well-recognized alternative method of amplifying nucleic acids.
LCR four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, which hybridize to the opposite strand are mixed and DNA ligase is added to the mixture.
ligase will covalently link each set of hybridized molecules.
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).
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.
Self-Sustained Synthetic Reaction (3SR/NASBA) 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' 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.
the use of 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.
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.
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 ofthe 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.
a similar 3'-mismatch strategy is used with greater effect to prevent ligation in the LCR.
the direct detection method may be, for example a cycling probe reaction (CPR) or a branched DNA analysis.
Cycling probe reaction 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 canied 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.
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, Denaturing/Temperature Gradient Gel Electrophoresis (DGGE/TGGE), Single-Strand Conformation Polymorphism (SSCP) analysis or Dideoxy fingerprinting (ddF).
RFLP analysis restriction fragment length polymorphism
ASO allele specific oligonucleotide
DGGE/TGGE Denaturing/Temperature Gradient Gel Electrophoresis
SSCP Single-Strand Conformation Polymorphism
ddF Dideoxy fingerprinting
test sample e.g., a bacterial isolate
amplified material e.g., PCR reaction products
This avoids the time and expense associated with cloning the segment of interest.
specialized equipment and highly trained personnel are required, and the method is too labor- intense and expensive to be practical and effective in the clinical setting.
a given segment of nucleic acid may be characterized on several other levels. At the lowest resolution, 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 ai 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 RFLP: For detection of single-base differences between like sequences, the requirements ofthe 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.
a change in the pattern of digestion can be used as a diagnostic tool (e.g., restriction fragment length polymo ⁇ hism [RFLP] analysis).
RFLP restriction fragment length polymo ⁇ hism
Single point mutations have been also detected by the creation or destruction of RFLPs. Mutations are detected and localized by the presence and size of the RNA fragments generated by cleavage at the mismatches.
Single nucleotide mismatches in DNA heteroduplexes are also recognized and cleaved by some chemicals, providing an alternative strategy to detect single base substitutions, generically named the "Mismatch Chemical Cleavage" (MCC).
MCC Mismatch Chemical Cleavage
RFLP analysis suffers from low sensitivity and requires a large amount of sample.
RFLP analysis is used for the 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.
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. With either of the techniques described above (i.e., RFLP and ASO), 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 is 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 requires the use of specialized equipment which can generate a temperamre gradient pe ⁇ endicularly 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 Polymo ⁇ hism” (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.
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 infra- molecular interactions can form and not be disturbed during the ran.
a DNA segment e.g., a PCR product
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 f ⁇ nge ⁇ 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 ebcfrophoresed on nondenaturing polyacrylamide gels to detect alterations in mobility of the termination segments as in SSCP analysis.
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).
all of these methods are limited as to the size of the nucleic acid fragment that can be analyzed.
sequences of greater than 600 base pairs require cloning, with the consequent delays and expense of either deletion sub-cloning or primer walking, in order to cover the entire fragment.
SSCP and DGGE have even more severe size limitations. Because of reduced sensitivity to sequence changes, these methods are not considered suitable for larger fragments.
SSCP is reportedly able to detect 90 % of single-base sub stitutions within a 200 base-pair fragment, the detection drops to less than 50 % for 400 base pair fragments. Similarly, the sensitivity of DGGE decreases as the length of the fragment reaches 500 base-pairs.
the ddF technique as a combination of direct sequencing and SSCP, is also limited by the relatively small size of the DNA that can be screened.
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 eaction, branched DNA, restriction fragment length polymo ⁇ hism 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 eaction, branched DNA, restriction fragment length polymo ⁇ hism analysis, mismatch chemical cleavage, heteroduplex analysis, allele-specific oligonu
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. Once the reaction is completed, 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 inco ⁇ orated into the nucleic acid, which is now bound to the probes attached to the chip.
the identity of the nucleic acid hybridized to a given probe can be determined. It will be appreciated that when utilized along with automated equipment, the above described detection methods can be used to screen multiple samples for a disease and/or pathological condition both rapidly and easily.
polypeptide amino acid sequences and peptides
polypeptide amino acid sequences and peptides
polypeptide polypeptide
peptide amino acid residues
protein 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 conesponding naturally occurring amino acid, as well as to naturally occuning amino acid polymers.
Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
polypeptide polypeptide
peptide and protein
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 Monow 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.
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 homology/identity is determined by using BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters, which preferably include using the DUST filter program, and dso 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, T ⁇ , Tyr and Phe may be substituted for synthetic non- natural acid such as Phenyl glycine, TIC, naphthylelanine (Nol), 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 omithine.
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.
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 natural 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.
Antibodies refers to a polypeptide ligand that is preferably substantially encoded by an immunoglobuhn gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen).
the recognized immunoglobuhn 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, CHI, 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' 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
Single chain antibody 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.
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 (19720].
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.
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, Lanick 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 non-human species
Fv framework residues of the human immunoglobulin are replaced by conesponding non-human residues.
Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323- 329 (1988); and Presta, Cun. 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 refened to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534- 1536 (1988)], by substituting rodent CDRs or CDR sequences for the conesponding 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 conesponding sequence from a non-human species.
humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and 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 ofthe antibody bound to the marker in the sample.
purified protein markers can be used.
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., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168.
a sample obtained from a subject can be contacted with the antibody that specifically binds the marker.
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 microtiter 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 hcubating 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.
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.
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- arker 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.
RIA Radio -immunoassay
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 in an alternate version of the RIA.
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). Presence of the substrate is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents.
Antibody binding reagents may be, for example, protein A, or other antibodies.
Antibody binding reagents may be radiolabelled 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 microscopy and subjective evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required.
Fluorescence activated cell sorting FACS: 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.
Radio-imaging Methods include but are not limited to, positron emission tomography (PET) single photon emission computed tomography (SPECT). Both of these techniques are non- invasive, and can be used to detect and/or measure a wide variety of tissue events and/or functions, such as detecting cancerous cells for example. Unlike PET, SPECT can optionally be used with two labels simultaneously. SPECT has some other advantages as well, for example with regard to cost and the types of labels that can be used. For example, US Patent No. 6,696,686 describes the use of SPECT for detection of breast cancer, and is hereby inco ⁇ orated 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
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.nlm.nih.gov/dbEST/; a general reference to dbEST, the EST database in GenBank, may be found in Boguski et al, Nat Genet.
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 inco ⁇ orated by reference as if fully set forth herein. Briefly, the software cleans the expressed sequences from repeats, vectors and immunoglobulins.
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.
SNPs sequence information
chromosomal information 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.
the potential markers were identified by a computational process that was designed to find genes and/or their splice variants that are specifically expressed in cardiac tissue, as opposed to other types of tissues and also particularly as opposed to muscle tissue, by using databases of expressed sequences.
EXAMPLE 1 Identification of differentially expressed gene products - Algorithm
an algorithm based on an analysis of frequencies was configured.
a specific algorithm for identification of transcripts specifically expressed in heart tissue is described hereinbelow.
EST analysis ESTs were taken from the following main sources: libraries contained in Genbank version 136 (June 15, 2003 ftp.ncbi.nih.gov/genbank release.notes/gbl36.release.notes) and Genbank version 139 (December 2003); and from the LifeSeq library of Incyte Co ⁇ oration (ESTs only; Wilmington, DE, USA). With regard to GenBank sequences, the human EST sequences from the EST (GBEST) section were used. Library annotation - EST libraries were manually classified according to: 1. Tissue origin 2.
Biological source - Examples of frequently used biological sources for construction of EST libraries include cancer cell- lines; normal tissues; cancer tissues; foetal 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 (described in the annotation available in Genbank). It will be appreciated that at times the protocol of library construction is not indicated in the information available about that library. The following rules were followed: EST libraries originating from identical biological samples were considered as a single library. EST libraries which included above-average levels of contamination, such as DNA contamination for example, were eliminated. The presence of such contamination was determined as follows. For each library, the number of unspliced ESTs that are not fully contained within other spliced sequences was counted.
heart tissue libraries/sequences were compared to the total number of libraries/sequences in the cluster and in Genebank, and to the relevant numbers for muscle tissue libraries/sequences.
Statistical tools were employed to identify clusters that were heart tissue specific, both as compared to all other tissues and also in comparison to muscle tissue.
the algorithm - for each tested tissue T and for each tested cluster the following were examined: 1.
Each cluster includes at least 2 libraries from the tissue T. At least 3 clones
n Tl N- T - M is the total number of ESTs available for a cluster
N is the total number of ESTs available in all of the libraries considered in the analysis (effectively all ESTs in Genbank, except for those that were rejected as belonging to contaminated libraries).
This ratio was preferably set to be at least about 8, although optionally the ratio could be set to be at least about 5.
the following equation was then used to determine heart tissue-specific expression vs.
This ratio was preferably set to be at least about 4, although optionally the ratio could be set to be at least about 2.
Fisher exact test P-values were computed for weighted clone counts to check that the counts are statistically significant according to the following function: F(t,T,n,N) which is the probability of a cluster actually being overexpressed in heart tissue, as compared to its overall level of expression. The P- value was preferably set to be less than about le-5, although optionally it could be set to be less than about le-3.
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. The following rules are followed: EST libraries originating from identical biological samples are considered as a single library. EST libraries which include above-average levels of DNA contamination are eliminated. Dry computation - development of engines which are capable of identifying genes and splice variants that are temporally and spacially expressed. Clusters (genes) having at least five sequences including at least two sequences from the tissue of interest are analyzed. EXAMPLE 2 Identification of genes over expressed in cancer. Two different scoring algorithms were developed.
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.
Library counting Small libraries (e.g., less than 1000 sequences) were excluded from consideration unless they participate in the cluster. For this reason, the total number of libraries is actually adjusted for each cluster. 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.
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”.
the algorithm - for each tested tissue T and for each tested cluster the following were examined: 1. 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 2. 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.
EXAMPLE 4 Identification of splice variants over expressed in cancer of clusters which are not over expressed in cancer Cancer-specific splice variants containing a unique region were identified. Identification of unique sequence regions in splice variants 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 refened also as “seg” or “node”) is defined as the shortest contiguous transcribed region without known splicing inside. Only reliable ESTs were considered for region and segment analysis.
An EST was defined as unreliable if: (i) Unspliced; (ii) Not covered by RNA; (iii) Not covered by spliced ESTs; and (iv) Alignment to the genome ends in proximity of long poly-A stretch or starts in proximity of long poly-T stretch. Only reliable regions were selected for further scoring. Unique sequence regions were considered reliable if: (i) Aligned to the genome; and (ii) Regions supported by more than 2 ESTs. The algorithm Each unique sequence region divides the set of transcripts into 2 groups: (i) Transcripts containing this region (group TA). (ii) Transcripts not containing this region (group TB).
the set of EST clones of every cluster is divided into 3 groups: (i) Supporting (originating from) transcripts of group TA (SI). (ii) Supporting transcripts of group TB (S2). (iii) Supporting transcripts from both groups (S3). Library and clones number scores described above were given to SI group. Fisher Exact Test P-values were used to check if: S 1 is significantly enriched by cancer EST clones compared to S2; and SI is significantly enriched by cancer EST clones compared to cluster background (S1+S2+S3). Identification of unique sequence regions and division ofthe group of transcripts accordingly is illustrated in Figure 2. Each of these unique sequence regions conesponds to a segment, also termed herein a "node”.
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.
EXAMPLE 5 Identification of cancer specific splice variants of genes over expressed in cancer
ii Genes shown to be over-expressed in cancer in published micro-anay experiments.
Reliable EST supported- regions were defined as supported by minimum of one of the following: (i) 3 spliced ESTs; or (ii) 2 spliced ESTs from 2 libraries; (iii) 10 unspliced ESTs from 2 libraries, or (iv) 3 libraries.
Microarray fabrication Microanays were printed by pin deposition using the MicroGrid II MGII 600 robot from BioRobotics Limited (Cambridge, UK). 50-mer oligonucleotides target sequences were designed by Compugen Ltd (Tel- Aviv, IL) as described by A. Shoshan et al, "Optical technologies and informatics", Proceedings of SPIE. Vol 4266, pp. 86-95 (2001).
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 CodeLink 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- 1 A Kibbutz Beit-Haemek, Israel) to a concentration of 50 ⁇ M.
the oligonucleotides were resuspended in 300mM sodium phosphate (pH 8.5) to final concentration of 150mM and printed at 35-40% relative humidity at 21°C.
Each slide contained a total of 9792 features in 32 subanays.
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-Coli genes which are commercially available in the Anay Control product (Anay control- sense oligo spots, Ambion Inc. Austin, TX. Cat #1781, Lot #112K06).
the slides were then rinsed twice with Ultra-pure DDW, followed by drying by centrifugation for 3 minutes at 800 ⁇ m.
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 mm/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 microanay 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).
FIG. 4 shows a schematic method for performing the microanay 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 conesponding tissues (for example, normal prostate tissue and cancerous prostate tissue).
conesponding tissues for example, normal prostate tissue and cancerous prostate tissue.
RNA 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” (microanay), 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.
SECTION 1 VARIANTS OF KNOWN SERUM MARKERS

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EP2167118A4 (en) *	2007-06-19	2010-12-08	Chempath Oy	TRANSMEMBRANE PROSTATIC ACID PHOSPHATASE
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